重组腺病毒（Ad-GFP）感染胚胎大鼠神经干细胞及耳蜗移植的实验研究
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摘要
第一部分胚胎大鼠神经干细胞的分离培养、鉴定及标记
目的探索胚胎大鼠神经干细胞(Neural stem cells ,NSCs)的分离培养、鉴定及标记的方法。
方法分离胚胎大鼠海马组织,用无血清培养技术培养NSCs,免疫组化荧光技术检测其巢蛋白(Nestin)的表达;5-溴脱氧尿嘧啶(Brdu)掺入试验,并用免疫荧光双标技术观测神经干细胞的增殖状况。采用荧光染料Hoechest33342标记NSCs并诱导其分化,用神经元特异性烯醇化酶(Neuron Specific Enolase, NSE)胶质纤维酸性蛋白(Glial Fibrillary Acidic Protein, GFAP)免疫组化荧光染色鉴定分化细胞。
结果1.获得了大量未分化呈巢状悬浮生长的NSCs团,并能分化为神经元和星型胶质细胞,且经传代培养10代后仍具干细胞特性。2.荧光染料的标记率可达97%,细胞传10代后荧光亮度无明显衰减,并且分化后的细胞核中仍有荧光表达。
结论成功地培养出胚胎大鼠NSCs,培养出的细胞具有增殖能力、自我更新能力和多潜能分化能力,可分化为神经元及星型胶质细胞;荧光染料的标记可获得较高的标记率,可作为NSCs移植试验研究的供体细胞。
第二部分胚胎大鼠神经干细胞的冷冻及复苏
目的研究胚胎大鼠神经干细胞(Neural stem cells ,NSCs)冻存复苏后的成活率及再培养生长情况,观察低温冻存对NSCs生物特性的影响。
方法含有10%BSA及7.5%DMSO的NSCs无血清培养液(不含神经生长因子)作为冻存液,将利用无血清培养基体外培养的原代、第三代、第六代胚胎大鼠NSCs进行冻存,于1周、4周、8周、12周、16分别复苏,计数活细胞比例并进行再培养、分化鉴定。
结果不同的细胞代数,不同的冻存时间对复苏后的细胞存活率都无明显影响。复苏后NSCs的成活率均达到60%-70%,并能分化为神经元和星型胶质细胞。
结论本实验成功地对胚胎大鼠NSCs进行了冻存复苏及再培养,为择期应用NSCs移植治疗中枢神经系统疾病的实验研究奠定了基础。第三部分携带绿色荧光蛋白基因的重组腺病毒构建及鉴定目的构建携带绿色荧光蛋白基因的重组腺病毒,为神经干细胞(Neural stem cells, NSCs)的基因转染实验提供条件。方法利用腺病毒细菌内同源重组方法,构建携带绿色荧光蛋白(green fluorescent protein, GFP)基因的复制缺陷型重组腺病毒Ad-GFP,通过反复感染大量扩增病毒后,测定病毒的滴度,-80℃保存备用。结果重组质粒经限制性内切酶酶切电泳证明重组成功;重组质粒转染293细胞后,随时间变化表达绿色荧光的细胞数目逐渐增多,荧光强度也逐渐增强;通过反复感染大量扩增病毒后,检测病毒滴度约为5×109PFU/ml。结论本试验通过细菌内同源重组方法,成功地构建了携带绿色荧光蛋白基因的重组腺病毒(Ad-GFP),为下一步NSCs的体外基因感染研究提供基础。
第四部分胚胎大鼠神经干细胞体外基因感染研究
目的:建立基因工程化神经干细胞(Neural stem cells ,NSCs)以便为治疗感音神经性耳聋的研究创造条件。
方法:分离、培养胎鼠海马组织NSCs后,将通过细菌内同源重组方法构建携带有报告基因绿色荧光蛋白(green fluorescent protein, GFP)的重组腺病毒(Ad-GFP)感染NSCs,并进行再培养及诱导分化,通过流式细胞仪检测及荧光显微镜观察了解病毒的感染效率及GFP的表达情况。
结果:97%以上的胚胎NSCs能被腺病毒感染,绿色荧光表达可持续4周以上;被腺病毒感染的NSCs分化为神经元和星型胶质细胞后仍可有效表达GFP报告基因。结论:重组腺病毒载体在体外可以高效率地感染胚胎NSCs,经培养传代及分化后仍具干细胞特性;经基因工程化的NSCs可以作为细胞移植治疗感音神经性耳聋试验研究的供体细胞。
第五部分感染重组腺病毒的神经干细胞耳蜗移植实验目的:探讨感染携带报告基因的重组腺病毒后的神经干细胞(Neural stem cells, NSCs)移植到正常大鼠耳蜗中存活和报告基因的表达情况及其对正常大鼠听力和耳蜗形态结构的影响。
