人骨髓间充质干细胞诱导分化神经元样细胞移植治疗局灶性脑缺血的实验研究
发布时间：2018-09-12 07:44
【摘要】： 目的:脑血管意外是世界范围内的三大死因之一,也是导致成人残疾的首位病因。我国年发病率约130-300万,死亡60-100万,存活者中75%有不同程度的残疾。尽管在脑血管意外的急救和早期康复方面取得了很大进展,但对脑血管意外后期严重的神经功能障碍缺乏有效的治疗方法。21世纪是再生医学的世纪,干细胞的研究则是当前生命科学的重中之重。与胚胎干细胞和神经干细胞相比,人骨髓间充质干细胞BMSCs具有以下优点:①取材方便,分离获取容易,可从自身骨髓提取;②在体外扩增迅速,具有多向分化潜能;③是来自中胚层的早期细胞,避免组织配型及免疫排斥等问题;④可避免伦理学方面的争议等。因此,受到越来越多的学者关注。但BMSCs移植后在体内微环境下分化为神经胶质细胞的比率较大而分化为神经元样细胞的比率较小,如果能将骨髓基质细胞在体外分化为神经元样细胞,再行细胞移植将会有事半功倍的效果。本研究拟探讨体外分离培养、纯化鉴定hBMSCs的方法,分析其生物学特性并定向诱导分化为神经元样细胞,并通过立体定向将hBMSCs源性神经元样细胞植入脑缺血大鼠脑内,观察移植后细胞存活、迁移、分化以及大鼠的神经功能障碍恢复情况,并与hBMSCs移植做一比较,探讨hBMSCs源性神经元样细胞移植对于局灶性脑缺血后神经功能缺失的修复作用及可能机制,以期为hBMSCs源性神经元样细胞移植治疗脑缺血提供理论依据。 方法:(1)用密度梯度离心结合贴壁培养法分离纯化hBMSCs,在体外观察hBMSCs生长特性、传代扩增,测定生长曲线,形态学观察,免疫细胞化学及图像分析测定细胞表面抗原表达情况;(2)将其与T淋巴细胞共培养,通过3H-TdR掺入、β液闪计数,分析其免疫原性;(3)采用全反式维甲酸(all-trans-retinoic acid,ATRA)、叔丁对甲氧酚(butylated hydroxyanisole,BHA)等对培养的细胞向神经细胞方向诱导分化,继而采用免疫细胞化学等方法对分化的细胞进行鉴定;(4)采用线栓法制备大鼠局灶性脑缺血模型。将脑缺血大鼠随机分为磷酸盐缓冲液(PBS)组、hBMSCs组及hBMSCs源性神经元样细胞组,分别将PBS、BrdU标记的hBMSCs及BrdU标记的源性神经元样细胞通过立体定向植入大鼠纹状体内。术前、术后1、3、6及8周进行神经损伤严重程度评分(NSS)、平衡木实验(BBT)、抬高身体摇摆实验(EBST)、一次性被动回避平台实验(Step-down Passive Avoidance)及水迷宫实验(Water Maze Test),观察各组术后神经功能改变情况;8周后处死大鼠取脑,HE染色、BrdU免疫组化染色以及BrdU/NSE, BrdU/GFAP免疫组化双染,观察植入BMSCs的存活、迁移、分化情况。 结果:(1)hBMSCs在体外可以大量增殖,原代培养需要2~3周,传代后速度加快,可以获取大量贴壁细胞。细胞呈均一的成纤维细胞样,应用流式细胞仪检测MSCs的表面抗原,均一表达CD29、CD44和CD105,不表达CD34、CD45、CD19、HLA-DR和CD106;(2)与T淋巴细胞体外共培养,无明显的促T细胞增殖反应,提示其具有低免疫原性的特点;(3)可诱导表达神经元标志抗原神经元特异性烯醇化酶(neuron specific enolase, NSE)、星型胶质细胞标志抗原胶质纤维酸性蛋白(glial fibrilament acidic protein,GFAP);(4)应用线栓法短暂闭塞大鼠右侧大脑中动脉后,大鼠出现左侧肢体不同程度偏瘫同时伴有右侧Honner氏征。TTC和HE染色均显示缺血区域位于右侧纹状体、额顶区皮层,位置和范围较为稳定;神经功能行为学检测显示:hBMSCs移植大鼠NSS、BBT、EBST在术后1、3、6、8周与PBS对照组比较有显著好转(P 0.01),在术后1、3周与hBMSCs源性神经元样细胞移植组比较有好转(P 0.05),6、8周NSS、BBT、EBST评分两组比较无统计学差异(P 0.05);hBMSCs源性神经元样细胞移植组大鼠NSS、BBT、EBST在术后3、6、8周较PBS对照组明显改善(P 0.01),在术后1、3周较hBMSCs移植组差(P 0.05),6、8周NSS、BBT、EBST评分两组比较无统计学差异(P 0.05);hBMSCs和hBMSCs源性神经元样细胞组较对照组一次性被动回避平台实验及水迷宫实验明显改善(P 0.01);hBMSCs源性神经元样细胞较hBMSCs组效果更好(P 0.05)。移植8周后,hBMSCs组和hBMSCs源性神经元样细胞组均可见较多BrdU表达阳性的细胞,细胞集中在移植位点及周围区域,有向缺血灶迁移的趋势,局部无胶质增生和淋巴细胞浸润,hBMSCs组Brdu阳性细胞群中,11.5-19.3%为GFAP阳性细胞,1.5-4.8%为NSE阳性细胞,hBMSCs源性神经元样细胞组Brdu阳性细胞群中,23.5-39.1%为NSE阳性细胞,9.8-17.6%为GFAP阳性细胞。 结论:hBMSCs属骨髓中单个核细胞,具有免疫原性低、可塑性好和扩增能力强等特性,密度梯度离心结合贴壁培养法能有效分离纯化hBMSCs;全反式维甲酸(ATRA)、叔丁对甲氧酚(BHA)等对培养的细胞向神经细胞方向诱导分化,可诱导表达神经元标志抗原神经元特异性烯醇化酶(NSE)的hBMSCs源性神经元样细胞;hBMSCs和hBMSCs源性神经元样细胞脑内移植均能有效改善局灶性脑缺血大鼠的神经功能症状,hBMSCs移植作用起效更快,hBMSCs源性神经元样细胞移植在学习、记忆功能方面改善更好;hBMSCs源性神经元样细胞在体内大部分表达NSE阳性,优于hBMSCs,局部无肿瘤生成及明显炎症反应。hBMSCs源性神经元样细胞脑内移植是治疗局灶性脑缺血的一种可能措施。
