c-Myc及CyclinA2基因诱导大鼠耳蜗前体细胞增殖的体外研究
|
摘要
在成年哺乳动物耳蜗中,听觉毛细胞和神经元损伤后不能再生,这成为感音神经性聋难以治愈的主要原因。近年来,自新生鼠耳蜗分离出前体细胞,在体外可定向分化为表达毛细胞和螺旋神经元表型的细胞。我们在体外比较了P1、P7和P14的大鼠耳蜗前体细胞的数量、增殖能力及超微结构,结果表明出生后耳蜗前体细胞大部分处于细胞周期的静止期,并逐步通过分化和凋亡彻底地退出了细胞周期,耳蜗前体细胞也并非真正意义上的干细胞,而是处于干细胞与成熟子代细胞之间的中间细胞或是处于干细胞未端的细胞。通过实验发现c-Myc可能参与调控耳蜗的发育以及前体细胞的增殖、分化。结合之前的工作基础,我们利用腺病毒技术将c-Myc和cyclin A2共转染至新生鼠耳蜗前体细胞,可使其增殖,并促进前体细胞重新进入细胞周期。初步研究其调控机制,表明为经典的CKI-cyclin-CDK途径。
实验一大鼠耳蜗前体细胞的分离、培养
目的对新生大鼠耳蜗前体细胞进行分离、培养。
方法从P7大鼠耳蜗中分离、培养前体细胞,用免疫细胞化学的方法对前体细胞进行鉴定;血清诱导分化后鉴定分化潜能,进一步了解其多向分化特性。
结果原代培养的细胞,培养1天后即可见“细胞球”,随着培养天数的增加,可见细胞球体积增大,所含细胞增多;培养5天后,少部分细胞球内的细胞出现分化及贴壁现象。细胞球内大部分细胞呈nestin、musashi1和BrdU阳性,表明其具有自我更新及有丝分裂的能力。细胞球经诱导分化14 d后,对分化细胞行免疫细胞化学鉴定,发现分化细胞表达毛细胞标志物myosin VIIA和phalloidin,表达成熟神经元标志物NeuN,表达不成熟神经元标志物Tuj1,表达星形胶质细胞标志物GFAP,表达少突胶质细胞标志物galactocerebroside,以及谷氨酸能神经元标志物GluR-1,证明其具有多向分化潜能。
结论本部分实验为研究耳蜗前体细胞成年后“沉默”的机制奠定基础,为研究通过基因治疗的方法促进前体细胞增殖提供了一种体外模型。
实验二不同日龄大鼠耳蜗前体细胞增殖能力及超微结构的比较
目的耳蜗前体细胞在成年后沉默或消失的具体机制尚不清楚,为探讨其原因,我们在体外比较了P1、P7和P14的大鼠耳蜗前体细胞的数量、增殖能力及超微结构,观察耳蜗前体细胞的转归。
方法对P1、P7和P14分离出的耳蜗前体细胞,经体外培养7天后进行计数,检测其数量变化,并每隔5天传代观察传代数;采用流式细胞技术检测细胞周期,来评估不同日龄新生大鼠耳蜗前体细胞增殖能力的变化;应用透射电镜观察不同日龄新生大鼠耳蜗前体细胞的超微结构。
结果出生1周后耳蜗前体细胞的数量急剧下降,出生2周后的耳蜗内未能分离出前体细胞;P7前体细胞的传代数较P1减少;P7较P1更多的前体细胞处于有丝分裂的静止期,增殖指数下降;P7的前体细胞超微结构中有更多分化细胞的特点。
结论耳蜗前体细胞出生后大部分处于细胞周期的静止期,并逐步通过分化和凋亡彻底地退出了细胞周期,他们并非真正意义上的干细胞,而是处于干细胞与成熟子代细胞之间的中间细胞或是处于干细胞未端的细胞。
实验三c-Myc在大鼠耳蜗组织发育和耳蜗前体细胞分化过程中的表达变化
目的原癌基因c-Myc具有调控G0/G1转换和去分化的双重作用。目前对于c-Myc在内耳中的功能尚未见报道。我们拟通过检测c-Myc在大鼠耳蜗组织发育及前体细胞分化过程中的表达情况,探讨其在哺乳动物耳蜗发育中的作用。
方法1、取E10、E15、P1、P7和P14的SD大鼠耳蜗组织,应用RT-PCR、Western blot的方法检测c-Myc在大鼠耳蜗组织发育过程中的表达情况。2、取耳蜗前体细胞和分化7天后的分化细胞,用RT-PCR、免疫细胞化学染色和Western blot的方法检测c-Myc在耳蜗前体细胞分化过程中的表达情况。结果从胚胎至出生后的耳蜗发育过程中,c-Myc表达量呈现逐渐下降趋势;在前体细胞的分化过程中c-Myc的表达量也呈现下降。
结论c-Myc的表达量在大鼠耳蜗组织发育及耳蜗前体细胞分化的过程中逐渐下降,这表明c-Myc可能参与调控大鼠耳蜗组织的发育及前体细胞的分化。
实验四Ad-c-Myc-EGFP和Ad-CyclinA2-EGFP的构建和转染耳蜗前体细胞的浓度确定
目的构建目的基因分别为c-Myc和CyclinA2的腺病毒载体,体外转染大鼠耳蜗前体细胞,观察转染效率及细胞活性,确定适当的转染浓度。方法1、构建复制缺陷重组腺病毒: Ad-CyclinA2-EGFP、Ad-c-Myc-EGFP及Ad-EGFP;2、在不同效靶比浓度下(MOI=50、100、150、200、250),Ad-EGFP转染耳蜗前体细胞,观察不同浓度转染时细胞形态变化,荧光显微镜观察EGFP表达情况,计算阳性细胞百分率;3、应用MTT观察细胞活性确定增殖点;4、确定MOI值,应用流式细胞仪检测转染效率。
结果毒种上清液经PCR鉴定,能够特异地扩增出预期大小的条带,证明该重组腺病毒携带目的片段。在不同效靶比浓度下,EGFP阳性细胞百分率随MOI值的升高而增大;当MOI=50,100,150时重组腺病毒对细胞的毒性作用与对照组相比无显著差异;当MOI值=200时,细胞状态不佳,出现生长抑制。根据光镜下细胞形态学证据,确定本实验采用MOI值为150。经MTT法测定,细胞生长曲线呈S形,转染后36小时做为增殖点,应用流式细胞技术检测转染效率为25.2%。
结论复制缺陷型腺病毒可有效转染原代培养的耳蜗前体细胞,为进一步研究c-Myc和CyclinA2基因对耳蜗前体细胞增殖能力的影响奠定了基础。
实验五c-Myc、CyclinA2基因对耳蜗前体细胞增殖能力的影响
目的耳蜗前体细胞在体外具有一定的增殖能力,可定向分化为表达毛细胞和神经元表型的细胞,但耳蜗前体细胞增殖能力差,并处于细胞周期的“静止”状态。我们用编码目的基因为c-Myc和CyclinA2的腺病毒表达载体转染耳蜗前体细胞,促进其增殖并重返细胞周期,从而为诱导耳蜗受损细胞再生提供新的思路和依据。
方法分别设立Ad-CyclinA2-EGFP转染组、Ad-c-Myc-EGFP转染组、Ad-EGFP转染组及Ad-CyclinA2-EGFP、Ad-c-Myc-EGFP共转染组;用复制缺陷腺病毒转染大鼠耳蜗前体细胞,MOI值为150,用流式细胞技术检测c-Myc和CyclinA2基因对耳蜗前体细胞周期的影响;BrdU孵育观察转染后有丝分裂能力的变化;观察各组传代能力变化,采用方差分析对数据进行统计学处理。
结果流式结果表明Ad-CyclinA2-EGFP转染组、Ad-c-Myc-EGFP转染组、Ad-EGFP转染组对耳蜗前体细胞周期无明显影响;而Ad-CyclinA2-EGFP、Ad-c-Myc-EGFP共转染组可使部分耳蜗前体细胞进入G1/S期和G2/M期,共转染组与对照组G0/G1期细胞比例分别为62.03±2.02%、78.27±6.09%(P﹤0.05);增殖指数分别为37.97±2.015、20.41±7.16(P﹤0.05)。共转染组中共表达绿色荧光(EGFP)、红色荧光(BrdU阳性)及蓝色荧光(Hoechst)的细胞球数量增多,表明共转染c-Myc、CyclinA2使更多的耳蜗前体细胞具有有丝分裂的能力;各组间传代能力无明显变化。
结论c-Myc基因和CyclinA2基因单独作用于耳蜗前体细胞,在体外对细胞增殖与细胞周期无明显影响。而c-Myc和CyclinA2基因共同作用于耳蜗前体细胞,可促进细胞增殖,使部分前体细胞重返细胞周期。
实验六c-Myc与CyclinA2基因促进耳蜗前体细胞增殖的作用机制
目的研究c-Myc和CyclinA2基因共同转染耳蜗前体细胞后,检测目的基因表达情况以及促进细胞增殖的机制。
方法应用real-time PCR和Western blot的方法观察转染Ad-EGFP与共转染Ad-CyclinA2-EGFP、Ad-c-Myc-EGFP时目的基因、PCNA、CDK2以及P27KIP1在细胞中的表达水平变化。
结果real-time PCR和Western blot结果证实携带目的基因的腺病毒转染耳蜗前体细胞后能有效表达出相应的mRNA和蛋白;并可升高PCNA与CDK2的表达水平,降低P27KIP1的表达水平。
结论复制缺陷型腺病毒可有效介导c-Myc和CyclinA2基因转染原代培养的耳蜗前体细胞,并通过上调细胞周期蛋白依赖激酶CDK2,下调CDK抑制蛋白P27KIP1,进而上调细胞DNA合成期标志蛋白PCNA来实现耳蜗前体细胞增殖的作用,其作用机制为经典的CKI-cyclin-CDK途径。
In adult mammalian cochlea, hair cells and spiral ganglion cells can not regenerate spontaneously after damaged. This is the major cause of permanent hearing loss. Cochlear progenitor cells have been isolated from the early postnatal rats and mice in previous studies and can differentiate into neurons, astrocytes, hair cells and supporting cells in vitro. We isolated the progenitor cells from the cochleae of 1-, 7-, and 14-day-old rats, and compared with the proliferative capacity and ultrastructure of the cells from each age group using flow cytometry and transmission electron microscopy, respectively. Our study suggests that the dormant state of the cochlear progenitor cells after birth. Although these progenitor cells displayed some features of stem cells, they lost their‘‘stemness’’and the capacity to robustly generate spheres. The cochlear progenitor cells are remnants of the stem cells that originally gave rise to the sensory epithelium. The disappearance of the cochlear progenitor cells in adult mammalian cochleae might result from their differentiation and/or apoptosis.
Our study suggests that it may be possible to keep these cells in a progenitor cell state or to activate the progenitor cells by triggering the cell cycle with the expression of cell cycle-related molecules. Hence, several cell cycle regulators, including c-myc and cyclin A2, may be involved in this progress. For better understanding of cochlea development mechanism, the level of c-myc was assessed in the embryonic and postnatal cochleae, the cochlear progenitor cells and the differentiated cells. Then, we established the adenoviral vector of c-myc and cyclinA2 gene and successfully delivered the target genes into the cochlear progenitor cells in vitro. The changes of the proliferative capacity were observed and the corresponding mechanism was further studied. We found c-myc and cyclinA2 gene may induce the progenitor cells proliferation and reenter into cell cycle. The mechanism is the classic way to CKI-cyclin-CDK.
