BDNF基因修饰骨髓间充质干细胞的表达及对豚鼠螺旋神经元保护作用
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摘要
第一章
BDNF基因修饰骨髓间充质干细胞的建立及体外表达
目的骨髓间充质干细胞(Mesenchymal Stem Cells,MSCs)易于分离扩增,具备多方向分化潜能,免疫源性低,可自体移植,并且有良好迁移性,暗示其在基因治疗中可能成为有价值的运载细胞。本章利用非病毒载体介导脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)转染MSCs,观察细胞转染特性和体外表达情况,建立基因工程细胞,为进一步体内研究提供基因工程细胞来源。
方法取豚鼠的骨髓进行MSCs的分离、培养和流式细胞仪鉴定。通过比较两种非病毒载体转染方法(脂质体法和电穿孔法),选择较好的方法介导pcDNA3.1(-)-BDNF转染MSCs并经优化浓度的G418筛选建立基因工程细胞,转染后48小时(瞬时表达)和筛选后(稳定表达)用免疫组化检测BDNF基因表达情况,RT-PCR进一步验证目的基因表达情况。
结果成功培养MSCs,流式细胞鉴定显示各细胞表面标志表达率分别为CD44:94.65%、CD34:4.37%、CD45:7.81%。免疫组化显示脂质体介导转染的BDNF瞬时表达率约为5.80%,电穿孔法瞬时表达阳性率约为24.29%。脂质体法转染筛选14天后细胞几乎全部死亡;电穿孔法转染成功扩大培养并建立工程细胞(BDNF-MSCs),免疫组化显示BDNF阳性表达率达90%,RT-PCR扩增产物电泳证实目的基因阳性条带。
结论用电穿孔法成功建立BDNF基因修饰MSCs的工程细胞,并用免疫组化和RT-PCR方法证实了工程细胞具备体外表达目的基因功能。
第二章
BDNF基因修饰骨髓间充质干细胞正常豚鼠内耳移植的实验研究
目的BDNF基因修饰MSCs(BDNF-MSCs)是治疗神经退变性疾病包括感音神经性聋的潜在有效方法,但首先要明确以下三个问题:一是手术及细胞移植对耳蜗功能及结构是否造成不良影响;二是基因修饰的MSCs在耳蜗内存活和分布情况如何;最后是移植细胞在耳蜗内是否能较长时间维持表达目的基因的能力。目前还没有转基因MSCs内耳移植的报道,在此部分我们将在正常豚鼠耳蜗中对这些问题进行初步的探讨。
方法正常豚鼠分3组,组Ⅰ:为空白对照组,组Ⅱ:经鼓阶开窗注射MSCs,组Ⅲ:经鼓阶开窗注射BDNF-MSCs。移植细胞均经DAPI荧光标记。3个组的动物均在术前1d、术后7d及术后28d分别行ABR检测,比较各组术前、术后的ABR阈值变化;分别取术后7d及28d各组动物的耳蜗石蜡切片HE染色观察耳蜗结构;荧光显微镜观察移植细胞存活及分布;免疫组化方法检测BDNF在耳蜗内的表达情况。
结果经鼓阶开窗移植细胞导致术后7d时ABR阈值的暂时性提高,术后28d后恢复至正常水平。耳蜗石蜡切片HE染色显示实验动物耳蜗内结构无明显异常改变。组Ⅱ和组Ⅲ术后7d耳蜗切片可见荧光信号多分布于耳蜗底周的外淋巴腔(鼓阶和前庭阶),极少量细胞能迁移到中阶及蜗轴,术后28d移植细胞存活数量有所下降,但两组动物的移植细胞存活时间均超过28d,并且BDNF基因修饰MSCs能提高移植细胞术后28d时的存活率。免疫组化显示组Ⅲ部分移植细胞表达BDNF,术后28d的阳性表达率比术后7d有显著性下降,而其余两组无阳性染色。
结论经鼓阶开窗移植MSCs对听力的损害是轻微(<10dB SPL)和暂时的(<28d)。移植后光镜下耳蜗形态结构没有明显变化。移植细胞能存活并分布于耳蜗内各周,主要在耳蜗底周的外淋巴腔,BDNF基因转染MSCs后可以提高移植细胞的存活率。基因修饰的MSCs能在正常耳蜗内维持表达BDNF至少28d,暗示了MSCs可用作细胞载体向内耳输送BDNF治疗感音神经性聋。
第三章
BDNF基因修饰骨髓间充质干细胞在致聋豚鼠内耳的表达及对螺旋神经元的保护作用
目的多种因素包括神经营养因子减少、噪音、缺血、耳毒性药物等均会导致内耳毛细胞和螺旋神经元(spiral ganglion neurons,SGNs)的直接或间接的死亡,细胞死亡主要以凋亡的形式。在前面已证明BDNF基因修饰MSCs具备体外及正常耳蜗内表达BDNF的能力,并可以在内耳中存活至少28d,本章中将进一步分析工程细胞在药物致聋豚鼠内耳的表达水平(荧光定量RT-PCR)及对SGNs的保护作用。
方法阿米卡星致聋的豚鼠随机分两组。实验组经鼓阶开窗注射BDNF-MSCs,对照组注射外淋巴液。每组均在7d及28d处死动物。荧光定量RT-PCR检测两组动物不同时间点的耳蜗组织BDNF的表达差异,并取耳蜗组织切片行TUNEL检测SGNs凋亡。
结果实验组在术后7d和28d的BDNF表达量均显著性高于对照组,实验组术后7d的BDNF表达量是对照组的107倍,术后28d下降为33倍。实验组术后7d及28d的耳蜗SGNs凋亡指数均比对照组有显著性下降。