方法:动物分为三组:对照组(人工外淋巴液注射组)15只,实验组(感染Ad-GFP的NSCs移植组)15只,正常组(未处理组)10只。对照组及实验组的手术径路均为经圆窗膜途径。两组均于移植前及移植后14天测试大鼠听觉脑干诱发电位(auditory brainstem response, ABR)了解其听力的改变;于移植后14天通过耳蜗中轴切片的免疫组化染色和荧光显微镜观察NSCs在耳蜗中的存活、分布及报告基因的表达情况;通过耳蜗基底膜铺片硝酸银染色和耳蜗扫描电镜观察毛细胞的数目及耳蜗形态学的变化。
结果:对照组及实验组大鼠耳蜗移植后ABR反应阈与移植前差异均无显著意义(P>0.05);两组大鼠耳蜗移植后ABR反应阈间的差异亦无显著意义(P>0.05)。实验组耳蜗中轴切片的免疫组化及荧光显微镜观察表明感染Ad-GFP的NSCs移植入耳蜗14天后,存活的干细胞主要分布在耳蜗底回的前庭阶和鼓阶,并且在耳蜗的其他各回的前庭阶和鼓阶中也有存活,同时表达强烈的绿色荧光;绿色荧光蛋白报告基因在各回血管纹、螺旋神经节细胞中也有较明显的表达,但在Corti器的毛细胞和支持细胞中无明显荧光表达。三组耳蜗基底膜铺片硝酸银染色和耳蜗扫描电镜观察,发现三组均出现耳蜗OHC的散在缺失,OHC的缺失在各组中均有从底回到顶回逐渐增加的趋势,三组OHC的总缺失率均未超过1%,并且三组间各回的OHC的缺失率及总缺失率之间的差异均无显著意义(P>0.05),这表明三组中出现的OHC的缺失是由于毛细胞的自然缺失所导致,而与手术无关。耳蜗各回均未见内毛细胞及静纤毛的缺失。
结论:感染携带报告基因的重组腺病毒(Ad-GFP)的胚胎NSCs移植到正常大鼠耳蜗中可以存活,并可有效表达绿色荧光蛋白报告基因,移植后对其听力和耳蜗形态结构无明显影响。
PartⅠ
Culture、identification and label of embryonic rat neural stem cells
Objective To explore the methods of culture、identification and label of embryonic rat neural stem cells.
Methods The cells isolated from fetal rat hippocampus were identified with nestin immunocytochemical fluorescent staining. The cellular multiplication was observed by immunocytochemical fluorescence co-label after accession of Brdu. The neural stem cells (NSCs) were marked with fluorescent dye, bisbenzimide (Hoechest33342) and induced to differentiate.The differentiated cells were detected respectively with Neuron Specific Enolase(NSE) and Glial Fibrillary Acidic Protein( GFAP)immunocytochemical fluorescent staining.
Results Nest-like clusters of neural stem cells were obtained in suspension and the cells could been differentiated into neurons and astrocytes which retaining the main characteristics of NSCs after 10 passages of culture. The label efficiency of cells with Hoechest33342 was 97% and no attenuation of fluorescent brightness was observed after 10 passages of culture. The cellular fluorescence was observed in the NSCs and the differentiated cells.