1. Isolation and culture of cochlear progenitor cells from newborn rats
Objective: To isolate and culture the CPCs. Methods: The CPCs were isolated from the P7 rat cochlea tissues and cultured. We identified progenitor cells in postnatal rat cochlea and their potential to differentiate into multiple lineages using immunocytochemistry. Results: After 1 day of culture, the acutely isolated cells formed floating otospheres. After a 3-day culture, the floating otospheres expanded gradually in both volume and cell number. Five days later, adherent and differential cells were found in some parts of the spheres. Acutely dissociated cells from the postnatal rat cochleae expressed nestin and musashi1 and incorporated BrdU after 7 days of culture. This demonstrates of these spheres not only expressed the specific markers of stem cells but also were actively undergoing mitosis. The progenitor cell-derived differentiated cells expressed myosin VIIA, phalloidin, NeuN, Tuj1, GFAP, galactocerebroside and GluR-1. The cells generated from the cochlea-derived spheres contained hair cells, neurons, neuroglial cells, and glutamic neurons. Conclusions: The progenitor cells were present in the cochlea of newborn SD rats. They had the capacity of self-renew and possess potential to differentiate into cell types of the inner ear in vitro. This experiment provided a good model in vitro for studying the mechanisms controlling the domant state of the CPCs in adult.
2. A comparison of the proliferative capacity and ultrastructure of progenitor cells from the cochleae of newborn rats of different ages
Objective: The progenitor cells that are capable of proliferation and regeneration are present in mammalian cochleae. However, none progenitor cells has been isolated from the adult cochlea. We examined the proliferative potential of cells derived from neonatal rats of various ages. The determination of the differences between the progenitor cells from rats of different ages may provide clues to the mechanisms controlling the destiny of these cells. Methods: The progenitor cells were isolated from the cochleae of 1-, 7-, and 14-day-old rats, and total cells and spheres were counted after 7 days of culture. The proliferative capacity and ultrastructure of the cells from each age group were assessed using flow cytometry and transmission electron microscopy, respectively. Results: During the first two postnatal weeks, the number of progenitor cells gradually fell to zero. This decrease occurred in parallel with the impairment of the proliferative capacity of the cells and the accumulation of progenitor cells in G0/G1. In addition, some of the cells exited the cell cycle by means of gradual maturity and apoptosis. Conclusions: Our study suggests that CPCs are remnants of the stem cells that originally gave rise to the sensory epithelium. The disappearance of the CPCs in adult mammalian cochleae may result from their differentiation and/or apoptosis.
3. Decreased level of c-myc in rat cochlea development and cochlear progenitor cell differentiation
Objective: The c-myc oncogene is a major regulator for cell proliferation, growth, and apoptosis. However, the role of this gene in the mammalian cochlea development is still unclear. To explore the role of the gene in cochleae, the c-myc expression was assessed in rat cochlea tissues of different ages, the CPCs and the progenitor cell-derived differentiated cells. Methods: We used RT-PCR to detect c-myc mRNA expression in the cochlea tissues, the CPCs and the differentiated cells. The cochlea tissues were excised from E10, E15, P1, P7 and P14 rats respectively. The differentiated cells were cultured for 7 days and the cell spheres were collected for further assay. Western blotting was used to detect c-myc protein expression in the CPCs, the differentiated cells cultured for 7 days, and the cochlea tissues (E10, E15, P1, P7 and P14). We also used immunocytochemistry to detect c-myc protein expression in the differentiated cells and the cell spheres. Results: The results indicated that c-myc level in cochlea tissues decreased gradually during embryo to newborn. Furthermore, c-myc level fell down after differentiation of CPCs. Conclusions: It was suggested that c-myc might be involved in the modulation of rat cochlea development and CPCs differentiation.
4. Construction and identification of recombinant adenovirus and determinating the suitable MOI in the transfection of the CPCs
Objective: We respectively established the adenoviral vector of c-myc and cyclinA2 gene. The adenovirus was used to transfect the CPCs in vitro. By observing the transfection efficiency and the cell viability, the suitable MOI was determined. Methods: Recombinant adenovirus were constructed. The complementary DNA sequence of c-myc and cyclinA2 gene was respectively obtained from GenBank. The sequence was subcloned into pDC316-CMV-EGFP and recombined with backbone pAdEasy-1 in BJ5183 bacteria. The adenovirus generation, amplifcation, and titer process was completed sequentially. The vector containing EGFP alone was also constructed. The CPCs were infected by the recombinant adenovirus with Enhance Green fluorescence protein (Ad-EGFP) in different MOI (50,100,150,200,250). After the CPCs re-cultured, we observed the expression of EGFP and the cell morphology through fluorescent microscopy. To determine the proliferation point, we used MTT to assess the cell viability. The MOI was ultimately determined and the infection rate was observed through Flow cytometry. Results: The recombinant adenovirus construction was testified through the detection of the purpose fragment in PCR. A positive dose-response relationship was observed between the expression of EGFP gene ranging from 50 ~250 MOI. The virus infecting rat CPCs at a MOI of 50, 100 and 150 can not impair the cell vitality and morphology. However, the cell growth inhibition occurs at a MOI of 200 and the cells necrosis occurs at a MOI of 250. According to the morphological evidence, we ultimately determined the MOI of 150. Conclusions: Adenovirus can efficiently transfect rat CPCs with EGFP gene in vitro. It will be a powerful tool for gene therapy and cell therapy based on CPCs.
5. The influence of c-myc and cyclinA2 on the gene-induced CPCs proliferation and cell cycle in Vitro
Objective: We established the adenoviral vector of c-myc and cyclinA2 gene and successfully delivered the target genes into the CPCs in vitro. Then, the changes of the proliferative capacity were observed. Methods: Plated on 6-well plates (Corning) at 2×106 cells/well, the CPCs were incubated with medium (1 ml) containing different adenoviruses (Ad-c-myc-EGFP, Ad-cyclinA2-EGFP, Ad-c-myc-EGFP and Ad-cyclinA2-EGFP, Ad-EGFP), each at the MOI of 150 using standard techniques. Flow cytometry was used for assessing the influence of the cell cycle and the capacity of proliferation. To assessing the capacity of mitosis, the CPCs cells after transfection were incorporated BrdU. Using immunocytochemistry we observed the expression of BrdU. We also observed the change of propagation. Results: Our data from flow cytometry analysis showed that the cell cycle distribution of the CPCs was significantly affected by the co-overexpression of cyclinA2 and c-myc protein. By contrast, those cells were transfected with Ad-EGFP, Ad-cyclinA2-EGFP and Ad-c-myc-EGFP respectively, and no significant differences were observed. The percentage of incorporated BrdU increased in the progenitor cells co-transfected with Ad-c-myc-EGFP and Ad-cyclinA2-EGFP. The capacity of propagation had no obviously change. Conclusions: These results suggested that cyclinA2 and c-myc induced the proliferation of CPCs and reentered into cell cycle together. However, these two genes had no alone significant effects on cell cycle and the capacity of proliferation.
6. The mechanism of c-myc and cyclinA2 inducing the CPCs proliferation
Objective: In order to examine the target gene expression and explore the underlying molecular mechanism of cyclinA2 and c-myc inducing the CPCs to reenter into the cell cycle, we detected the expression of target gene, cell cycle-related molecules and PCNA. Methods: We used real-time PCR and westen bloting to detect c-myc, cyclinA2, PCNA, CDK2 and P27kip1 mRNA and protein expression between the Ad-EGFP transfection group and the co-transfect c-myc and cyclinA2 group. Results: The expression of c-myc and cylinA2 protein and the mRNA levels in the Ad-c-myc-EGFP and the Ad-cyclinA2-EGFP infected the CPCs also showed obviously increasing trend than that of the in Ad-EGFP infected cells. At the same time, the up-regulation of cyclinA2 and c-myc protein was associated with the exaltation of CDK2 and PCNA but with the reduction of p27Kip1 in mRNA level and protein expression. Conclusions: These results suggested a successful transfection to the progenitor cells and a significant expression of target gene in these cells, including mRNA level and protein levels. The mechanism of cyclinA2 and c-myc gene inducing the CPCs proliferation and reentering into the cell cycle is the classic way through CKI-cyclin-CDK.