结论药物致聋豚鼠内耳移植的BDNF基因工程细胞能维持表达长达28d,并能显著减少SGNs的凋亡。
PartⅠ
Establishment of BDNF-engineered mesenchymal stem cells and its expression in vitro
Objective Cell replacement and gene therapy are attractive research giving new hope to cure sensorineural hearin loss.One of the key points for long-term expression of target gene in vivo is that what kind of vector to be selected.Bone marrow mesenchymal stem cells(MSCs) do possess easy-manipulation,differentiated multipotention,agility immigration,relatively immuno-privilege,which potentially enable their allogeneic therapeutic and autotransplantation.MSCs have gained rapidly increasing attention as a therapeutic tool for gene delivery.This part is aim to establish genetic engineering cell using brain-derived neurotrophic factor(BDNF) gene transected MSCs by means of non-virus vector,and explore its characteristics and expression in vitro,provide engineering cell population for subsequent research.
Methods The MSCs were obtained from the tibias and femurs of the guinea pigs.After cultured for free passages,flow cytometry(FCM) analysis was used to detect markers CD44,CD34,and CD45.By comparison of two non-virus vectors as lipofectamine and electroporation,the better was choice to transfer pcDNA3.1(-)-BDNF to MSCs and stable DNA integration were selected by culturing with G418 of optimized concentration.Expressed BDNF were detected by immunohistochemistry 48h post-tranfection and after selection.The expression of BDNF also confirmed by RT-PCR following by electrophoresis.
Results MSCs population were isolated and expanded successful with distinct expression of CD44,CD34 and CD45 by 94.65%,4.37%,7.81% respectively.Immunohistochemistry transient expressed BDNF was 5%by lipofectamine and 25%by electroporation.Stable expression cell lines of BDNF engineered MSCs,with 90%expression rate of BDNF by immunohistochemistry and expression of BDNF mRNA confirmed by RT-PCR, were successfully established by electroporation whereas failed by lipofectamine.