[Abstract]:Objective:Cerebrovascular accident is one of the three leading causes of death worldwide and the first cause of disability in adults.The annual incidence of cerebrovascular accident in China is about 130-3 million,the death rate is 600-1 million,75% of the survivors have different degrees of disability.Although great progress has been made in the first aid and early rehabilitation of cerebrovascular accident,it is serious in the late stage of cerebrovascular accident. Compared with embryonic stem cells and neural stem cells, human bone marrow mesenchymal stem cells (BMSCs) have the following advantages: (1) easy extraction, easy isolation and acquisition, and can be extracted from their own bone marrow; In vitro amplification is rapid and has the potential of multidirectional differentiation; (3) early cells from the mesoderm, avoiding tissue matching and immune rejection; (4) avoiding ethical controversy and so on. In order to reduce the ratio of neuron-like cells, bone marrow stromal cells can be differentiated into neuron-like cells in vitro, and then transplanted with cells. This study aims to explore the methods of isolation, culture, purification and identification of hBMSCs in vitro, analyze their biological characteristics, and induce differentiation into neuron-like cells, and establish a new method. Neuron-like cells derived from hBMSCs were implanted into the brain of rats with focal cerebral ischemia in vivo. The survival, migration, differentiation and recovery of neurological dysfunction were observed after transplantation. The effects of transplantation of hBMSCs-derived neuron-like cells on the repair of neurological deficits after focal cerebral ischemia were compared with that of transplantation of hBMSCs. The mechanism provides a theoretical basis for the treatment of cerebral ischemia by hBMSCs derived neuron like cell transplantation.