实验一大鼠耳蜗前体细胞的分离、培养
目的对新生大鼠耳蜗前体细胞进行分离、培养。
方法从P7大鼠耳蜗中分离、培养前体细胞,用免疫细胞化学的方法对前体细胞进行鉴定;血清诱导分化后鉴定分化潜能,进一步了解其多向分化特性。
结果原代培养的细胞,培养1天后即可见“细胞球”,随着培养天数的增加,可见细胞球体积增大,所含细胞增多;培养5天后,少部分细胞球内的细胞出现分化及贴壁现象。细胞球内大部分细胞呈nestin、musashi1和BrdU阳性,表明其具有自我更新及有丝分裂的能力。细胞球经诱导分化14 d后,对分化细胞行免疫细胞化学鉴定,发现分化细胞表达毛细胞标志物myosin VIIA和phalloidin,表达成熟神经元标志物NeuN,表达不成熟神经元标志物Tuj1,表达星形胶质细胞标志物GFAP,表达少突胶质细胞标志物galactocerebroside,以及谷氨酸能神经元标志物GluR-1,证明其具有多向分化潜能。
结论本部分实验为研究耳蜗前体细胞成年后“沉默”的机制奠定基础,为研究通过基因治疗的方法促进前体细胞增殖提供了一种体外模型。
实验二不同日龄大鼠耳蜗前体细胞增殖能力及超微结构的比较
目的耳蜗前体细胞在成年后沉默或消失的具体机制尚不清楚,为探讨其原因,我们在体外比较了P1、P7和P14的大鼠耳蜗前体细胞的数量、增殖能力及超微结构,观察耳蜗前体细胞的转归。
方法对P1、P7和P14分离出的耳蜗前体细胞,经体外培养7天后进行计数,检测其数量变化,并每隔5天传代观察传代数;采用流式细胞技术检测细胞周期,来评估不同日龄新生大鼠耳蜗前体细胞增殖能力的变化;应用透射电镜观察不同日龄新生大鼠耳蜗前体细胞的超微结构。
结果出生1周后耳蜗前体细胞的数量急剧下降,出生2周后的耳蜗内未能分离出前体细胞;P7前体细胞的传代数较P1减少;P7较P1更多的前体细胞处于有丝分裂的静止期,增殖指数下降;P7的前体细胞超微结构中有更多分化细胞的特点。
结论耳蜗前体细胞出生后大部分处于细胞周期的静止期,并逐步通过分化和凋亡彻底地退出了细胞周期,他们并非真正意义上的干细胞,而是处于干细胞与成熟子代细胞之间的中间细胞或是处于干细胞未端的细胞。
实验三c-Myc在大鼠耳蜗组织发育和耳蜗前体细胞分化过程中的表达变化
目的原癌基因c-Myc具有调控G0/G1转换和去分化的双重作用。目前对于c-Myc在内耳中的功能尚未见报道。我们拟通过检测c-Myc在大鼠耳蜗组织发育及前体细胞分化过程中的表达情况,探讨其在哺乳动物耳蜗发育中的作用。
方法1、取E10、E15、P1、P7和P14的SD大鼠耳蜗组织,应用RT-PCR、Western blot的方法检测c-Myc在大鼠耳蜗组织发育过程中的表达情况。2、取耳蜗前体细胞和分化7天后的分化细胞,用RT-PCR、免疫细胞化学染色和Western blot的方法检测c-Myc在耳蜗前体细胞分化过程中的表达情况。结果从胚胎至出生后的耳蜗发育过程中,c-Myc表达量呈现逐渐下降趋势;在前体细胞的分化过程中c-Myc的表达量也呈现下降。
结论c-Myc的表达量在大鼠耳蜗组织发育及耳蜗前体细胞分化的过程中逐渐下降,这表明c-Myc可能参与调控大鼠耳蜗组织的发育及前体细胞的分化。
实验四Ad-c-Myc-EGFP和Ad-CyclinA2-EGFP的构建和转染耳蜗前体细胞的浓度确定
目的构建目的基因分别为c-Myc和CyclinA2的腺病毒载体,体外转染大鼠耳蜗前体细胞,观察转染效率及细胞活性,确定适当的转染浓度。方法1、构建复制缺陷重组腺病毒: Ad-CyclinA2-EGFP、Ad-c-Myc-EGFP及Ad-EGFP;2、在不同效靶比浓度下(MOI=50、100、150、200、250),Ad-EGFP转染耳蜗前体细胞,观察不同浓度转染时细胞形态变化,荧光显微镜观察EGFP表达情况,计算阳性细胞百分率;3、应用MTT观察细胞活性确定增殖点;4、确定MOI值,应用流式细胞仪检测转染效率。
结果毒种上清液经PCR鉴定,能够特异地扩增出预期大小的条带,证明该重组腺病毒携带目的片段。在不同效靶比浓度下,EGFP阳性细胞百分率随MOI值的升高而增大;当MOI=50,100,150时重组腺病毒对细胞的毒性作用与对照组相比无显著差异;当MOI值=200时,细胞状态不佳,出现生长抑制。根据光镜下细胞形态学证据,确定本实验采用MOI值为150。经MTT法测定,细胞生长曲线呈S形,转染后36小时做为增殖点,应用流式细胞技术检测转染效率为25.2%。
结论复制缺陷型腺病毒可有效转染原代培养的耳蜗前体细胞,为进一步研究c-Myc和CyclinA2基因对耳蜗前体细胞增殖能力的影响奠定了基础。
实验五c-Myc、CyclinA2基因对耳蜗前体细胞增殖能力的影响
目的耳蜗前体细胞在体外具有一定的增殖能力,可定向分化为表达毛细胞和神经元表型的细胞,但耳蜗前体细胞增殖能力差,并处于细胞周期的“静止”状态。我们用编码目的基因为c-Myc和CyclinA2的腺病毒表达载体转染耳蜗前体细胞,促进其增殖并重返细胞周期,从而为诱导耳蜗受损细胞再生提供新的思路和依据。
方法分别设立Ad-CyclinA2-EGFP转染组、Ad-c-Myc-EGFP转染组、Ad-EGFP转染组及Ad-CyclinA2-EGFP、Ad-c-Myc-EGFP共转染组;用复制缺陷腺病毒转染大鼠耳蜗前体细胞,MOI值为150,用流式细胞技术检测c-Myc和CyclinA2基因对耳蜗前体细胞周期的影响;BrdU孵育观察转染后有丝分裂能力的变化;观察各组传代能力变化,采用方差分析对数据进行统计学处理。
结果流式结果表明Ad-CyclinA2-EGFP转染组、Ad-c-Myc-EGFP转染组、Ad-EGFP转染组对耳蜗前体细胞周期无明显影响;而Ad-CyclinA2-EGFP、Ad-c-Myc-EGFP共转染组可使部分耳蜗前体细胞进入G1/S期和G2/M期,共转染组与对照组G0/G1期细胞比例分别为62.03±2.02%、78.27±6.09%(P﹤0.05);增殖指数分别为37.97±2.015、20.41±7.16(P﹤0.05)。共转染组中共表达绿色荧光(EGFP)、红色荧光(BrdU阳性)及蓝色荧光(Hoechst)的细胞球数量增多,表明共转染c-Myc、CyclinA2使更多的耳蜗前体细胞具有有丝分裂的能力;各组间传代能力无明显变化。
结论c-Myc基因和CyclinA2基因单独作用于耳蜗前体细胞,在体外对细胞增殖与细胞周期无明显影响。而c-Myc和CyclinA2基因共同作用于耳蜗前体细胞,可促进细胞增殖,使部分前体细胞重返细胞周期。
实验六c-Myc与CyclinA2基因促进耳蜗前体细胞增殖的作用机制
目的研究c-Myc和CyclinA2基因共同转染耳蜗前体细胞后,检测目的基因表达情况以及促进细胞增殖的机制。
方法应用real-time PCR和Western blot的方法观察转染Ad-EGFP与共转染Ad-CyclinA2-EGFP、Ad-c-Myc-EGFP时目的基因、PCNA、CDK2以及P27KIP1在细胞中的表达水平变化。
结果real-time PCR和Western blot结果证实携带目的基因的腺病毒转染耳蜗前体细胞后能有效表达出相应的mRNA和蛋白;并可升高PCNA与CDK2的表达水平,降低P27KIP1的表达水平。
结论复制缺陷型腺病毒可有效介导c-Myc和CyclinA2基因转染原代培养的耳蜗前体细胞,并通过上调细胞周期蛋白依赖激酶CDK2,下调CDK抑制蛋白P27KIP1,进而上调细胞DNA合成期标志蛋白PCNA来实现耳蜗前体细胞增殖的作用,其作用机制为经典的CKI-cyclin-CDK途径。
In adult mammalian cochlea, hair cells and spiral ganglion cells can not regenerate spontaneously after damaged. This is the major cause of permanent hearing loss. Cochlear progenitor cells have been isolated from the early postnatal rats and mice in previous studies and can differentiate into neurons, astrocytes, hair cells and supporting cells in vitro. We isolated the progenitor cells from the cochleae of 1-, 7-, and 14-day-old rats, and compared with the proliferative capacity and ultrastructure of the cells from each age group using flow cytometry and transmission electron microscopy, respectively. Our study suggests that the dormant state of the cochlear progenitor cells after birth. Although these progenitor cells displayed some features of stem cells, they lost their‘‘stemness’’and the capacity to robustly generate spheres. The cochlear progenitor cells are remnants of the stem cells that originally gave rise to the sensory epithelium. The disappearance of the cochlear progenitor cells in adult mammalian cochleae might result from their differentiation and/or apoptosis.
Our study suggests that it may be possible to keep these cells in a progenitor cell state or to activate the progenitor cells by triggering the cell cycle with the expression of cell cycle-related molecules. Hence, several cell cycle regulators, including c-myc and cyclin A2, may be involved in this progress. For better understanding of cochlea development mechanism, the level of c-myc was assessed in the embryonic and postnatal cochleae, the cochlear progenitor cells and the differentiated cells. Then, we established the adenoviral vector of c-myc and cyclinA2 gene and successfully delivered the target genes into the cochlear progenitor cells in vitro. The changes of the proliferative capacity were observed and the corresponding mechanism was further studied. We found c-myc and cyclinA2 gene may induce the progenitor cells proliferation and reenter into cell cycle. The mechanism is the classic way to CKI-cyclin-CDK.
1. Isolation and culture of cochlear progenitor cells from newborn rats
Objective: To isolate and culture the CPCs. Methods: The CPCs were isolated from the P7 rat cochlea tissues and cultured. We identified progenitor cells in postnatal rat cochlea and their potential to differentiate into multiple lineages using immunocytochemistry. Results: After 1 day of culture, the acutely isolated cells formed floating otospheres. After a 3-day culture, the floating otospheres expanded gradually in both volume and cell number. Five days later, adherent and differential cells were found in some parts of the spheres. Acutely dissociated cells from the postnatal rat cochleae expressed nestin and musashi1 and incorporated BrdU after 7 days of culture. This demonstrates of these spheres not only expressed the specific markers of stem cells but also were actively undergoing mitosis. The progenitor cell-derived differentiated cells expressed myosin VIIA, phalloidin, NeuN, Tuj1, GFAP, galactocerebroside and GluR-1. The cells generated from the cochlea-derived spheres contained hair cells, neurons, neuroglial cells, and glutamic neurons. Conclusions: The progenitor cells were present in the cochlea of newborn SD rats. They had the capacity of self-renew and possess potential to differentiate into cell types of the inner ear in vitro. This experiment provided a good model in vitro for studying the mechanisms controlling the domant state of the CPCs in adult.
2. A comparison of the proliferative capacity and ultrastructure of progenitor cells from the cochleae of newborn rats of different ages
Objective: The progenitor cells that are capable of proliferation and regeneration are present in mammalian cochleae. However, none progenitor cells has been isolated from the adult cochlea. We examined the proliferative potential of cells derived from neonatal rats of various ages. The determination of the differences between the progenitor cells from rats of different ages may provide clues to the mechanisms controlling the destiny of these cells. Methods: The progenitor cells were isolated from the cochleae of 1-, 7-, and 14-day-old rats, and total cells and spheres were counted after 7 days of culture. The proliferative capacity and ultrastructure of the cells from each age group were assessed using flow cytometry and transmission electron microscopy, respectively. Results: During the first two postnatal weeks, the number of progenitor cells gradually fell to zero. This decrease occurred in parallel with the impairment of the proliferative capacity of the cells and the accumulation of progenitor cells in G0/G1. In addition, some of the cells exited the cell cycle by means of gradual maturity and apoptosis. Conclusions: Our study suggests that CPCs are remnants of the stem cells that originally gave rise to the sensory epithelium. The disappearance of the CPCs in adult mammalian cochleae may result from their differentiation and/or apoptosis.
3. Decreased level of c-myc in rat cochlea development and cochlear progenitor cell differentiation
Objective: The c-myc oncogene is a major regulator for cell proliferation, growth, and apoptosis. However, the role of this gene in the mammalian cochlea development is still unclear. To explore the role of the gene in cochleae, the c-myc expression was assessed in rat cochlea tissues of different ages, the CPCs and the progenitor cell-derived differentiated cells. Methods: We used RT-PCR to detect c-myc mRNA expression in the cochlea tissues, the CPCs and the differentiated cells. The cochlea tissues were excised from E10, E15, P1, P7 and P14 rats respectively. The differentiated cells were cultured for 7 days and the cell spheres were collected for further assay. Western blotting was used to detect c-myc protein expression in the CPCs, the differentiated cells cultured for 7 days, and the cochlea tissues (E10, E15, P1, P7 and P14). We also used immunocytochemistry to detect c-myc protein expression in the differentiated cells and the cell spheres. Results: The results indicated that c-myc level in cochlea tissues decreased gradually during embryo to newborn. Furthermore, c-myc level fell down after differentiation of CPCs. Conclusions: It was suggested that c-myc might be involved in the modulation of rat cochlea development and CPCs differentiation.