Conclusion The cells from embryonic rat hippocampus possessed ability of division、multiplication and self-renew, which were believed to be NSCs of the central nervous system. The cells could be efficiently labeled with fluorescent dye, they could be used as donor cells in experimental research with transplantion of cells.
PartⅡCryopreservation and resuscitation of neural stem cells from fetal rat Objective To explore the survival rate of neural stem cells(NSCs) from fetal rat and its biological properties after cryopreservation and thawing.
Methods Different passages NSCs (the first passage、the third passage and the sixth passage)from fetal rat cultivated with serum-free medium in vitro were cryopreserved in the cryogen,which were neurosphere culture medium with 10%BSA and 7.5%DMSO(without neural growth factor).The cryopreserved cells were resuscitated at 1 week, 4 week, 8 week,12 week and 16 week respectively. The survival rate of cells were calculated and the cells were incubated and differentiated again.
Result Different time of cryopreservation、different passages did not affect NSCs survival (P>0.05)after cryopreservation .The survival rate of NSCs was from 60% to 70% after resuscitated, which were differentiated into neurons and astrocytes in 10% embryonic bovine serum(without growth factor).
Conclusion NSCs were successfully cryopreserved, resuscitated and re-cultured, which would create bases for the experimental study on the selected-date application of NSCs transplantation for nervous system disease.
PartⅢ
Construction and identification of recombinant adenovirus with Green fluorescence protein
Objective To construct recombinant adenovirus with Green fluorescence protein (Ad-GFP) and to create bases for the experimental study on gene transfer into the NSCs. Methods After the Ad-GFP was constructed by homologous recombination in bacteria, the adenovirus was amplified and the viral titer was determined by plaque assays, then the virus was conserved in refrigeratory(-80℃).
Result The constructed recombination was confirmed correct on agarose gel after restriction digestion. After transfected by the plasmid DNA, the package 293 cells began to express green fluorescence after 48 hours. The intensity of fluorescence increased gradually along with the extension of time, and the amounts of cells expressing the fluorescence enhanced gradually. The viral titer of 5×109 plaque forming units (pfu)/ml was achieved after amplification.
Conclusion The recombinant adenovirus carried green fluorescent protein gene (Ad-GFP) has been constructed successfully.
PartⅣThe study of gene transfer into neural stem cells in vitro
Objective To establish the genetically engineered neural stem cells (NSCs) to create bases for the experimental study on treating sensorineural deafness .
Methods After isolated and cultured from fetal rat hippocampus,the NSCs were infected by the recombinant adenovirus with Green fluorescence protein(Ad-GFP)which was constructed by homologous recombination in bacteria. After re-cultured and induced to differentiate, we observed the infection rate and the expression of GFP through Flow cytometry and fluorescent microscopy.
Result Above 97% of NSCs were infected by Ad-GFP and the expression of GFP could last for above 4 weeks, After infected by Ad-GFP, the NSCs were induced to differentiate. The differentiated cells could efficiently expression the GFP.
Conclusion The recombinant adenovirus could more effectively infect the NSCs from fetal rat in vitro, which retained their characteristics and differentiation ability after infected. They can be used as donor cells to experimental research on treating sensorineural deafness with transplantation of cells.
PartⅤ
The experimental study on embryonic neural stem cells infected by Ad-GFP transplantation into natural rat cochlea
Objective To explore the survival of neural stem cells (NSCs) infected by Ad-GFP in natural rat cochlea and to observed the changes of auditory electrophysiology and inner ear pathology of rat cochlea after NSCs transplantation.
Methods NSCs infected by Ad-GFP were transplanted into rat cochlea in the study group through round window, in the same way, the artificial perilymph were injected in control group. Auditory function was monitored by measuring thresholds of auditory brain stem responses (ABR) in two groups before transplantation and after transplantation; At 14 days after transplantation, the survival of cells and the expression of GFP was observed by immunohistochemistry and fluorescent microscopy; the changes of inner ear pathology of rat cochlea was determined by HC nuclei stained and scanning electron microscopy (SEM).