Conclusions Genetic engineering cells using BDNF transected MSCs were established by electroporation whereas failed by lipofectamine,and the expressed BDNF was confirmed by immunohistochemistry and RT-PCR in vitro.
PartⅡ
Transplantation of bone BDNF-engineered mesenchymal stem cells into the cochlea of normal guinea pigs
Objective BDNF-engineered MSCs has the potential to treat an array of degenerative neurologic disorders including sensorineural hearing impairment. But the following questions must be identified in its practice.First is the functional and structural influence to recipients inner ear by cells transplantation;Second is the distribution and survival of grafted MSCs.Third is that how long can the BDNF-engineered MSCs maintain the expression of the target gene in vovo.By now there are no studies on transgened MSCs transplanted into the guinea pig cochleas.The main purpose of this prospective animal study was to reveal the questions as mentioned above, serving as control for the studies on deafened models.
Methods the guinea pigs of normal hearing serving as recipients were performed an intraperilymphatic transplantation of different mixture with scala tympani injection.GroupⅠserved as a negative control without any intervention,groupⅡwere injected MSCs,and groupⅢinjected BDNF-engineered MSCs,grafted cells were labeled by DAPI.Preoperative and postoperative ABR audibility thresholds were compared between and within group.Cochlear structure was evaluated by microscopic examination of paraffin sections cut through the cochlea of the recipient animals.The distribution and survival of transplanted cells was evaluated by fluorescence microscope.Expressed BDNF was detected by immunohistochemistry.
Results An identical significant elevated ARB thresholds were found in 7 days postoperatively at frequency 4kHz,8 kHz,16 kHz in groupⅡandⅢ, but the impairment was transcent and recovered within 28 days.NO distinct structural alternation was found in cochlea postoperatively.7 days postoperatively,most transplanted cells labeled by DAPI in groupⅢandⅢwere found in the scala tympani and scala vestibule,even a small number located in the scala media and modiolus.28 days postoperatively,labeled cells still exist in perilymphatic space despite significantly decrement. BDNF-engineered MSCs had higher survivals in inner ears compare to bare MSCs.The positive immunohistochemical reaction was detected in groupⅢin 7 and 28 days postoperatively,differed from the other two groups which were negative.
Conclusions The trancent hearing impairment was found when using MSCs transplantation with scala tympani injection,which could recover within 28 days.NO distinct structural alternation was found in cochlea postoperatively.The grafted cells can survive at least for 28 days in the cochlea and have the potential to migrate.BDNF transfection can elevated survivals of grafted MSCs.Thus,BDNF-engineered MSCs can be used as vehicles to deliver the BDNF gene into the cochlea with implications for the treatment of sensorineural deafness.
PartⅢ
The expression of BDNF-engineered MSCs transplanted into the cochlea of the deafened guinea pigs and its protection to the spiral ganglion neurons in vovo
Objective BDNF plays an important role in regulating the survival and differentiation of various neuronal populations including spiral ganglion neurons(SGNs).Based on these salutary effects of BDNF and the expression of BDNF-engineered MSCs in vitro and in normal cochlea,its expression level and efficacy in preventing the spiral ganglion neurons(SGNs) apoptosis in the deafened guinea pigs cochlea were evaluated in this part.
Methods 24 guinea pig deafened by amikacin subcutaneous injection were randomly divided into two group,BDNF-engineered MSCs were transplanted into the cochlea of the experimental group with scala tympani injection,while the control group were injected artificial perilymphatic fluid.Total RNA were extracted for semiquantitatve real time RT- PCR,and TUNEL was used to identify the apoptotic neurons.
Results The BDNF expressive level in experimental group was significant higher than in the control group,respectively,107 and 28 times higher at 7 days and 28 days postoperatively.The decreased apoptotic index of SGNs were detected in the experimental group compared to the control group.
Conclusions BDNF-engineered MSCs can maintain high level expressed BDNF for at least 28 days after transplanted into deafened cochlea of the guinea pigs,and provide neurotrophic effect on SGNs by preventing its apoptosis.