4. Construction and identification of recombinant adenovirus and determinating the suitable MOI in the transfection of the CPCs
Objective: We respectively established the adenoviral vector of c-myc and cyclinA2 gene. The adenovirus was used to transfect the CPCs in vitro. By observing the transfection efficiency and the cell viability, the suitable MOI was determined. Methods: Recombinant adenovirus were constructed. The complementary DNA sequence of c-myc and cyclinA2 gene was respectively obtained from GenBank. The sequence was subcloned into pDC316-CMV-EGFP and recombined with backbone pAdEasy-1 in BJ5183 bacteria. The adenovirus generation, amplifcation, and titer process was completed sequentially. The vector containing EGFP alone was also constructed. The CPCs were infected by the recombinant adenovirus with Enhance Green fluorescence protein (Ad-EGFP) in different MOI (50,100,150,200,250). After the CPCs re-cultured, we observed the expression of EGFP and the cell morphology through fluorescent microscopy. To determine the proliferation point, we used MTT to assess the cell viability. The MOI was ultimately determined and the infection rate was observed through Flow cytometry. Results: The recombinant adenovirus construction was testified through the detection of the purpose fragment in PCR. A positive dose-response relationship was observed between the expression of EGFP gene ranging from 50 ~250 MOI. The virus infecting rat CPCs at a MOI of 50, 100 and 150 can not impair the cell vitality and morphology. However, the cell growth inhibition occurs at a MOI of 200 and the cells necrosis occurs at a MOI of 250. According to the morphological evidence, we ultimately determined the MOI of 150. Conclusions: Adenovirus can efficiently transfect rat CPCs with EGFP gene in vitro. It will be a powerful tool for gene therapy and cell therapy based on CPCs.
5. The influence of c-myc and cyclinA2 on the gene-induced CPCs proliferation and cell cycle in Vitro
Objective: We established the adenoviral vector of c-myc and cyclinA2 gene and successfully delivered the target genes into the CPCs in vitro. Then, the changes of the proliferative capacity were observed. Methods: Plated on 6-well plates (Corning) at 2×106 cells/well, the CPCs were incubated with medium (1 ml) containing different adenoviruses (Ad-c-myc-EGFP, Ad-cyclinA2-EGFP, Ad-c-myc-EGFP and Ad-cyclinA2-EGFP, Ad-EGFP), each at the MOI of 150 using standard techniques. Flow cytometry was used for assessing the influence of the cell cycle and the capacity of proliferation. To assessing the capacity of mitosis, the CPCs cells after transfection were incorporated BrdU. Using immunocytochemistry we observed the expression of BrdU. We also observed the change of propagation. Results: Our data from flow cytometry analysis showed that the cell cycle distribution of the CPCs was significantly affected by the co-overexpression of cyclinA2 and c-myc protein. By contrast, those cells were transfected with Ad-EGFP, Ad-cyclinA2-EGFP and Ad-c-myc-EGFP respectively, and no significant differences were observed. The percentage of incorporated BrdU increased in the progenitor cells co-transfected with Ad-c-myc-EGFP and Ad-cyclinA2-EGFP. The capacity of propagation had no obviously change. Conclusions: These results suggested that cyclinA2 and c-myc induced the proliferation of CPCs and reentered into cell cycle together. However, these two genes had no alone significant effects on cell cycle and the capacity of proliferation.
6. The mechanism of c-myc and cyclinA2 inducing the CPCs proliferation
Objective: In order to examine the target gene expression and explore the underlying molecular mechanism of cyclinA2 and c-myc inducing the CPCs to reenter into the cell cycle, we detected the expression of target gene, cell cycle-related molecules and PCNA. Methods: We used real-time PCR and westen bloting to detect c-myc, cyclinA2, PCNA, CDK2 and P27kip1 mRNA and protein expression between the Ad-EGFP transfection group and the co-transfect c-myc and cyclinA2 group. Results: The expression of c-myc and cylinA2 protein and the mRNA levels in the Ad-c-myc-EGFP and the Ad-cyclinA2-EGFP infected the CPCs also showed obviously increasing trend than that of the in Ad-EGFP infected cells. At the same time, the up-regulation of cyclinA2 and c-myc protein was associated with the exaltation of CDK2 and PCNA but with the reduction of p27Kip1 in mRNA level and protein expression. Conclusions: These results suggested a successful transfection to the progenitor cells and a significant expression of target gene in these cells, including mRNA level and protein levels. The mechanism of cyclinA2 and c-myc gene inducing the CPCs proliferation and reentering into the cell cycle is the classic way through CKI-cyclin-CDK.
引文
1. Hawkins JE, Johnsson L-G, Stebbins WC, Moody DB, Coombs SL. Hearing Loss and Cochlear Pathology in Monkeys After Noise Exposure. Acta Oto-laryngologica 1976;81(3-6):337-343.
2. Saunders JC, Dear SP, Schneider ME. The anatomical consequences of acoustic injury: A review and tutorial. J Acoust Soc Am 1985;78(3):833-860.
3. Schacht J. Molecular mechanisms of drug-induced hearing loss. Hear Res 1986;22:297-304.
4. Forge A, Li L, Corwin JT, Nevill G. Ultrastructural evidence for hair cell regeneration in the mammalian inner ear. Science 1993;259(5101):1616-1619.
5. Lim DJ. Functional structure of the organ of Corti: a review. Hear Res 1986;22:117-146.
6. D.J. L, J R. Structural development of the cochlea. In: Roman R, editor. Development of auditory and vestibular systems 2. Oxford: Elsevier; 1992. p. 33-58.
7. Malgrange B, Thiry M, Van De Water TR, Nguyen L, Moonen G, Lefebvre PP. Epithelial supporting cells can differentiate into outer hair cells and Deiters' cells in the cultured organ of Corti. Cell Mol Life Sci 2002;59(10):1744-1757.
8. Breuskin I, Bodson M, Thelen N, Thiry M, Nguyen L, Belachew S, et al. Strategies to regenerate hair cells: identification of progenitors and critical genes. Hear Res 2008;236(1-2):1-10.
9. Zhai S, Shi L, Wang BE, Zheng G, Song W, Hu Y, et al. Isolation and culture of hair cell progenitors from postnatal rat cochleae. J Neurobiol 2005;65(3):282-293.
10. Doyle KL, Kazda A, Hort Y, McKay SM, Oleskevich S. Differentiation of adult mouse olfactory precursor cells into hair cells in vitro. Stem Cells 2007;25(3):621-627.
11. Jeon SJ, Oshima K, Heller S, Edge AS. Bone marrow mesenchymal stem cells are progenitors in vitro for inner ear hair cells. Mol Cell Neurosci 2007;34(1):59-68.
12. Li H, Roblin G, Liu H, Heller S. Generation of hair cells by stepwise differentiation of embryonic stem cells. Proc Natl Acad Sci U S A 2003;100(23):13495-13500.
13. Li H, Corrales CE, Edge A, Heller S. Stem cells as therapy for hearing loss. Trends Mol Med 2004;10(7):309-315.
14. Rivolta MN, Li H, Heller S. Generation of inner ear cell types from embryonic stem cells. Methods Mol Biol 2006;330:71-92.
15. Doetzlhofer A, White PM, Johnson JE, Segil N, Groves AK. In vitro growth and differentiation of mammalian sensory hair cell progenitors: a requirement for EGF and periotic mesenchyme. Dev Biol 2004;272(2):432-447.
16. Sobkowicz HM, Loftus JM, Slapnick SM. Tissue culture of the organ of Corti. Acta Otolaryngol Suppl 1993;502:3-36.
17. Sobkowicz HM, Holy J, Scott GL. Ultrastructure of the intercalated body, a novel organelleassociated with fluid forming cells in the organ of Corti. J Electron Microsc Tech 1990;15(3):301-315.
18. Baird RA, Burton MD, Lysakowski A, Fashena DS, Naeger RA. Hair cell recovery in mitotically blocked cultures of the bullfrog saccule. Proc Natl Acad Sci U S A 2000;97(22):11722-11729.
19. Minoda R, Izumikawa M, Kawamoto K, Raphael Y. Strategies for replacing lost cochlear hair cells. Neuroreport 2004;15(7):1089-1092.
20. Raff M. Adult stem cell plasticity: fact or artifact? Annu Rev Cell Dev Biol 2003;19:1-22.
21. Lendahl U, Zimmerman LB, McKay RD. CNS stem cells express a new class of intermediate filament protein. Cell 1990;60(4):585-595.
22. Kojima K, Tamura S, Nishida AT, Ito J. Generation of inner ear hair cell immunophenotypes from neurospheres obtained from fetal rat central nervous system in vitro. Acta Otolaryngol Suppl 2004;551(551):26-30.
23. Malgrange B, Belachew S, Thiry M, Nguyen L, Rogister B, Alvarez ML, et al. Proliferative generation of mammalian auditory hair cells in culture. Mech Dev 2002;112(1-2):79-88.
24. Abdouh A, Despres G, Romand R. Hair cell overproduction in the developing mammalian cochlea in culture. Neuroreport 1993;5(1):33-36.
25. Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 1999;13(12):1501-1512.
26. Zhu L, Skoultchi AI. Coordinating cell proliferation and differentiation. Curr Opin Genet Dev 2001;11(1):91-97.
27. Weinberg RA. The retinoblastoma protein and cell cycle control. Cell 1995;81(3):323-330.
28. Sherr CJ, Roberts JM. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 1995;9(10):1149-1163.
29. Chen P, Zindy F, Abdala C, Liu F, Li X, Roussel MF, et al. Progressive hearing loss in mice lacking the cyclin-dependent kinase inhibitor Ink4d. Nat Cell Biol 2003;5(5):422-426.
30. Lee YS, Liu F, Segil N. A morphogenetic wave of p27Kip1 transcription directs cell cycle exit during organ of Corti development. Development 2006;133(15):2817-2826.
31. Bermingham NA, Hassan BA, Price SD, Vollrath MA, Ben-Arie N, Eatock RA, et al. Math1: an essential gene for the generation of inner ear hair cells. Science 1999;284(5421):1837-1841.
32. Chen P, Johnson JE, Zoghbi HY, Segil N. The role of Math1 in inner ear development: Uncoupling the establishment of the sensory primordium from hair cell fate determination. Development 2002;129(10):2495-2505.
33. Woods C, Montcouquiol M, Kelley MW. Math1 regulates development of the sensory epithelium in the mammalian cochlea. Nat Neurosci 2004;7(12):1310-1318.
34. Jones JM, Montcouquiol M, Dabdoub A, Woods C, Kelley MW. Inhibitors of differentiation and DNA binding (Ids) regulate Math1 and hair cell formation during the development of the organ of Corti. J Neurosci 2006;26(2):550-558.
35. Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal integration in development. Science 1999;284(5415):770-776.
36. Lewis AK, Frantz GD, Carpenter DA, de Sauvage FJ, Gao WQ. Distinct expression patterns of notch family receptors and ligands during development of the mammalian inner ear. Mech Dev 1998;78(1-2):159-163.
37. Iso T, Kedes L, Hamamori Y. HES and HERP families: multiple effectors of the Notch signaling pathway. J Cell Physiol 2003;194(3):237-255.
38. Weinmaster G. Notch signaling: direct or what? Curr Opin Genet Dev 1998;8(4):436-442.
39. Kiernan AE, Cordes R, Kopan R, Gossler A, Gridley T. The Notch ligands DLL1 and JAG2 act synergistically to regulate hair cell development in the mammalian inner ear. Development 2005;132(19):4353-4362.