Result There was not significant difference in thresholds of ABR of the two groups between pre-transplantation and post-transplantation; At 14 days after transplantation, there was also not significant difference in thresholds of ABR between the two groups. In study group, the graft cells located mostly in the scala vestibule and the scala tympani of the basal turn of cochlea at 14 days after transplantation, and the survival cells were also observed in the scala vestibule and the scala tympani of other turns of cochlea, which all expressed strong green fluorescence; The expression of GFP was also obvious in stria vascularis and spiral ganglion cells but no obvious expression in cochlear hair cell(HC) and support cells. There were dispersive loss of out hair cell(OHC) in all turns of cochlea of every group, the loss rates of OHC increasd gradually from the first turn to the third turn and the total loss rate of every group were all under 1%. There was not significant difference in hair cells loss among these three groups(P>0.05). The inner hair cells (IHC) and the stereocilia of IHC were normal in all turns of cochlea of every group.So the loss of OHC was natural loss and was foreign to the surgery.
Conclusion The embryonic NSCs infected by Ad-GFP could survive in normal rat cochlea after transplantation, which could express GFP efficiently, and NSCs infected by Ad-GFP transplantation had not obvious affection to auditory function and inner ear pathology of rat cochlea.
目的探索胚胎大鼠神经干细胞(Neural stem cells ,NSCs)的分离培养、鉴定及标记的方法。
方法分离胚胎大鼠海马组织,用无血清培养技术培养NSCs,免疫组化荧光技术检测其巢蛋白(Nestin)的表达;5-溴脱氧尿嘧啶(Brdu)掺入试验,并用免疫荧光双标技术观测神经干细胞的增殖状况。采用荧光染料Hoechest33342标记NSCs并诱导其分化,用神经元特异性烯醇化酶(Neuron Specific Enolase, NSE)胶质纤维酸性蛋白(Glial Fibrillary Acidic Protein, GFAP)免疫组化荧光染色鉴定分化细胞。
结果1.获得了大量未分化呈巢状悬浮生长的NSCs团,并能分化为神经元和星型胶质细胞,且经传代培养10代后仍具干细胞特性。2.荧光染料的标记率可达97%,细胞传10代后荧光亮度无明显衰减,并且分化后的细胞核中仍有荧光表达。
结论成功地培养出胚胎大鼠NSCs,培养出的细胞具有增殖能力、自我更新能力和多潜能分化能力,可分化为神经元及星型胶质细胞;荧光染料的标记可获得较高的标记率,可作为NSCs移植试验研究的供体细胞。
第二部分胚胎大鼠神经干细胞的冷冻及复苏
目的研究胚胎大鼠神经干细胞(Neural stem cells ,NSCs)冻存复苏后的成活率及再培养生长情况,观察低温冻存对NSCs生物特性的影响。