BDNF基因修饰骨髓间充质干细胞的建立及体外表达
目的骨髓间充质干细胞(Mesenchymal Stem Cells,MSCs)易于分离扩增,具备多方向分化潜能,免疫源性低,可自体移植,并且有良好迁移性,暗示其在基因治疗中可能成为有价值的运载细胞。本章利用非病毒载体介导脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)转染MSCs,观察细胞转染特性和体外表达情况,建立基因工程细胞,为进一步体内研究提供基因工程细胞来源。
方法取豚鼠的骨髓进行MSCs的分离、培养和流式细胞仪鉴定。通过比较两种非病毒载体转染方法(脂质体法和电穿孔法),选择较好的方法介导pcDNA3.1(-)-BDNF转染MSCs并经优化浓度的G418筛选建立基因工程细胞,转染后48小时(瞬时表达)和筛选后(稳定表达)用免疫组化检测BDNF基因表达情况,RT-PCR进一步验证目的基因表达情况。
结果成功培养MSCs,流式细胞鉴定显示各细胞表面标志表达率分别为CD44:94.65%、CD34:4.37%、CD45:7.81%。免疫组化显示脂质体介导转染的BDNF瞬时表达率约为5.80%,电穿孔法瞬时表达阳性率约为24.29%。脂质体法转染筛选14天后细胞几乎全部死亡;电穿孔法转染成功扩大培养并建立工程细胞(BDNF-MSCs),免疫组化显示BDNF阳性表达率达90%,RT-PCR扩增产物电泳证实目的基因阳性条带。
结论用电穿孔法成功建立BDNF基因修饰MSCs的工程细胞,并用免疫组化和RT-PCR方法证实了工程细胞具备体外表达目的基因功能。
第二章
BDNF基因修饰骨髓间充质干细胞正常豚鼠内耳移植的实验研究
目的BDNF基因修饰MSCs(BDNF-MSCs)是治疗神经退变性疾病包括感音神经性聋的潜在有效方法,但首先要明确以下三个问题:一是手术及细胞移植对耳蜗功能及结构是否造成不良影响;二是基因修饰的MSCs在耳蜗内存活和分布情况如何;最后是移植细胞在耳蜗内是否能较长时间维持表达目的基因的能力。目前还没有转基因MSCs内耳移植的报道,在此部分我们将在正常豚鼠耳蜗中对这些问题进行初步的探讨。
方法正常豚鼠分3组,组Ⅰ:为空白对照组,组Ⅱ:经鼓阶开窗注射MSCs,组Ⅲ:经鼓阶开窗注射BDNF-MSCs。移植细胞均经DAPI荧光标记。3个组的动物均在术前1d、术后7d及术后28d分别行ABR检测,比较各组术前、术后的ABR阈值变化;分别取术后7d及28d各组动物的耳蜗石蜡切片HE染色观察耳蜗结构;荧光显微镜观察移植细胞存活及分布;免疫组化方法检测BDNF在耳蜗内的表达情况。
结果经鼓阶开窗移植细胞导致术后7d时ABR阈值的暂时性提高,术后28d后恢复至正常水平。耳蜗石蜡切片HE染色显示实验动物耳蜗内结构无明显异常改变。组Ⅱ和组Ⅲ术后7d耳蜗切片可见荧光信号多分布于耳蜗底周的外淋巴腔(鼓阶和前庭阶),极少量细胞能迁移到中阶及蜗轴,术后28d移植细胞存活数量有所下降,但两组动物的移植细胞存活时间均超过28d,并且BDNF基因修饰MSCs能提高移植细胞术后28d时的存活率。免疫组化显示组Ⅲ部分移植细胞表达BDNF,术后28d的阳性表达率比术后7d有显著性下降,而其余两组无阳性染色。
结论经鼓阶开窗移植MSCs对听力的损害是轻微(<10dB SPL)和暂时的(<28d)。移植后光镜下耳蜗形态结构没有明显变化。移植细胞能存活并分布于耳蜗内各周,主要在耳蜗底周的外淋巴腔,BDNF基因转染MSCs后可以提高移植细胞的存活率。基因修饰的MSCs能在正常耳蜗内维持表达BDNF至少28d,暗示了MSCs可用作细胞载体向内耳输送BDNF治疗感音神经性聋。
第三章
BDNF基因修饰骨髓间充质干细胞在致聋豚鼠内耳的表达及对螺旋神经元的保护作用
目的多种因素包括神经营养因子减少、噪音、缺血、耳毒性药物等均会导致内耳毛细胞和螺旋神经元(spiral ganglion neurons,SGNs)的直接或间接的死亡,细胞死亡主要以凋亡的形式。在前面已证明BDNF基因修饰MSCs具备体外及正常耳蜗内表达BDNF的能力,并可以在内耳中存活至少28d,本章中将进一步分析工程细胞在药物致聋豚鼠内耳的表达水平(荧光定量RT-PCR)及对SGNs的保护作用。
方法阿米卡星致聋的豚鼠随机分两组。实验组经鼓阶开窗注射BDNF-MSCs,对照组注射外淋巴液。每组均在7d及28d处死动物。荧光定量RT-PCR检测两组动物不同时间点的耳蜗组织BDNF的表达差异,并取耳蜗组织切片行TUNEL检测SGNs凋亡。
结果实验组在术后7d和28d的BDNF表达量均显著性高于对照组,实验组术后7d的BDNF表达量是对照组的107倍,术后28d下降为33倍。实验组术后7d及28d的耳蜗SGNs凋亡指数均比对照组有显著性下降。
结论药物致聋豚鼠内耳移植的BDNF基因工程细胞能维持表达长达28d,并能显著减少SGNs的凋亡。
PartⅠ
Establishment of BDNF-engineered mesenchymal stem cells and its expression in vitro