Methods: (1) hBMSCs were isolated and purified by density gradient centrifugation combined with adherent culture, and the growth characteristics of hBMSCs were observed in vitro, subcultured and amplified, the growth curve, morphological observation, immunocytochemistry and image analysis were used to determine the expression of cell surface antigen; (2) hBMSCs were co-cultured with T lymphocytes, and were analyzed by 3H-TdR incorporation and beta liquid scintillation counting. Its immunogenicity; (3) All-trans-retinoic acid (ATRA), butylated hydroxyanisole (BHA) were used to induce the differentiation of cultured cells into neural cells, and then the differentiated cells were identified by immunocytochemical methods; (4) Focal cerebral ischemia model was prepared by thread embolization in rats. Rats with cerebral ischemia were randomly divided into phosphate buffer (PBS) group, hBMSCs group and hBMSCs-derived neuron-like cells group. PBS, BrdU-labeled hBMSCs and BrdU-labeled neuron-like cells were implanted into the striatum by stereotactic implantation. The nerve injury severity score (NSS) and balance beam were evaluated before operation, 1, 3, 6 and 8 weeks after operation. The changes of neurological function were observed by BBT, EBST, Step-down Passive Avoidance and Water Maze Test, and the rats were sacrificed 8 weeks later for brain harvesting, HE staining, BrdU immunohistochemical staining and BrdU/NSE, BrdU/GFAP immunohistochemical double staining. The survival, migration and differentiation of SCs.
Results: (1) hBMSCs could proliferate in vitro, and need 2-3 weeks for primary culture. After passage, a large number of adherent cells could be obtained. The cells were homogeneous fibroblast-like, and the surface antigens of MSCs were detected by flow cytometry. CD29, CD44 and CD105 were uniformly expressed, but CD34, CD45, CD19, HLA-DR and CD106 were not expressed; (2) and T lymphocyte bodies were detected by flow cytometry. In vitro co-culture, there was no obvious T cell proliferation response, suggesting that it has low immunogenicity; (3) neuron specific enolase (NSE), astrocyte marker antigen glial fibrillary acidic protein (GFAP) could be induced to express; (4) thread embolism was used. TTC and HE staining showed that the ischemic area was located in the right striatum, and the frontal-parietal cortex, and its location and range were stable. Neurological and behavioral tests showed that NSS, BBT, EBST were transplanted with hBMSCs in rats at 1, 3, 6 after operation. Compared with the PBS control group, the NSS, BBT and EBST scores were significantly improved at 8 weeks (P 0.01) and 1, 3 weeks after operation (P 0.05) and 6, 8 weeks after operation (P 0.05), but the NSS, BBT and EBST scores were not significantly different between the two groups (P 0.05). There was no significant difference in NSS, BBT and EBST scores between the two groups at 1 and 3 weeks after operation (P 0.05), but the effect of hBMSCs and hBMSCs-derived neuron-like cells was better than that of the control group in one-off passive avoidance platform test and water maze test (P 0.01); the effect of hBMSCs-derived neuron-like cells was better than that of hBMSCs-derived neuron-like cells (P 0.05). After 8 weeks of transplantation, a large number of BrdU-positive cells were found in both hBMSCs and hBMSCs-derived neuron-like cells. The cells were concentrated in the transplantation site and surrounding areas, and migrated to ischemic focus. There was no glial proliferation and lymphocyte infiltration in the region. In the hBMSCs group, 11.5-19.3% of Brdu-positive cells were GFAP-positive cells, and 1.5-4.8% were NSE-positive cells. Among the Brdu-positive cells in sex cells and hBMSCs-derived neuron-like cells, 23.5-39.1% were NSE-positive cells and 9.8-17.6% were GFAP-positive cells.