方法含有10%BSA及7.5%DMSO的NSCs无血清培养液(不含神经生长因子)作为冻存液,将利用无血清培养基体外培养的原代、第三代、第六代胚胎大鼠NSCs进行冻存,于1周、4周、8周、12周、16分别复苏,计数活细胞比例并进行再培养、分化鉴定。
结果不同的细胞代数,不同的冻存时间对复苏后的细胞存活率都无明显影响。复苏后NSCs的成活率均达到60%-70%,并能分化为神经元和星型胶质细胞。
结论本实验成功地对胚胎大鼠NSCs进行了冻存复苏及再培养,为择期应用NSCs移植治疗中枢神经系统疾病的实验研究奠定了基础。第三部分携带绿色荧光蛋白基因的重组腺病毒构建及鉴定目的构建携带绿色荧光蛋白基因的重组腺病毒,为神经干细胞(Neural stem cells, NSCs)的基因转染实验提供条件。方法利用腺病毒细菌内同源重组方法,构建携带绿色荧光蛋白(green fluorescent protein, GFP)基因的复制缺陷型重组腺病毒Ad-GFP,通过反复感染大量扩增病毒后,测定病毒的滴度,-80℃保存备用。结果重组质粒经限制性内切酶酶切电泳证明重组成功;重组质粒转染293细胞后,随时间变化表达绿色荧光的细胞数目逐渐增多,荧光强度也逐渐增强;通过反复感染大量扩增病毒后,检测病毒滴度约为5×109PFU/ml。结论本试验通过细菌内同源重组方法,成功地构建了携带绿色荧光蛋白基因的重组腺病毒(Ad-GFP),为下一步NSCs的体外基因感染研究提供基础。
第四部分胚胎大鼠神经干细胞体外基因感染研究
目的:建立基因工程化神经干细胞(Neural stem cells ,NSCs)以便为治疗感音神经性耳聋的研究创造条件。
方法:分离、培养胎鼠海马组织NSCs后,将通过细菌内同源重组方法构建携带有报告基因绿色荧光蛋白(green fluorescent protein, GFP)的重组腺病毒(Ad-GFP)感染NSCs,并进行再培养及诱导分化,通过流式细胞仪检测及荧光显微镜观察了解病毒的感染效率及GFP的表达情况。
结果:97%以上的胚胎NSCs能被腺病毒感染,绿色荧光表达可持续4周以上;被腺病毒感染的NSCs分化为神经元和星型胶质细胞后仍可有效表达GFP报告基因。结论:重组腺病毒载体在体外可以高效率地感染胚胎NSCs,经培养传代及分化后仍具干细胞特性;经基因工程化的NSCs可以作为细胞移植治疗感音神经性耳聋试验研究的供体细胞。
第五部分感染重组腺病毒的神经干细胞耳蜗移植实验目的:探讨感染携带报告基因的重组腺病毒后的神经干细胞(Neural stem cells, NSCs)移植到正常大鼠耳蜗中存活和报告基因的表达情况及其对正常大鼠听力和耳蜗形态结构的影响。
方法:动物分为三组:对照组(人工外淋巴液注射组)15只,实验组(感染Ad-GFP的NSCs移植组)15只,正常组(未处理组)10只。对照组及实验组的手术径路均为经圆窗膜途径。两组均于移植前及移植后14天测试大鼠听觉脑干诱发电位(auditory brainstem response, ABR)了解其听力的改变;于移植后14天通过耳蜗中轴切片的免疫组化染色和荧光显微镜观察NSCs在耳蜗中的存活、分布及报告基因的表达情况;通过耳蜗基底膜铺片硝酸银染色和耳蜗扫描电镜观察毛细胞的数目及耳蜗形态学的变化。
结果:对照组及实验组大鼠耳蜗移植后ABR反应阈与移植前差异均无显著意义(P>0.05);两组大鼠耳蜗移植后ABR反应阈间的差异亦无显著意义(P>0.05)。实验组耳蜗中轴切片的免疫组化及荧光显微镜观察表明感染Ad-GFP的NSCs移植入耳蜗14天后,存活的干细胞主要分布在耳蜗底回的前庭阶和鼓阶,并且在耳蜗的其他各回的前庭阶和鼓阶中也有存活,同时表达强烈的绿色荧光;绿色荧光蛋白报告基因在各回血管纹、螺旋神经节细胞中也有较明显的表达,但在Corti器的毛细胞和支持细胞中无明显荧光表达。三组耳蜗基底膜铺片硝酸银染色和耳蜗扫描电镜观察,发现三组均出现耳蜗OHC的散在缺失,OHC的缺失在各组中均有从底回到顶回逐渐增加的趋势,三组OHC的总缺失率均未超过1%,并且三组间各回的OHC的缺失率及总缺失率之间的差异均无显著意义(P>0.05),这表明三组中出现的OHC的缺失是由于毛细胞的自然缺失所导致,而与手术无关。耳蜗各回均未见内毛细胞及静纤毛的缺失。
结论:感染携带报告基因的重组腺病毒(Ad-GFP)的胚胎NSCs移植到正常大鼠耳蜗中可以存活,并可有效表达绿色荧光蛋白报告基因,移植后对其听力和耳蜗形态结构无明显影响。
PartⅠ
Culture、identification and label of embryonic rat neural stem cells
Objective To explore the methods of culture、identification and label of embryonic rat neural stem cells.