40. Lanford PJ, Lan Y, Jiang R, Lindsell C, Weinmaster G, Gridley T, et al. Notch signalling pathway mediates hair cell development in mammalian cochlea. Nat Genet 1999;21(3):289-292.
41. Brooker R, Hozumi K, Lewis J. Notch ligands with contrasting functions: Jagged1 and Delta1 in the mouse inner ear. Development 2006;133(7):1277-1286.
42. Kawamoto K, Ishimoto S, Minoda R, Brough DE, Raphael Y. Math1 gene transfer generates new cochlear hair cells in mature guinea pigs in vivo. J Neurosci 2003;23(11):4395-4400.
43. Chen J, Wang F, Gao X, Zha D, Xue T, Cheng X, et al. Decreased level of cyclin A2 in rat cochlea development and cochlear stem cell differentiation. Neurosci Lett 2009;453(3):166-169.
44. Ge RS, Hardy MP. Decreased cyclin A2 and increased cyclin G1 levels coincide with loss of proliferative capacity in rat Leydig cells during pubertal development. Endocrinology 1997;138(9):3719-3726.
45. Ravnik SE, Wolgemuth DJ. The developmentally restricted pattern of expression in the male germ line of a murine cyclin A, cyclin A2, suggests roles in both mitotic and meiotic cell cycles. Dev Biol 1996;173(1):69-78.
46. Yoshizumi M, Lee WS, Hsieh CM, Tsai JC, Li J, Perrella MA, et al. Disappearance of cyclin A correlates with permanent withdrawal of cardiomyocytes from the cell cycle in human and rat hearts. J Clin Invest 1995;95(5):2275-2280.
47. Girard F, Strausfeld U, Fernandez A, Lamb NJ. Cyclin A is required for the onset of DNA replication in mammalian fibroblasts. Cell 1991;67(6):1169-1179.
48. Pagano M, Pepperkok R, Verde F, Ansorge W, Draetta G. Cyclin A is required at two points in the human cell cycle. Embo J 1992;11(3):961-971.
49. Woo YJ, Panlilio CM, Cheng RK, Liao GP, Atluri P, Hsu VM, et al. Therapeutic delivery of cyclin A2 induces myocardial regeneration and enhances cardiac function in ischemic heart failure. Circulation 2006;114(1 Suppl):I206-213.
50. Laine H, Sulg M, Kirjavainen A, Pirvola U. Cell cycle regulation in the inner ear sensory epithelia: role of cyclin D1 and cyclin-dependent kinase inhibitors. Dev Biol2010;337(1):134-146.
51. Rocha-Sanchez SM, Beisel KW. Pocket proteins and cell cycle regulation in inner ear development. Int J Dev Biol 2007;51(6-7):585-595.
52. Sage C, Huang M, Karimi K, Gutierrez G, Vollrath MA, Zhang DS, et al. Proliferation of functional hair cells in vivo in the absence of the retinoblastoma protein. Science 2005;307(5712):1114-1118.
53. Mantela J, Jiang Z, Ylikoski J, Fritzsch B, Zacksenhaus E, Pirvola U. The retinoblastoma gene pathway regulates the postmitotic state of hair cells of the mouse inner ear. Development 2005;132(10):2377-2388.
54. Weber T, Corbett MK, Chow LM, Valentine MB, Baker SJ, Zuo J. Rapid cell-cycle reentry and cell death after acute inactivation of the retinoblastoma gene product in postnatal cochlear hair cells. Proc Natl Acad Sci U S A 2008;105(2):781-785.
55. Sage C, Huang M, Vollrath MA, Brown MC, Hinds PW, Corey DP, et al. Essential role of retinoblastoma protein in mammalian hair cell development and hearing. Proc Natl Acad Sci U S A 2006;103(19):7345-7350.
56. Novitch BG, Spicer DB, Kim PS, Cheung WL, Lassar AB. pRb is required for MEF2-dependent gene expression as well as cell-cycle arrest during skeletal muscle differentiation. Curr Biol 1999;9(9):449-459.
57. Thomas DM, Carty SA, Piscopo DM, Lee JS, Wang WF, Forrester WC, et al. The retinoblastoma protein acts as a transcriptional coactivator required for osteogenic differentiation. Mol Cell 2001;8(2):303-316.
58. Wu X, Gao J, Guo Y, Zuo J. Hearing threshold elevation precedes hair-cell loss in prestin knockout mice. Brain Res Mol Brain Res 2004;126(1):30-37.
59. Morris EJ, Dyson NJ. Retinoblastoma protein partners. Adv Cancer Res 2001;82:1-54.
60. Assoian RK. Stopping and going with p27kip1. Dev Cell 2004;6(4):458-459.
61. Fero ML, Rivkin M, Tasch M, Porter P, Carow CE, Firpo E, et al. A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice. Cell 1996;85(5):733-744.
62. Chen P, Segil N. p27(Kip1) links cell proliferation to morphogenesis in the developing organ of Corti. Development 1999;126(8):1581-1590.
63. Kanzaki S, Beyer LA, Swiderski DL, Izumikawa M, Stover T, Kawamoto K, et al. p27(Kip1) deficiency causes organ of Corti pathology and hearing loss. Hear Res 2006;214(1-2):28-36.
64. Lowenheim H, Furness DN, Kil J, Zinn C, Gultig K, Fero ML, et al. Gene disruption of p27(Kip1) allows cell proliferation in the postnatal and adult organ of corti. Proc Natl Acad Sci U S A 1999;96(7):4084-4088.
65. Besson A, Gurian-West M, Schmidt A, Hall A, Roberts JM. p27Kip1 modulates cell migration through the regulation of RhoA activation. Genes Dev 2004;18(8):862-876.
66. Kiernan AE, Pelling AL, Leung KK, Tang AS, Bell DM, Tease C, et al. Sox2 is required forsensory organ development in the mammalian inner ear. Nature 2005;434(7036):1031-1035.
67. Kiernan AE, Xu J, Gridley T. The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear. PLoS Genet 2006;2(1):e4.
68. Zine A, Van De Water TR, de Ribaupierre F. Notch signaling regulates the pattern of auditory hair cell differentiation in mammals. Development 2000;127(15):3373-3383.
69. Besson A, Dowdy SF, Roberts JM. CDK inhibitors: cell cycle regulators and beyond. Dev Cell 2008;14(2):159-169.
70. Chu I, Sun J, Arnaout A, Kahn H, Hanna W, Narod S, et al. p27 phosphorylation by Src regulates inhibition of cyclin E-Cdk2. Cell 2007;128(2):281-294.
71. Chu IM, Hengst L, Slingerland JM. The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy. Nat Rev Cancer 2008;8(4):253-267.
72. Grimmler M, Wang Y, Mund T, Cilensek Z, Keidel EM, Waddell MB, et al. Cdk-inhibitory activity and stability of p27Kip1 are directly regulated by oncogenic tyrosine kinases. Cell 2007;128(2):269-280.
73. Laine H, Doetzlhofer A, Mantela J, Ylikoski J, Laiho M, Roussel MF, et al. p19(Ink4d) and p21(Cip1) collaborate to maintain the postmitotic state of auditory hair cells, their codeletion leading to DNA damage and p53-mediated apoptosis. J Neurosci 2007;27(6):1434-1444.
74. Cafaro J, Lee GS, Stone JS. Atoh1 expression defines activated progenitors and differentiating hair cells during avian hair cell regeneration. Dev Dyn 2007;236(1):156-170.
75. Hirose K, Westrum LE, Cunningham DE, Rubel EW. Electron microscopy of degenerative changes in the chick basilar papilla after gentamicin exposure. J Comp Neurol 2004;470(2):164-180.
76. Martinez-Monedero R, Edge AS. Stem cells for the replacement of inner ear neurons and hair cells. Int J Dev Biol 2007;51(6-7):655-661.
77. Stone JS, Cotanche DA. Hair cell regeneration in the avian auditory epithelium. Int J Dev Biol 2007;51(6-7):633-647.
78. White PM, Doetzlhofer A, Lee YS, Groves AK, Segil N. Mammalian cochlear supporting cells can divide and trans-differentiate into hair cells. Nature 2006;441(7096):984-987.
79. Izumikawa M, Minoda R, Kawamoto K, Abrashkin KA, Swiderski DL, Dolan DF, et al. Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals. Nat Med 2005;11(3):271-276.
80. Corwin JT, Cotanche DA. Regeneration of sensory hair cells after acoustic trauma. Science 1988;240(4860):1772-1774.
81. Ryals BM, Rubel EW. Hair cell regeneration after acoustic trauma in adult Coturnix quail. Science 1988;240(4860):1774-1776.
82. Montcouquiol M, Corwin JT. Brief treatments with forskolin enhance s-phase entry in balance epithelia from the ears of rats. J Neurosci 2001;21(3):974-982.
83. Corwin JT, Oberholtzer JC. Fish n' chicks: model recipes for hair-cell regeneration? Neuron 1997;19(5):951-954.
84. Kelley MW, Talreja DR, Corwin JT. Replacement of hair cells after laser microbeam irradiation in cultured organs of corti from embryonic and neonatal mice. J Neurosci 1995;15(4):3013-3026.
85. Lambert PR. Inner ear hair cell regeneration in a mammal: identification of a triggering factor. Laryngoscope 1994;104(6 Pt 1):701-718.
86. Li H, Liu H, Heller S. Pluripotent stem cells from the adult mouse inner ear. Nat Med 2003;9(10):1293-1299.
87. Oshima K, Grimm CM, Corrales CE, Senn P, Martinez Monedero R, Geleoc GS, et al. Differential distribution of stem cells in the auditory and vestibular organs of the inner ear. J Assoc Res Otolaryngol 2007;8(1):18-31.
88. Liberman MC, Kiang NY. Acoustic trauma in cats. Cochlear pathology and auditory-nerve activity. Acta Otolaryngol Suppl 1978;358:1-63.
89. Horner PJ, Thallmair M, Gage FH. Defining the NG2-expressing cell of the adult CNS. J Neurocytol 2002;31(6-7):469-480.
90. Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992;255(5052):1707-1710.
91. Lopez IA, Zhao PM, Yamaguchi M, de Vellis J, Espinosa-Jeffrey A. Stem/progenitor cells in the postnatal inner ear of the GFP-nestin transgenic mouse. Int J Dev Neurosci 2004;22(4):205-213.
92. Zheng JL, Gao WQ. Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears. Nat Neurosci 2000;3(6):580-586.
93. Zimmermann CE, Burgess BJ, Nadol JB, Jr. Patterns of degeneration in the human cochlear nerve. Hear Res 1995;90(1-2):192-201.
94. Varga R, Kelley PM, Keats BJ, Starr A, Leal SM, Cohn E, et al. Non-syndromic recessive auditory neuropathy is the result of mutations in the otoferlin (OTOF) gene. J Med Genet 2003;40(1):45-50.
95. Kujawa SG, Liberman MC. Acceleration of age-related hearing loss by early noise exposure: evidence of a misspent youth. J Neurosci 2006;26(7):2115-2123.
96. Sekiya T, Shimamura N, Yagihashi A, Suzuki S. Effect of topically applied basic fibroblast growth factor on injured cochlear nerve. Neurosurgery 2003;52(4):900-907; discussion 907.