CONCLUSION: HBMSCs are mononuclear cells in bone marrow, which have the characteristics of low immunogenicity, good plasticity and strong amplification ability. Density gradient centrifugation combined with adherent culture can effectively isolate and purify hBMSCs. All-trans retinoic acid (ATRA) and tert-butylcresol (BHA) can induce the differentiation of cultured cells into neural cells and induce the expression of neurons. Neuron-like cells derived from hBMSCs, labeled with neuron-specific enolase (NSE), and neuron-like cells derived from hBMSCs and hBMSCs can effectively improve the neurological symptoms of rats with focal cerebral ischemia. The transplantation of hBMSCs has a faster effect, and the transplantation of neuron-like cells derived from hBMSCs can improve the learning and memory functions. Most of the neuron-like cells derived from hBMSCs expressed NSE positive in vivo, which was superior to that of hBMSCs. There was no local tumor formation and obvious inflammatory reaction. Intracerebral transplantation of hBMSCs-derived neuron-like cells was a possible treatment for focal cerebral ischemia.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R329
本文编号：2238362
[Abstract]:Objective:Cerebrovascular accident is one of the three leading causes of death worldwide and the first cause of disability in adults.The annual incidence of cerebrovascular accident in China is about 130-3 million,the death rate is 600-1 million,75% of the survivors have different degrees of disability.Although great progress has been made in the first aid and early rehabilitation of cerebrovascular accident,it is serious in the late stage of cerebrovascular accident. Compared with embryonic stem cells and neural stem cells, human bone marrow mesenchymal stem cells (BMSCs) have the following advantages: (1) easy extraction, easy isolation and acquisition, and can be extracted from their own bone marrow; In vitro amplification is rapid and has the potential of multidirectional differentiation; (3) early cells from the mesoderm, avoiding tissue matching and immune rejection; (4) avoiding ethical controversy and so on. In order to reduce the ratio of neuron-like cells, bone marrow stromal cells can be differentiated into neuron-like cells in vitro, and then transplanted with cells. This study aims to explore the methods of isolation, culture, purification and identification of hBMSCs in vitro, analyze their biological characteristics, and induce differentiation into neuron-like cells, and establish a new method. Neuron-like cells derived from hBMSCs were implanted into the brain of rats with focal cerebral ischemia in vivo. The survival, migration, differentiation and recovery of neurological dysfunction were observed after transplantation. The effects of transplantation of hBMSCs-derived neuron-like cells on the repair of neurological deficits after focal cerebral ischemia were compared with that of transplantation of hBMSCs. The mechanism provides a theoretical basis for the treatment of cerebral ischemia by hBMSCs derived neuron like cell transplantation.
Methods: (1) hBMSCs were isolated and purified by density gradient centrifugation combined with adherent culture, and the growth characteristics of hBMSCs were observed in vitro, subcultured and amplified, the growth curve, morphological observation, immunocytochemistry and image analysis were used to determine the expression of cell surface antigen; (2) hBMSCs were co-cultured with T lymphocytes, and were analyzed by 3H-TdR incorporation and beta liquid scintillation counting. Its immunogenicity; (3) All-trans-retinoic acid (ATRA), butylated hydroxyanisole (BHA) were used to induce the differentiation of cultured cells into neural cells, and then the differentiated cells were identified by immunocytochemical methods; (4) Focal cerebral ischemia model was prepared by thread embolization in rats. Rats with cerebral ischemia were randomly divided into phosphate buffer (PBS) group, hBMSCs group and hBMSCs-derived neuron-like cells group. PBS, BrdU-labeled hBMSCs and BrdU-labeled neuron-like cells were implanted into the striatum by stereotactic implantation. The nerve injury severity score (NSS) and balance beam were evaluated before operation, 1, 3, 6 and 8 weeks after operation. The changes of neurological function were observed by BBT, EBST, Step-down Passive Avoidance and Water Maze Test, and the rats were sacrificed 8 weeks later for brain harvesting, HE staining, BrdU immunohistochemical staining and BrdU/NSE, BrdU/GFAP immunohistochemical double staining. The survival, migration and differentiation of SCs.