Methods The cells isolated from fetal rat hippocampus were identified with nestin immunocytochemical fluorescent staining. The cellular multiplication was observed by immunocytochemical fluorescence co-label after accession of Brdu. The neural stem cells (NSCs) were marked with fluorescent dye, bisbenzimide (Hoechest33342) and induced to differentiate.The differentiated cells were detected respectively with Neuron Specific Enolase(NSE) and Glial Fibrillary Acidic Protein( GFAP)immunocytochemical fluorescent staining.
Results Nest-like clusters of neural stem cells were obtained in suspension and the cells could been differentiated into neurons and astrocytes which retaining the main characteristics of NSCs after 10 passages of culture. The label efficiency of cells with Hoechest33342 was 97% and no attenuation of fluorescent brightness was observed after 10 passages of culture. The cellular fluorescence was observed in the NSCs and the differentiated cells.
Conclusion The cells from embryonic rat hippocampus possessed ability of division、multiplication and self-renew, which were believed to be NSCs of the central nervous system. The cells could be efficiently labeled with fluorescent dye, they could be used as donor cells in experimental research with transplantion of cells.
PartⅡCryopreservation and resuscitation of neural stem cells from fetal rat Objective To explore the survival rate of neural stem cells(NSCs) from fetal rat and its biological properties after cryopreservation and thawing.
Methods Different passages NSCs (the first passage、the third passage and the sixth passage)from fetal rat cultivated with serum-free medium in vitro were cryopreserved in the cryogen,which were neurosphere culture medium with 10%BSA and 7.5%DMSO(without neural growth factor).The cryopreserved cells were resuscitated at 1 week, 4 week, 8 week,12 week and 16 week respectively. The survival rate of cells were calculated and the cells were incubated and differentiated again.
Result Different time of cryopreservation、different passages did not affect NSCs survival (P>0.05)after cryopreservation .The survival rate of NSCs was from 60% to 70% after resuscitated, which were differentiated into neurons and astrocytes in 10% embryonic bovine serum(without growth factor).
Conclusion NSCs were successfully cryopreserved, resuscitated and re-cultured, which would create bases for the experimental study on the selected-date application of NSCs transplantation for nervous system disease.
PartⅢ
Construction and identification of recombinant adenovirus with Green fluorescence protein
Objective To construct recombinant adenovirus with Green fluorescence protein (Ad-GFP) and to create bases for the experimental study on gene transfer into the NSCs. Methods After the Ad-GFP was constructed by homologous recombination in bacteria, the adenovirus was amplified and the viral titer was determined by plaque assays, then the virus was conserved in refrigeratory(-80℃).
Result The constructed recombination was confirmed correct on agarose gel after restriction digestion. After transfected by the plasmid DNA, the package 293 cells began to express green fluorescence after 48 hours. The intensity of fluorescence increased gradually along with the extension of time, and the amounts of cells expressing the fluorescence enhanced gradually. The viral titer of 5×109 plaque forming units (pfu)/ml was achieved after amplification.
Conclusion The recombinant adenovirus carried green fluorescent protein gene (Ad-GFP) has been constructed successfully.
PartⅣThe study of gene transfer into neural stem cells in vitro
Objective To establish the genetically engineered neural stem cells (NSCs) to create bases for the experimental study on treating sensorineural deafness .
Methods After isolated and cultured from fetal rat hippocampus,the NSCs were infected by the recombinant adenovirus with Green fluorescence protein(Ad-GFP)which was constructed by homologous recombination in bacteria. After re-cultured and induced to differentiate, we observed the infection rate and the expression of GFP through Flow cytometry and fluorescent microscopy.
Result Above 97% of NSCs were infected by Ad-GFP and the expression of GFP could last for above 4 weeks, After infected by Ad-GFP, the NSCs were induced to differentiate. The differentiated cells could efficiently expression the GFP.
Conclusion The recombinant adenovirus could more effectively infect the NSCs from fetal rat in vitro, which retained their characteristics and differentiation ability after infected. They can be used as donor cells to experimental research on treating sensorineural deafness with transplantation of cells.