97. Nadol JB, Jr. Patterns of neural degeneration in the human cochlea and auditory nerve: implications for cochlear implantation. Otolaryngol Head Neck Surg 1997;117(3 Pt 1):220-228.
98. Rask-Andersen H, Bostrom M, Gerdin B, Kinnefors A, Nyberg G, Engstrand T, et al. Regeneration of human auditory nerve. In vitro/in video demonstration of neural progenitor cells in adult human and guinea pig spiral ganglion. Hear Res 2005;203(1-2):180-191.
99. Corrales CE, Pan L, Li H, Liberman MC, Heller S, Edge AS. Engraftment and differentiationof embryonic stem cell-derived neural progenitor cells in the cochlear nerve trunk: growth of processes into the organ of Corti. J Neurobiol 2006;66(13):1489-1500.
100. Martinez-Monedero R, Corrales CE, Cuajungco MP, Heller S, Edge AS. Reinnervation of hair cells by auditory neurons after selective removal of spiral ganglion neurons. J Neurobiol 2006;66(4):319-331.
101. Coleman B, Hardman J, Coco A, Epp S, Silva Md, Crook J, et al. Fate of embryonic stem cells transplanted into the deafened mammalian cochlea. Cell Transplant 2006;15(5):369-380.
102. Brigande JV, Heller S. Quo vadis, hair cell regeneration? Nat Neurosci 2009;12(6):679-685.
103. Gubbels SP, Woessner DW, Mitchell JC, Ricci AJ, Brigande JV. Functional auditory hair cells produced in the mammalian cochlea by in utero gene transfer. Nature 2008;455(7212):537-541.
104. Hayashi T, Cunningham D, Bermingham-McDonogh O. Loss of Fgfr3 leads to excess hair cell development in the mouse organ of Corti. Dev Dyn 2007;236(2):525-533.
105. Raft S, Koundakjian EJ, Quinones H, Jayasena CS, Goodrich LV, Johnson JE, et al. Cross-regulation of Ngn1 and Math1 coordinates the production of neurons and sensory hair cells during inner ear development. Development 2007;134(24):4405-4415.
106. Konishi M, Kawamoto K, Izumikawa M, Kuriyama H, Yamashita T. Gene transfer into guinea pig cochlea using adeno-associated virus vectors. J Gene Med 2008;10(6):610-618.
107.陈俊.新生大鼠耳蜗前体细胞分化的分子机制及微环境的初步研究.耳鼻咽喉头颈外科.西安:第四军医大学; 2009.
108. Strojnik T, Rosland GV, Sakariassen PO, Kavalar R, Lah T. Neural stem cell markers, nestin and musashi proteins, in the progression of human glioma: correlation of nestin with prognosis of patient survival. Surg Neurol 2007;68(2):133-143; discussion 143-134.
109. Kaneko Y, Sakakibara S, Imai T, Suzuki A, Nakamura Y, Sawamoto K, et al. Musashi1: an evolutionally conserved marker for CNS progenitor cells including neural stem cells. Dev Neurosci 2000;22(1-2):139-153.
110. Lawlor P, Marcotti W, Rivolta MN, Kros CJ, Holley MC. Differentiation of mammalian vestibular hair cells from conditionally immortal, postnatal supporting cells. J Neurosci 1999;19(21):9445-9458.
111. Nordang L, Cestreicher E, Arnold W, Anniko M. Glutamate is the afferent neurotransmitter in the human cochlea. Acta Otolaryngol 2000;120(3):359-362.
112. Diensthuber M, Oshima K, Heller S. Stem/progenitor cells derived from the cochlear sensory epithelium give rise to spheres with distinct morphologies and features. J Assoc Res Otolaryngol 2009;10(2):173-190.
113. Liu Z, Zuo J. Cell cycle regulation in hair cell development and regeneration in the mouse cochlea. Cell Cycle 2008;7(14):2129-2133.
114. Feghali JG, Lefebvre PP, Staecker H, Kopke R, Frenz DA, Malgrange B, et al. Mammalian auditory hair cell regeneration/repair and protection: a review and future directions. EarNose Throat J 1998;77(4):276, 280, 282-275.
115. Stone JS, Oesterle EC, Rubel EW. Recent insights into regeneration of auditory and vestibular hair cells. Curr Opin Neurol 1998;11(1):17-24.
116. Zhong C, Han Y, Qiu J, Lu L, Chen Y, Chen J, et al. A comparison of the proliferative capacity and ultrastructure of proliferative cells from the cochleae of newborn rats of different ages. International Journal of Pediatric Otorhinolaryngology 2010;74(2):192-197.
117. Simile MM, Miglio MRD, Muroni MR, Frau M, Asara G, Serra S, et al. Down-regulation of c-myc and Cyclin D1 genes by antisense oligodeoxy nucleotides inhibits the expression of E2F1 and in vitro growth of HepG2 and Morris 5123 liver cancer cells. Carcinogenesis 2004;25(3):333-341.
118. Minoda R, Izumikawa M, Kawamoto K, Zhang H, Raphael Y. Manipulating cell cycle regulation in the mature cochlea. Hear Res 2007;232(1-2):44-51.
119. Lemaitre JM, Buckle RS, Mechali M. c-Myc in the control of cell proliferation and embryonic development. Adv Cancer Res 1996;70:95-144.
120. Romand R, Hirning-Folz U, Ehret G. N-myc expression in the embryonic cochlea of the mouse. Hear Res 1994;72(1-2):53-58.
121.李华伟.雏鸡听毛细胞损伤后c-myc mRNA及其产物表达的研究.临床耳鼻咽喉科杂志1999;13(4):173-175.
122.高春生.人胎儿内耳c-myc、c-fos蛋白的定位表达.中山大学学报(医学科学版) 2003;24(6):607-610.
123. Kelley MW. Exposing the roots of hair cell regeneration in the ear. Nat Med 2003;9(10):1257-1259.
124. Holt JR, Johns DC, Wang S, Chen ZY, Dunn RJ, Marban E, et al. Functional expression of exogenous proteins in mammalian sensory hair cells infected with adenoviral vectors. J Neurophysiol 1999;81(4):1881-1888.
125. Karpati G, Lochmuller H, Nalbantoglu J, Durham H. The principles of gene therapy for the nervous system. Trends Neurosci 1996;19(2):49-54.
126. Holt JR. Viral-mediated gene transfer to study the molecular physiology of the Mammalian inner ear. Audiol Neurootol 2002;7(3):157-160.
127.王亭忠.大鼠骨髓间充质干细胞的分离培养及GFP转染标记.西安交通大学学报(医学版) 2005;26(5):431-434.
128. Han Y, Zhong C, Hong L, Wang Y, Qiao L, Qiu J. Effect of c-myc on the ultrastructural structure of cochleae in guinea pigs with noise induced hearing loss. Biochem Biophys Res Commun 2009;390(3):458-462.
129. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126(4):663-676.
130. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131(5):861-872.
131. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007;318(5858):1917-1920.
132. Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 2007;448(7151):318-324.
133. Lowry WE, Richter L, Yachechko R, Pyle AD, Tchieu J, Sridharan R, et al. Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc Natl Acad Sci U S A 2008;105(8):2883-2888.
134. Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature 2007;448(7151):313-317.
135. Wernig M, Lengner CJ, Hanna J, Lodato MA, Steine E, Foreman R, et al. A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types. Nat Biotechnol 2008;26(8):916-924.
136. Zhou W, Freed CR. Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells. Stem Cells 2009;27(11):2667-2674.
137.张四清,王永潮.细胞周期调控. In:翟中和, editor.细胞生物学动态(第一卷).北京:北京师范大学出版社; 1996.
138. Bandara LR, Adamczewski JP, Hunt T, La Thangue NB. Cyclin A and the retinoblastoma gene product complex with a common transcription factor. Nature 1991; 352(6332):249-251.
139. Toyoshima H, Hunter T. p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell 1994;78(1):67-74.
2. Saunders JC, Dear SP, Schneider ME. The anatomical consequences of acoustic injury: A review and tutorial. J Acoust Soc Am 1985;78(3):833-860.
3. Schacht J. Molecular mechanisms of drug-induced hearing loss. Hear Res 1986;22:297-304.
4. Forge A, Li L, Corwin JT, Nevill G. Ultrastructural evidence for hair cell regeneration in the mammalian inner ear. Science 1993;259(5101):1616-1619.
5. Lim DJ. Functional structure of the organ of Corti: a review. Hear Res 1986;22:117-146.
6. D.J. L, J R. Structural development of the cochlea. In: Roman R, editor. Development of auditory and vestibular systems 2. Oxford: Elsevier; 1992. p. 33-58.
7. Malgrange B, Thiry M, Van De Water TR, Nguyen L, Moonen G, Lefebvre PP. Epithelial supporting cells can differentiate into outer hair cells and Deiters' cells in the cultured organ of Corti. Cell Mol Life Sci 2002;59(10):1744-1757.
8. Breuskin I, Bodson M, Thelen N, Thiry M, Nguyen L, Belachew S, et al. Strategies to regenerate hair cells: identification of progenitors and critical genes. Hear Res 2008;236(1-2):1-10.
9. Zhai S, Shi L, Wang BE, Zheng G, Song W, Hu Y, et al. Isolation and culture of hair cell progenitors from postnatal rat cochleae. J Neurobiol 2005;65(3):282-293.
10. Doyle KL, Kazda A, Hort Y, McKay SM, Oleskevich S. Differentiation of adult mouse olfactory precursor cells into hair cells in vitro. Stem Cells 2007;25(3):621-627.
11. Jeon SJ, Oshima K, Heller S, Edge AS. Bone marrow mesenchymal stem cells are progenitors in vitro for inner ear hair cells. Mol Cell Neurosci 2007;34(1):59-68.
12. Li H, Roblin G, Liu H, Heller S. Generation of hair cells by stepwise differentiation of embryonic stem cells. Proc Natl Acad Sci U S A 2003;100(23):13495-13500.
13. Li H, Corrales CE, Edge A, Heller S. Stem cells as therapy for hearing loss. Trends Mol Med 2004;10(7):309-315.
14. Rivolta MN, Li H, Heller S. Generation of inner ear cell types from embryonic stem cells. Methods Mol Biol 2006;330:71-92.
15. Doetzlhofer A, White PM, Johnson JE, Segil N, Groves AK. In vitro growth and differentiation of mammalian sensory hair cell progenitors: a requirement for EGF and periotic mesenchyme. Dev Biol 2004;272(2):432-447.
16. Sobkowicz HM, Loftus JM, Slapnick SM. Tissue culture of the organ of Corti. Acta Otolaryngol Suppl 1993;502:3-36.
17. Sobkowicz HM, Holy J, Scott GL. Ultrastructure of the intercalated body, a novel organelleassociated with fluid forming cells in the organ of Corti. J Electron Microsc Tech 1990;15(3):301-315.
18. Baird RA, Burton MD, Lysakowski A, Fashena DS, Naeger RA. Hair cell recovery in mitotically blocked cultures of the bullfrog saccule. Proc Natl Acad Sci U S A 2000;97(22):11722-11729.
19. Minoda R, Izumikawa M, Kawamoto K, Raphael Y. Strategies for replacing lost cochlear hair cells. Neuroreport 2004;15(7):1089-1092.