Objective Cell replacement and gene therapy are attractive research giving new hope to cure sensorineural hearin loss.One of the key points for long-term expression of target gene in vivo is that what kind of vector to be selected.Bone marrow mesenchymal stem cells(MSCs) do possess easy-manipulation,differentiated multipotention,agility immigration,relatively immuno-privilege,which potentially enable their allogeneic therapeutic and autotransplantation.MSCs have gained rapidly increasing attention as a therapeutic tool for gene delivery.This part is aim to establish genetic engineering cell using brain-derived neurotrophic factor(BDNF) gene transected MSCs by means of non-virus vector,and explore its characteristics and expression in vitro,provide engineering cell population for subsequent research.
Methods The MSCs were obtained from the tibias and femurs of the guinea pigs.After cultured for free passages,flow cytometry(FCM) analysis was used to detect markers CD44,CD34,and CD45.By comparison of two non-virus vectors as lipofectamine and electroporation,the better was choice to transfer pcDNA3.1(-)-BDNF to MSCs and stable DNA integration were selected by culturing with G418 of optimized concentration.Expressed BDNF were detected by immunohistochemistry 48h post-tranfection and after selection.The expression of BDNF also confirmed by RT-PCR following by electrophoresis.
Results MSCs population were isolated and expanded successful with distinct expression of CD44,CD34 and CD45 by 94.65%,4.37%,7.81% respectively.Immunohistochemistry transient expressed BDNF was 5%by lipofectamine and 25%by electroporation.Stable expression cell lines of BDNF engineered MSCs,with 90%expression rate of BDNF by immunohistochemistry and expression of BDNF mRNA confirmed by RT-PCR, were successfully established by electroporation whereas failed by lipofectamine.
Conclusions Genetic engineering cells using BDNF transected MSCs were established by electroporation whereas failed by lipofectamine,and the expressed BDNF was confirmed by immunohistochemistry and RT-PCR in vitro.