PartⅤ
The experimental study on embryonic neural stem cells infected by Ad-GFP transplantation into natural rat cochlea
Objective To explore the survival of neural stem cells (NSCs) infected by Ad-GFP in natural rat cochlea and to observed the changes of auditory electrophysiology and inner ear pathology of rat cochlea after NSCs transplantation.
Methods NSCs infected by Ad-GFP were transplanted into rat cochlea in the study group through round window, in the same way, the artificial perilymph were injected in control group. Auditory function was monitored by measuring thresholds of auditory brain stem responses (ABR) in two groups before transplantation and after transplantation; At 14 days after transplantation, the survival of cells and the expression of GFP was observed by immunohistochemistry and fluorescent microscopy; the changes of inner ear pathology of rat cochlea was determined by HC nuclei stained and scanning electron microscopy (SEM).
Result There was not significant difference in thresholds of ABR of the two groups between pre-transplantation and post-transplantation; At 14 days after transplantation, there was also not significant difference in thresholds of ABR between the two groups. In study group, the graft cells located mostly in the scala vestibule and the scala tympani of the basal turn of cochlea at 14 days after transplantation, and the survival cells were also observed in the scala vestibule and the scala tympani of other turns of cochlea, which all expressed strong green fluorescence; The expression of GFP was also obvious in stria vascularis and spiral ganglion cells but no obvious expression in cochlear hair cell(HC) and support cells. There were dispersive loss of out hair cell(OHC) in all turns of cochlea of every group, the loss rates of OHC increasd gradually from the first turn to the third turn and the total loss rate of every group were all under 1%. There was not significant difference in hair cells loss among these three groups(P>0.05). The inner hair cells (IHC) and the stereocilia of IHC were normal in all turns of cochlea of every group.So the loss of OHC was natural loss and was foreign to the surgery.
Conclusion The embryonic NSCs infected by Ad-GFP could survive in normal rat cochlea after transplantation, which could express GFP efficiently, and NSCs infected by Ad-GFP transplantation had not obvious affection to auditory function and inner ear pathology of rat cochlea.
引文
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12. Reynold BA, Tetzlaff W, Weiss S.A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes.J Neurosci. 1992, 12, 4565- 4574
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14. Cao QL, Zhang YP, Howard RM, et al. Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage.Exp Neurol.2001 Jan;167(1): 48-58.
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1. Keirstead H.Stem cell transplantation into the central nervous system and centrol of differentiation. J Neurosci Res.2001;63(3):233-236
2. Cao QL , Zhang YP , Howard RM, Walters WM, Tsoulfas P Whittemore SR. Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage. Exp Neurol 2001;167 :48 - 58.
3. Christopher I , Lu XB , Lu PH , Xu XM. Neural stem cell transplantation in the repair of spinal cord injury. Prog Nat Sci 2001 ;11(7) :490 - 502.
4. Christopher RH , Jonathon PW, Nevin AL , Condie BG.Neuronal differentiation of cryopreserved neural progenitor cells derived from mouse embryonic stem cells. Biochem Biophys Res Commun 2000 ;271(2) : 418 - 421.
5.叶韵斌,陈强,陈明永,等.外周血干细胞冻存的探讨[J].中国肿瘤临床与康复, 2000;7(4):38-40.
6.周燕琼,刘小辉,刘幸平,等.人肝癌细胞冷冻保存方法研究[J].世界华人消化杂志,2000;8(6):714-715.
7. Richter E , Eichler H , Raske D , et al . 5 % Me2SO is sufficient to preserve stem cells derived from cord blood [ J ] . Bone Marrow Transplant , 1998; 22 suppll:16-18.
8. Tornford WW, Duff GP, Mankin HJ . Experimental freezepreservation of chondrocytes [J] . Clin Orthop , 1985;197:11.
9.马政文,富赛里,尹岚,等.大鼠胚胎神经干细胞的冷冻复苏.中国神经科学杂志.2003; 19 (2):122-126.
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