20. Raff M. Adult stem cell plasticity: fact or artifact? Annu Rev Cell Dev Biol 2003;19:1-22.
21. Lendahl U, Zimmerman LB, McKay RD. CNS stem cells express a new class of intermediate filament protein. Cell 1990;60(4):585-595.
22. Kojima K, Tamura S, Nishida AT, Ito J. Generation of inner ear hair cell immunophenotypes from neurospheres obtained from fetal rat central nervous system in vitro. Acta Otolaryngol Suppl 2004;551(551):26-30.
23. Malgrange B, Belachew S, Thiry M, Nguyen L, Rogister B, Alvarez ML, et al. Proliferative generation of mammalian auditory hair cells in culture. Mech Dev 2002;112(1-2):79-88.
24. Abdouh A, Despres G, Romand R. Hair cell overproduction in the developing mammalian cochlea in culture. Neuroreport 1993;5(1):33-36.
25. Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 1999;13(12):1501-1512.
26. Zhu L, Skoultchi AI. Coordinating cell proliferation and differentiation. Curr Opin Genet Dev 2001;11(1):91-97.
27. Weinberg RA. The retinoblastoma protein and cell cycle control. Cell 1995;81(3):323-330.
28. Sherr CJ, Roberts JM. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 1995;9(10):1149-1163.
29. Chen P, Zindy F, Abdala C, Liu F, Li X, Roussel MF, et al. Progressive hearing loss in mice lacking the cyclin-dependent kinase inhibitor Ink4d. Nat Cell Biol 2003;5(5):422-426.
30. Lee YS, Liu F, Segil N. A morphogenetic wave of p27Kip1 transcription directs cell cycle exit during organ of Corti development. Development 2006;133(15):2817-2826.
31. Bermingham NA, Hassan BA, Price SD, Vollrath MA, Ben-Arie N, Eatock RA, et al. Math1: an essential gene for the generation of inner ear hair cells. Science 1999;284(5421):1837-1841.
32. Chen P, Johnson JE, Zoghbi HY, Segil N. The role of Math1 in inner ear development: Uncoupling the establishment of the sensory primordium from hair cell fate determination. Development 2002;129(10):2495-2505.
33. Woods C, Montcouquiol M, Kelley MW. Math1 regulates development of the sensory epithelium in the mammalian cochlea. Nat Neurosci 2004;7(12):1310-1318.
34. Jones JM, Montcouquiol M, Dabdoub A, Woods C, Kelley MW. Inhibitors of differentiation and DNA binding (Ids) regulate Math1 and hair cell formation during the development of the organ of Corti. J Neurosci 2006;26(2):550-558.
35. Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal integration in development. Science 1999;284(5415):770-776.
36. Lewis AK, Frantz GD, Carpenter DA, de Sauvage FJ, Gao WQ. Distinct expression patterns of notch family receptors and ligands during development of the mammalian inner ear. Mech Dev 1998;78(1-2):159-163.
37. Iso T, Kedes L, Hamamori Y. HES and HERP families: multiple effectors of the Notch signaling pathway. J Cell Physiol 2003;194(3):237-255.
38. Weinmaster G. Notch signaling: direct or what? Curr Opin Genet Dev 1998;8(4):436-442.
39. Kiernan AE, Cordes R, Kopan R, Gossler A, Gridley T. The Notch ligands DLL1 and JAG2 act synergistically to regulate hair cell development in the mammalian inner ear. Development 2005;132(19):4353-4362.
40. Lanford PJ, Lan Y, Jiang R, Lindsell C, Weinmaster G, Gridley T, et al. Notch signalling pathway mediates hair cell development in mammalian cochlea. Nat Genet 1999;21(3):289-292.
41. Brooker R, Hozumi K, Lewis J. Notch ligands with contrasting functions: Jagged1 and Delta1 in the mouse inner ear. Development 2006;133(7):1277-1286.
42. Kawamoto K, Ishimoto S, Minoda R, Brough DE, Raphael Y. Math1 gene transfer generates new cochlear hair cells in mature guinea pigs in vivo. J Neurosci 2003;23(11):4395-4400.
43. Chen J, Wang F, Gao X, Zha D, Xue T, Cheng X, et al. Decreased level of cyclin A2 in rat cochlea development and cochlear stem cell differentiation. Neurosci Lett 2009;453(3):166-169.
44. Ge RS, Hardy MP. Decreased cyclin A2 and increased cyclin G1 levels coincide with loss of proliferative capacity in rat Leydig cells during pubertal development. Endocrinology 1997;138(9):3719-3726.
45. Ravnik SE, Wolgemuth DJ. The developmentally restricted pattern of expression in the male germ line of a murine cyclin A, cyclin A2, suggests roles in both mitotic and meiotic cell cycles. Dev Biol 1996;173(1):69-78.
46. Yoshizumi M, Lee WS, Hsieh CM, Tsai JC, Li J, Perrella MA, et al. Disappearance of cyclin A correlates with permanent withdrawal of cardiomyocytes from the cell cycle in human and rat hearts. J Clin Invest 1995;95(5):2275-2280.
47. Girard F, Strausfeld U, Fernandez A, Lamb NJ. Cyclin A is required for the onset of DNA replication in mammalian fibroblasts. Cell 1991;67(6):1169-1179.
48. Pagano M, Pepperkok R, Verde F, Ansorge W, Draetta G. Cyclin A is required at two points in the human cell cycle. Embo J 1992;11(3):961-971.
49. Woo YJ, Panlilio CM, Cheng RK, Liao GP, Atluri P, Hsu VM, et al. Therapeutic delivery of cyclin A2 induces myocardial regeneration and enhances cardiac function in ischemic heart failure. Circulation 2006;114(1 Suppl):I206-213.
50. Laine H, Sulg M, Kirjavainen A, Pirvola U. Cell cycle regulation in the inner ear sensory epithelia: role of cyclin D1 and cyclin-dependent kinase inhibitors. Dev Biol2010;337(1):134-146.
51. Rocha-Sanchez SM, Beisel KW. Pocket proteins and cell cycle regulation in inner ear development. Int J Dev Biol 2007;51(6-7):585-595.
52. Sage C, Huang M, Karimi K, Gutierrez G, Vollrath MA, Zhang DS, et al. Proliferation of functional hair cells in vivo in the absence of the retinoblastoma protein. Science 2005;307(5712):1114-1118.
53. Mantela J, Jiang Z, Ylikoski J, Fritzsch B, Zacksenhaus E, Pirvola U. The retinoblastoma gene pathway regulates the postmitotic state of hair cells of the mouse inner ear. Development 2005;132(10):2377-2388.
54. Weber T, Corbett MK, Chow LM, Valentine MB, Baker SJ, Zuo J. Rapid cell-cycle reentry and cell death after acute inactivation of the retinoblastoma gene product in postnatal cochlear hair cells. Proc Natl Acad Sci U S A 2008;105(2):781-785.
55. Sage C, Huang M, Vollrath MA, Brown MC, Hinds PW, Corey DP, et al. Essential role of retinoblastoma protein in mammalian hair cell development and hearing. Proc Natl Acad Sci U S A 2006;103(19):7345-7350.
56. Novitch BG, Spicer DB, Kim PS, Cheung WL, Lassar AB. pRb is required for MEF2-dependent gene expression as well as cell-cycle arrest during skeletal muscle differentiation. Curr Biol 1999;9(9):449-459.
57. Thomas DM, Carty SA, Piscopo DM, Lee JS, Wang WF, Forrester WC, et al. The retinoblastoma protein acts as a transcriptional coactivator required for osteogenic differentiation. Mol Cell 2001;8(2):303-316.
58. Wu X, Gao J, Guo Y, Zuo J. Hearing threshold elevation precedes hair-cell loss in prestin knockout mice. Brain Res Mol Brain Res 2004;126(1):30-37.
59. Morris EJ, Dyson NJ. Retinoblastoma protein partners. Adv Cancer Res 2001;82:1-54.
60. Assoian RK. Stopping and going with p27kip1. Dev Cell 2004;6(4):458-459.
61. Fero ML, Rivkin M, Tasch M, Porter P, Carow CE, Firpo E, et al. A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice. Cell 1996;85(5):733-744.
62. Chen P, Segil N. p27(Kip1) links cell proliferation to morphogenesis in the developing organ of Corti. Development 1999;126(8):1581-1590.
63. Kanzaki S, Beyer LA, Swiderski DL, Izumikawa M, Stover T, Kawamoto K, et al. p27(Kip1) deficiency causes organ of Corti pathology and hearing loss. Hear Res 2006;214(1-2):28-36.
64. Lowenheim H, Furness DN, Kil J, Zinn C, Gultig K, Fero ML, et al. Gene disruption of p27(Kip1) allows cell proliferation in the postnatal and adult organ of corti. Proc Natl Acad Sci U S A 1999;96(7):4084-4088.
65. Besson A, Gurian-West M, Schmidt A, Hall A, Roberts JM. p27Kip1 modulates cell migration through the regulation of RhoA activation. Genes Dev 2004;18(8):862-876.
66. Kiernan AE, Pelling AL, Leung KK, Tang AS, Bell DM, Tease C, et al. Sox2 is required forsensory organ development in the mammalian inner ear. Nature 2005;434(7036):1031-1035.
67. Kiernan AE, Xu J, Gridley T. The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear. PLoS Genet 2006;2(1):e4.
68. Zine A, Van De Water TR, de Ribaupierre F. Notch signaling regulates the pattern of auditory hair cell differentiation in mammals. Development 2000;127(15):3373-3383.
69. Besson A, Dowdy SF, Roberts JM. CDK inhibitors: cell cycle regulators and beyond. Dev Cell 2008;14(2):159-169.
70. Chu I, Sun J, Arnaout A, Kahn H, Hanna W, Narod S, et al. p27 phosphorylation by Src regulates inhibition of cyclin E-Cdk2. Cell 2007;128(2):281-294.
71. Chu IM, Hengst L, Slingerland JM. The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy. Nat Rev Cancer 2008;8(4):253-267.
72. Grimmler M, Wang Y, Mund T, Cilensek Z, Keidel EM, Waddell MB, et al. Cdk-inhibitory activity and stability of p27Kip1 are directly regulated by oncogenic tyrosine kinases. Cell 2007;128(2):269-280.
73. Laine H, Doetzlhofer A, Mantela J, Ylikoski J, Laiho M, Roussel MF, et al. p19(Ink4d) and p21(Cip1) collaborate to maintain the postmitotic state of auditory hair cells, their codeletion leading to DNA damage and p53-mediated apoptosis. J Neurosci 2007;27(6):1434-1444.
74. Cafaro J, Lee GS, Stone JS. Atoh1 expression defines activated progenitors and differentiating hair cells during avian hair cell regeneration. Dev Dyn 2007;236(1):156-170.
75. Hirose K, Westrum LE, Cunningham DE, Rubel EW. Electron microscopy of degenerative changes in the chick basilar papilla after gentamicin exposure. J Comp Neurol 2004;470(2):164-180.
76. Martinez-Monedero R, Edge AS. Stem cells for the replacement of inner ear neurons and hair cells. Int J Dev Biol 2007;51(6-7):655-661.