Results: (1) hBMSCs could proliferate in vitro, and need 2-3 weeks for primary culture. After passage, a large number of adherent cells could be obtained. The cells were homogeneous fibroblast-like, and the surface antigens of MSCs were detected by flow cytometry. CD29, CD44 and CD105 were uniformly expressed, but CD34, CD45, CD19, HLA-DR and CD106 were not expressed; (2) and T lymphocyte bodies were detected by flow cytometry. In vitro co-culture, there was no obvious T cell proliferation response, suggesting that it has low immunogenicity; (3) neuron specific enolase (NSE), astrocyte marker antigen glial fibrillary acidic protein (GFAP) could be induced to express; (4) thread embolism was used. TTC and HE staining showed that the ischemic area was located in the right striatum, and the frontal-parietal cortex, and its location and range were stable. Neurological and behavioral tests showed that NSS, BBT, EBST were transplanted with hBMSCs in rats at 1, 3, 6 after operation. Compared with the PBS control group, the NSS, BBT and EBST scores were significantly improved at 8 weeks (P 0.01) and 1, 3 weeks after operation (P 0.05) and 6, 8 weeks after operation (P 0.05), but the NSS, BBT and EBST scores were not significantly different between the two groups (P 0.05). There was no significant difference in NSS, BBT and EBST scores between the two groups at 1 and 3 weeks after operation (P 0.05), but the effect of hBMSCs and hBMSCs-derived neuron-like cells was better than that of the control group in one-off passive avoidance platform test and water maze test (P 0.01); the effect of hBMSCs-derived neuron-like cells was better than that of hBMSCs-derived neuron-like cells (P 0.05). After 8 weeks of transplantation, a large number of BrdU-positive cells were found in both hBMSCs and hBMSCs-derived neuron-like cells. The cells were concentrated in the transplantation site and surrounding areas, and migrated to ischemic focus. There was no glial proliferation and lymphocyte infiltration in the region. In the hBMSCs group, 11.5-19.3% of Brdu-positive cells were GFAP-positive cells, and 1.5-4.8% were NSE-positive cells. Among the Brdu-positive cells in sex cells and hBMSCs-derived neuron-like cells, 23.5-39.1% were NSE-positive cells and 9.8-17.6% were GFAP-positive cells.
CONCLUSION: HBMSCs are mononuclear cells in bone marrow, which have the characteristics of low immunogenicity, good plasticity and strong amplification ability. Density gradient centrifugation combined with adherent culture can effectively isolate and purify hBMSCs. All-trans retinoic acid (ATRA) and tert-butylcresol (BHA) can induce the differentiation of cultured cells into neural cells and induce the expression of neurons. Neuron-like cells derived from hBMSCs, labeled with neuron-specific enolase (NSE), and neuron-like cells derived from hBMSCs and hBMSCs can effectively improve the neurological symptoms of rats with focal cerebral ischemia. The transplantation of hBMSCs has a faster effect, and the transplantation of neuron-like cells derived from hBMSCs can improve the learning and memory functions. Most of the neuron-like cells derived from hBMSCs expressed NSE positive in vivo, which was superior to that of hBMSCs. There was no local tumor formation and obvious inflammatory reaction. Intracerebral transplantation of hBMSCs-derived neuron-like cells was a possible treatment for focal cerebral ischemia.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R329
【参考文献】
相关期刊论文 前3条
1 许予明,宋波,秦洁,黄悦,张化彪,张苏明;骨髓间充质干细胞对大鼠脑缺血再灌注损伤的保护机制[J];国外医学(脑血管疾病分册);2004年09期
2 宋书欣,邓志锋,汪泱,赖贤良,李明;骨髓间充质干细胞移植治疗脑缺血大鼠的实验研究[J];中国微侵袭神经外科杂志;2005年02期
3 杨华静,徐光锦,褚倩,张苏明;对比研究经颈动脉和立体定位注射骨髓间充质干细胞治疗缺血性脑卒中[J];卒中与神经疾病;2005年02期
，本文编号：2238362
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2238362.html
最近更新
教材专著