PartⅡ
Transplantation of bone BDNF-engineered mesenchymal stem cells into the cochlea of normal guinea pigs
Objective BDNF-engineered MSCs has the potential to treat an array of degenerative neurologic disorders including sensorineural hearing impairment. But the following questions must be identified in its practice.First is the functional and structural influence to recipients inner ear by cells transplantation;Second is the distribution and survival of grafted MSCs.Third is that how long can the BDNF-engineered MSCs maintain the expression of the target gene in vovo.By now there are no studies on transgened MSCs transplanted into the guinea pig cochleas.The main purpose of this prospective animal study was to reveal the questions as mentioned above, serving as control for the studies on deafened models.
Methods the guinea pigs of normal hearing serving as recipients were performed an intraperilymphatic transplantation of different mixture with scala tympani injection.GroupⅠserved as a negative control without any intervention,groupⅡwere injected MSCs,and groupⅢinjected BDNF-engineered MSCs,grafted cells were labeled by DAPI.Preoperative and postoperative ABR audibility thresholds were compared between and within group.Cochlear structure was evaluated by microscopic examination of paraffin sections cut through the cochlea of the recipient animals.The distribution and survival of transplanted cells was evaluated by fluorescence microscope.Expressed BDNF was detected by immunohistochemistry.
Results An identical significant elevated ARB thresholds were found in 7 days postoperatively at frequency 4kHz,8 kHz,16 kHz in groupⅡandⅢ, but the impairment was transcent and recovered within 28 days.NO distinct structural alternation was found in cochlea postoperatively.7 days postoperatively,most transplanted cells labeled by DAPI in groupⅢandⅢwere found in the scala tympani and scala vestibule,even a small number located in the scala media and modiolus.28 days postoperatively,labeled cells still exist in perilymphatic space despite significantly decrement. BDNF-engineered MSCs had higher survivals in inner ears compare to bare MSCs.The positive immunohistochemical reaction was detected in groupⅢin 7 and 28 days postoperatively,differed from the other two groups which were negative.
Conclusions The trancent hearing impairment was found when using MSCs transplantation with scala tympani injection,which could recover within 28 days.NO distinct structural alternation was found in cochlea postoperatively.The grafted cells can survive at least for 28 days in the cochlea and have the potential to migrate.BDNF transfection can elevated survivals of grafted MSCs.Thus,BDNF-engineered MSCs can be used as vehicles to deliver the BDNF gene into the cochlea with implications for the treatment of sensorineural deafness.
PartⅢ
The expression of BDNF-engineered MSCs transplanted into the cochlea of the deafened guinea pigs and its protection to the spiral ganglion neurons in vovo
Objective BDNF plays an important role in regulating the survival and differentiation of various neuronal populations including spiral ganglion neurons(SGNs).Based on these salutary effects of BDNF and the expression of BDNF-engineered MSCs in vitro and in normal cochlea,its expression level and efficacy in preventing the spiral ganglion neurons(SGNs) apoptosis in the deafened guinea pigs cochlea were evaluated in this part.
Methods 24 guinea pig deafened by amikacin subcutaneous injection were randomly divided into two group,BDNF-engineered MSCs were transplanted into the cochlea of the experimental group with scala tympani injection,while the control group were injected artificial perilymphatic fluid.Total RNA were extracted for semiquantitatve real time RT- PCR,and TUNEL was used to identify the apoptotic neurons.
Results The BDNF expressive level in experimental group was significant higher than in the control group,respectively,107 and 28 times higher at 7 days and 28 days postoperatively.The decreased apoptotic index of SGNs were detected in the experimental group compared to the control group.
Conclusions BDNF-engineered MSCs can maintain high level expressed BDNF for at least 28 days after transplanted into deafened cochlea of the guinea pigs,and provide neurotrophic effect on SGNs by preventing its apoptosis.
引文
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