【作者】 陆华；

【导师】 惠国桢；

【作者基本信息】 苏州大学 ， 神经外科， 2007， 博士

【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。更多还原

【Abstract】 Objective:The cerebral vascular accident is one of three severe causes of death in the world and is also the first pathogeny of the adult disability.The year incidence of the cerebral vascular accident in our country is approximately 130-300 tenthousand, and 60-100 tenthousand are dead among them, and seventy-five percent survivors had some disability. Although great progress about the first aid and early healing to the cerebral vascular accident had been obtained, there were short of effective treatment for the later severely neurological deficiencies of accident. 21st century is the century of regeneration. The study of stem-cell is the focus of life medicine. Compare with the embryo-stem cell and the neuro-stem cell, BMSCs have many merits, including:①get and separate the material conveniently and easily, we can withdraw it from our own bone marrow.②Expands and increases in vitro rapidly and has multi-potential.③BMSCs are early cells from mesoblast, which can avoid tissue type and immuno-exclusion.④can avoid the ethical dispute and so on. More and more scholars had paid attention to the BMSCs. Because the transplanted BMSCs have more probability to translate to the neuro-glial cell than neuron-like cell in internal-entironment, so if we can translate the BMSCs to neuron-like cell in vitro, we can have twice the result with half the effort to transplant the neuron-like cells.By establishing the cell-culturing system in vitro of hBMSCs and neuron-like cell from hBMSCs early and observing the biological properties, hBMSCs and the neuron-like cells differentiated from hBMSCs are considered to be good resources of cell transplant and the cell treatment. The objective of this study is to explore a new method for the isolation, purification and amplification in vitro of hBMSCs from human bone marrow. We observe their biological character and directional differentiate to neuron- type cell, and observed the survival, migration, differentiation and the recovery of neurological deficits after transplantation of hBMSCs neuron- type cell into ischemic rats by stereotactic operation. Then we explored the effects of hBMSCs neuron- type cell transplantation on the repair of neurological functions and possible mechanism, so that theoretical assistance for clinical therapy of cerebral ischemia with hBMSCs neuron- type cell transplantation could be provided.Method:(1) BMSCs were isolated and purified from the bone marrow of human by density gradient centrifugation and by adhering to the culture plastic. After successive subculture and amplification, the growth curve was drawn, the morphology and growth characteristics were observed under phase contrast microscope , and the cell surface antigens were examined by immunocytochemistry and image analysis in vitro; (2) BMSCs were co-cultured with T lymphocytes with 3H-TdR were mixed to analysis its immunogenicity.(3) BMSCs were induced towards neuro- cells with all-trans-retinoic acid (ATRA) and butylated hydroxyanisole(BHA), then identified with immunocytochemistry. (4) Focal cerebral ischemia was induced by transient MCA with a monofilament suture in adult Wistar rats. The cerebral ischemia rats were divided into three groups including group PBS, group hBMSCs and group of neuron-like cells from BMSC randomly. We transplant the hBMSCs marked by PBS and BrdU and neuron-like cells from BMSC marked by BrdU into rats’striatum by stereotactic operation. We used the NSS, BBT, EBST, SDPAT and WMT to evaluate the rats at pro-operation and 1st, 3rd,6th,8th week post-operation. These rats were killed at 8th week after cerebral ischemia. In the same time, HE staining, immunohistochemical staining for BrdU, and immunohistochemical doublestaining for BrdU/NSE and BrdU/GFAP were processed to observe the survival, migration and differentiation of grafted BMSCs.Result: (1) Human bone marrow mesenchymal stem cells can generate with large quantity in vitro and primary culture require 2~3 weeks under conventional culture, the growth velocity speed up after passage. We can get a number of cells adhering to the culture plastic.Uiform fibroblast-like cells were got and the surface antigen of MSCs detected by flow cytometry show it is positive for CD44、CD29、CD105 and negative for CD45、CD106、CD34、HLA-DR. (2) When BMSCs were cocultured with T lymphocytes, the proliferation of T cells was not found. It suggested the low immunogenicity of the BMSCs. (3) BMSCs could be induced to neuron-like cells, which developed rounded cell bodies with multiple neuron-like extension as well as several neuronal proteins such as neuron specific enolased and astrocyte marker glial fibrillary acid protein. (4) Left hemiplegia and right Honner’s sign were observed after transient right MCA with a monofilament suture. TTC and HE staining showed that ischemic regions were located in the right striatum and frontoparietal cortex. The position and area were relatively stable each time. The NSS, BBT、EBST of group hBMSCs are much better than the group PBS at the 1st,3rd,6th,8th week post-operation (P<0.01), and better than the group of neuron-like cells from hBMSCs at 1st,3rd week post-operation(P<0.05), but there is no statistical significance between the group hBMSCs and group of neuron-like cells from hBMSCs(P>0.05); The NSS, BBT、EBST of group of neuron-like cells from hBMSCs are much better than the group PBS at the 3rd,6th,8th week post-operation (P<0.01), is lower than the group hBMSCs at 1st,3rd week post-operation(P<0.05), and there is no statistical significance between the two groups at the 6th ,8th week (P>0.05) .The SDPAT and WMT of the two groups of neuron-like cells and hBMSCs are much better than the control group, and the effection of group hBMSCs is worse than the group of neuron-like cells from hBMSCs (P<0.05). After the 8th week of transplantation, numerous cells labeled with BrdU were present at both of the group hBMSCs and group neuron-like cells from BMSCs, which were present in the injecting points and surrounding areas, and had the trend to migration to the ischemia areas. There were no significance glial-cell proliferation and lymphocyte infiltration. Among all the Brdu reactive cells of group hBMSCs, 11.5-19.3% were reactive for the astrocyte marker glial fibrillary acidic protein(GFAP), 1.5-4.8% expressed the neuronal markers neuron specific enolase(NSE). Among all the Brdu reactive cells of group of neuron-like cells from hBMSCs, 23.5-39.1% were reactive for the neuronal markers neuron specific enolase(NSE), 9.8-17.6% expressed the astrocyte marker glial fibrillary acidic protein(GFAP).Conclusion:hBMSCs are mono-neuclear cells in the bone marrow,and have much charactistics such as low inmunogenicity, good plasticity, strong generation et.al , and we can isolate and purify the hBMSCs by density gradient centrifugation combined with adhering to the culture plastic effectively . The cultured cells can induced by the all-trans-retinoic acid (ATRA) and butylated hydroxyanisole(BHA) to nerve cells, and can be induced to neuron-like cells from hBMSCs wich expressed the neuron-spesific protein—neuron specific enolased. The two groups of hBMSCs and neuron-like cells from hBMSCs both can improve the neurological function of the focal cerebral ischemia rats effectively, but hBMSCs have faster effection than another. The transplantion effection on improving study and memory of group of neuron-like cells from hBMSCs is better than another group. The most of neuron-like cells from hBMSCs in vivo are expressed NSE which is better than the group of hBMSCs, and there are no tumors appear and inflammation responds on local regions. The intra-cerebral transplantation of the neuron-like cells from hBMSCs would be one of measures for treatment of local cerebral ischemia.更多还原