77. Stone JS, Cotanche DA. Hair cell regeneration in the avian auditory epithelium. Int J Dev Biol 2007;51(6-7):633-647.
78. White PM, Doetzlhofer A, Lee YS, Groves AK, Segil N. Mammalian cochlear supporting cells can divide and trans-differentiate into hair cells. Nature 2006;441(7096):984-987.
79. Izumikawa M, Minoda R, Kawamoto K, Abrashkin KA, Swiderski DL, Dolan DF, et al. Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals. Nat Med 2005;11(3):271-276.
80. Corwin JT, Cotanche DA. Regeneration of sensory hair cells after acoustic trauma. Science 1988;240(4860):1772-1774.
81. Ryals BM, Rubel EW. Hair cell regeneration after acoustic trauma in adult Coturnix quail. Science 1988;240(4860):1774-1776.
82. Montcouquiol M, Corwin JT. Brief treatments with forskolin enhance s-phase entry in balance epithelia from the ears of rats. J Neurosci 2001;21(3):974-982.
83. Corwin JT, Oberholtzer JC. Fish n' chicks: model recipes for hair-cell regeneration? Neuron 1997;19(5):951-954.
84. Kelley MW, Talreja DR, Corwin JT. Replacement of hair cells after laser microbeam irradiation in cultured organs of corti from embryonic and neonatal mice. J Neurosci 1995;15(4):3013-3026.
85. Lambert PR. Inner ear hair cell regeneration in a mammal: identification of a triggering factor. Laryngoscope 1994;104(6 Pt 1):701-718.
86. Li H, Liu H, Heller S. Pluripotent stem cells from the adult mouse inner ear. Nat Med 2003;9(10):1293-1299.
87. Oshima K, Grimm CM, Corrales CE, Senn P, Martinez Monedero R, Geleoc GS, et al. Differential distribution of stem cells in the auditory and vestibular organs of the inner ear. J Assoc Res Otolaryngol 2007;8(1):18-31.
88. Liberman MC, Kiang NY. Acoustic trauma in cats. Cochlear pathology and auditory-nerve activity. Acta Otolaryngol Suppl 1978;358:1-63.
89. Horner PJ, Thallmair M, Gage FH. Defining the NG2-expressing cell of the adult CNS. J Neurocytol 2002;31(6-7):469-480.
90. Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992;255(5052):1707-1710.
91. Lopez IA, Zhao PM, Yamaguchi M, de Vellis J, Espinosa-Jeffrey A. Stem/progenitor cells in the postnatal inner ear of the GFP-nestin transgenic mouse. Int J Dev Neurosci 2004;22(4):205-213.
92. Zheng JL, Gao WQ. Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears. Nat Neurosci 2000;3(6):580-586.
93. Zimmermann CE, Burgess BJ, Nadol JB, Jr. Patterns of degeneration in the human cochlear nerve. Hear Res 1995;90(1-2):192-201.
94. Varga R, Kelley PM, Keats BJ, Starr A, Leal SM, Cohn E, et al. Non-syndromic recessive auditory neuropathy is the result of mutations in the otoferlin (OTOF) gene. J Med Genet 2003;40(1):45-50.
95. Kujawa SG, Liberman MC. Acceleration of age-related hearing loss by early noise exposure: evidence of a misspent youth. J Neurosci 2006;26(7):2115-2123.
96. Sekiya T, Shimamura N, Yagihashi A, Suzuki S. Effect of topically applied basic fibroblast growth factor on injured cochlear nerve. Neurosurgery 2003;52(4):900-907; discussion 907.
97. Nadol JB, Jr. Patterns of neural degeneration in the human cochlea and auditory nerve: implications for cochlear implantation. Otolaryngol Head Neck Surg 1997;117(3 Pt 1):220-228.
98. Rask-Andersen H, Bostrom M, Gerdin B, Kinnefors A, Nyberg G, Engstrand T, et al. Regeneration of human auditory nerve. In vitro/in video demonstration of neural progenitor cells in adult human and guinea pig spiral ganglion. Hear Res 2005;203(1-2):180-191.
99. Corrales CE, Pan L, Li H, Liberman MC, Heller S, Edge AS. Engraftment and differentiationof embryonic stem cell-derived neural progenitor cells in the cochlear nerve trunk: growth of processes into the organ of Corti. J Neurobiol 2006;66(13):1489-1500.
100. Martinez-Monedero R, Corrales CE, Cuajungco MP, Heller S, Edge AS. Reinnervation of hair cells by auditory neurons after selective removal of spiral ganglion neurons. J Neurobiol 2006;66(4):319-331.
101. Coleman B, Hardman J, Coco A, Epp S, Silva Md, Crook J, et al. Fate of embryonic stem cells transplanted into the deafened mammalian cochlea. Cell Transplant 2006;15(5):369-380.
102. Brigande JV, Heller S. Quo vadis, hair cell regeneration? Nat Neurosci 2009;12(6):679-685.
103. Gubbels SP, Woessner DW, Mitchell JC, Ricci AJ, Brigande JV. Functional auditory hair cells produced in the mammalian cochlea by in utero gene transfer. Nature 2008;455(7212):537-541.
104. Hayashi T, Cunningham D, Bermingham-McDonogh O. Loss of Fgfr3 leads to excess hair cell development in the mouse organ of Corti. Dev Dyn 2007;236(2):525-533.
105. Raft S, Koundakjian EJ, Quinones H, Jayasena CS, Goodrich LV, Johnson JE, et al. Cross-regulation of Ngn1 and Math1 coordinates the production of neurons and sensory hair cells during inner ear development. Development 2007;134(24):4405-4415.
106. Konishi M, Kawamoto K, Izumikawa M, Kuriyama H, Yamashita T. Gene transfer into guinea pig cochlea using adeno-associated virus vectors. J Gene Med 2008;10(6):610-618.
107.陈俊.新生大鼠耳蜗前体细胞分化的分子机制及微环境的初步研究.耳鼻咽喉头颈外科.西安:第四军医大学; 2009.
108. Strojnik T, Rosland GV, Sakariassen PO, Kavalar R, Lah T. Neural stem cell markers, nestin and musashi proteins, in the progression of human glioma: correlation of nestin with prognosis of patient survival. Surg Neurol 2007;68(2):133-143; discussion 143-134.
109. Kaneko Y, Sakakibara S, Imai T, Suzuki A, Nakamura Y, Sawamoto K, et al. Musashi1: an evolutionally conserved marker for CNS progenitor cells including neural stem cells. Dev Neurosci 2000;22(1-2):139-153.
110. Lawlor P, Marcotti W, Rivolta MN, Kros CJ, Holley MC. Differentiation of mammalian vestibular hair cells from conditionally immortal, postnatal supporting cells. J Neurosci 1999;19(21):9445-9458.
111. Nordang L, Cestreicher E, Arnold W, Anniko M. Glutamate is the afferent neurotransmitter in the human cochlea. Acta Otolaryngol 2000;120(3):359-362.
112. Diensthuber M, Oshima K, Heller S. Stem/progenitor cells derived from the cochlear sensory epithelium give rise to spheres with distinct morphologies and features. J Assoc Res Otolaryngol 2009;10(2):173-190.
113. Liu Z, Zuo J. Cell cycle regulation in hair cell development and regeneration in the mouse cochlea. Cell Cycle 2008;7(14):2129-2133.
114. Feghali JG, Lefebvre PP, Staecker H, Kopke R, Frenz DA, Malgrange B, et al. Mammalian auditory hair cell regeneration/repair and protection: a review and future directions. EarNose Throat J 1998;77(4):276, 280, 282-275.
115. Stone JS, Oesterle EC, Rubel EW. Recent insights into regeneration of auditory and vestibular hair cells. Curr Opin Neurol 1998;11(1):17-24.
116. Zhong C, Han Y, Qiu J, Lu L, Chen Y, Chen J, et al. A comparison of the proliferative capacity and ultrastructure of proliferative cells from the cochleae of newborn rats of different ages. International Journal of Pediatric Otorhinolaryngology 2010;74(2):192-197.
117. Simile MM, Miglio MRD, Muroni MR, Frau M, Asara G, Serra S, et al. Down-regulation of c-myc and Cyclin D1 genes by antisense oligodeoxy nucleotides inhibits the expression of E2F1 and in vitro growth of HepG2 and Morris 5123 liver cancer cells. Carcinogenesis 2004;25(3):333-341.
118. Minoda R, Izumikawa M, Kawamoto K, Zhang H, Raphael Y. Manipulating cell cycle regulation in the mature cochlea. Hear Res 2007;232(1-2):44-51.
119. Lemaitre JM, Buckle RS, Mechali M. c-Myc in the control of cell proliferation and embryonic development. Adv Cancer Res 1996;70:95-144.
120. Romand R, Hirning-Folz U, Ehret G. N-myc expression in the embryonic cochlea of the mouse. Hear Res 1994;72(1-2):53-58.
121.李华伟.雏鸡听毛细胞损伤后c-myc mRNA及其产物表达的研究.临床耳鼻咽喉科杂志1999;13(4):173-175.
122.高春生.人胎儿内耳c-myc、c-fos蛋白的定位表达.中山大学学报(医学科学版) 2003;24(6):607-610.
123. Kelley MW. Exposing the roots of hair cell regeneration in the ear. Nat Med 2003;9(10):1257-1259.
124. Holt JR, Johns DC, Wang S, Chen ZY, Dunn RJ, Marban E, et al. Functional expression of exogenous proteins in mammalian sensory hair cells infected with adenoviral vectors. J Neurophysiol 1999;81(4):1881-1888.
125. Karpati G, Lochmuller H, Nalbantoglu J, Durham H. The principles of gene therapy for the nervous system. Trends Neurosci 1996;19(2):49-54.
126. Holt JR. Viral-mediated gene transfer to study the molecular physiology of the Mammalian inner ear. Audiol Neurootol 2002;7(3):157-160.
127.王亭忠.大鼠骨髓间充质干细胞的分离培养及GFP转染标记.西安交通大学学报(医学版) 2005;26(5):431-434.
128. Han Y, Zhong C, Hong L, Wang Y, Qiao L, Qiu J. Effect of c-myc on the ultrastructural structure of cochleae in guinea pigs with noise induced hearing loss. Biochem Biophys Res Commun 2009;390(3):458-462.
129. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126(4):663-676.
130. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131(5):861-872.
131. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007;318(5858):1917-1920.
132. Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 2007;448(7151):318-324.
133. Lowry WE, Richter L, Yachechko R, Pyle AD, Tchieu J, Sridharan R, et al. Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc Natl Acad Sci U S A 2008;105(8):2883-2888.
134. Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature 2007;448(7151):313-317.
135. Wernig M, Lengner CJ, Hanna J, Lodato MA, Steine E, Foreman R, et al. A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types. Nat Biotechnol 2008;26(8):916-924.
136. Zhou W, Freed CR. Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells. Stem Cells 2009;27(11):2667-2674.
137.张四清,王永潮.细胞周期调控. In:翟中和, editor.细胞生物学动态(第一卷).北京:北京师范大学出版社; 1996.
138. Bandara LR, Adamczewski JP, Hunt T, La Thangue NB. Cyclin A and the retinoblastoma gene product complex with a common transcription factor. Nature 1991; 352(6332):249-251.
139. Toyoshima H, Hunter T. p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell 1994;78(1):67-74.