【作者】 黄振华；

【导师】 莫雪安；

【作者基本信息】 广西医科大学 ， 神经病学， 2009， 硕士

【摘要】 第一部分大鼠骨髓间充质干细胞的分离、培养和鉴定【目的】体外分离培养大鼠骨髓间充质干细胞(Mesenchymal stem cells,MSCs),观察其生物学特性并对其进行鉴定和标记。【方法】采用全骨髓贴壁法在体外分离培养大鼠MSCs,相差显微镜观察MSCs细胞形态,透射电镜观察MSCs超微结构并绘制生长曲线,分别使用成骨细胞诱导液和脂肪细胞诱导液诱导其向成骨细胞和脂肪细胞两个方向分化。用BrdU对MSCs进行体外标记,通过免疫组化染色观察并计算BrdU阳性细胞标记率。【结果】分离培养的MSCs为贴壁生长的成纤维样细胞,传代后的细胞均一性好。超微结构显示MSCs核质比大,胞浆可见较丰富的粗面内质网、线粒体等,细胞表面有大量微绒毛。第3代MSCs生长曲线呈S形,经历潜伏期、对数增殖期和停滞期。经诱导的MSCs能分化为成骨细胞和脂肪细胞,茜素红S和油红O染色阳性。10μmol/L BrdU体外孵育48h后标记率为90%。【结论】体外MSCs易于分离、培养和扩增。体外培养的MSCs有独特的超微结构特点。MSCs具有多向分化潜能,可通过诱导向成骨细胞和脂肪细胞分化。用BrdU标记MSCs的浓度为10μmol/L,时间为48h。第二部分大鼠骨髓间充质干细胞对血管性痴呆大鼠学习记忆功能及对BDNF表达的影响【目的】建立2-VO血管性痴呆大鼠模型。探讨MSCs在VD大鼠脑组织中的存活情况,MSCs对VD大鼠空间学习记忆功能、病理改变及脑组织内BDNF表达的影响。【方法】①将11～13月龄的SD大鼠随机分成3组:假手术组(n＝10),暴露双侧颈总动脉但不结扎;模型对照组(n＝15),结扎双侧颈总动脉(2-VO),30d后脑立体定位将10μl PBS注入大鼠海马内;MSCs治疗组(n＝15),2-VO 30d后脑立体定位将1×10~6 MSCs移植入大鼠海马内。②饲养4w后以Morris水迷宫检测各组大鼠空间学习记忆能力。③HE染色后光镜下观察各组大鼠海马区和额叶的病理变化。④免疫组织化学染色观察BrdU标记的MSCs在VD大鼠脑组织中的存活情况及BDNF在各组大鼠脑组织中的表达情况并进行分析。【结果】①造模前各组大鼠Morris水迷宫检测成绩差别无统计学意义,海马定位移植4w后,模型对照组与假手术组比较,平均逃避潜伏期长(P＜0.01),且60s内在原平台象限滞留时间短(P＜0.01)。而MSCs治疗组测试成绩优于模型对照组。②光镜下可见模型对照组细胞核固缩,胞质密度增高,细胞脱失。而MSCs治疗组上述病理改变减轻。③MSCs移植4w后可在海马及额叶观察到BrdU标记的MSCs。④海马定位移植4w后,模型对照组海马及额叶的BDNF表达较假手术组少(P＜0.05),而MSCs治疗组BDNF表达较模型对照组高(P＜0.05)。【结论】①MSCs移植4W后,VD大鼠空间学习记忆能力得到改善。②BrdU免疫组化染色证实MSCs能够在移植鼠脑内存活并迁移。③MSCs可以增加VD大鼠脑内BDNF的表达,提示MSCs可能通过增加BDNF的表达改善VD大鼠空间学习记忆能力。更多还原

【Abstract】 Part 1 Isolation,cultivation and identification of rat bone marrow-derived Mesenchymai Stem Cells(MSCs)[Objective]To study the isolation,purification of rat MSCs in vitro. Detecte the biological features of MSCs.Identify and label the MSCs in the study.[Methods]We selected suitable culture medium and FBS concentration, separated and purified the MSCs with adherent method.The morphology of MSCs was observed with invert-microscopy constantly.The ultrastructure of MSCs was observed by transmission electron microscope.MSCs’curve of growth was depicted.MSCs were induced to differentiate into osteoblasts and adipocytes in suitable induction medium.MSCs were marked in vitro by BrdU. Immunohistochemical stain and light microscope were used for identification BrdU marker ratio.[Results]MSCs could be isolated and proliferated by adherent method in vitro.The appearence of MSCs were fibroblast-like cells and its growth curve was like shape of S.Under the electron microscope MSCs had plentiful rough endoplasmic reticula.The ratio of the nucleus to cytoplasm was high,and the surface of MSCs had a lot of microvilli.MSCs could be differentiated into osteoblasts and adipocytes in vitro.The incubation with 10μmol/L BrdU for 48h achieved over 90%of the labeling rate.[Conclutions]MSCs can be isolated,pured and expanded easily by adherent method.MSCs have the characters of earlier immature cells.MSCs have mutipotency which can be induced to differentiate into osteoblasts and adipocytes.The optimal BrdU labeling concentration and time period are 10μmol/L and 48h.Part 2 Transplantation of MSCs improved cognition of VD rats and enhanced the expression of BDNF[Objective]To construct the model of VD rats.To observe of MSCs improve cognition of VD rats.To observe the pathology and the expression of BDNF in rats’ brain tissues after transplantation of MSCs.[Methods]①The rats were randomly divided into a sham group(n＝10);a model group(n＝15),10μ1 PBS was injected into rats’hippocampus at 30 days after 2-VO;a treatment group(n＝15),1×10~6 MSCs was transplanted into rats’ hippocampus at 30 days after 2-VO.②The performance of the rats in Morris water maze were tested at 4 weeks after transplantation.③Hematoxylin eosin staining was used to observe the pathology of rats’ brain tissues.④Immunohistochemical staining was used to identify MSCs in VD rats’brains and analysesed the expression of BDNF in brain tissues of rats in all groups.[Results]①There was no marked difference in the sham group,model group and treatment group before 2-VO.4w after transplantation,compared with the sham group,the average escape latency of model group was longer(P＜0.01),and the lingering time in platform area was shorter(P＜0.01).But the performance of treatment group had obvious improvement compared with the model group.②Light microscope assessment showed that the brain tissue in the model group had the pathology changes such as pyknosis and condensation of the cytoplasm.These damage was lighten in the treatment group compared to the model group.③BrdU positive cells were found in the brain tissue of rats in the treatment group by immunohistochemical assessment.④4w after transplantation,the number of BDNF positive cells in model group was less than sham group(P＜0.05).But BDNF increased in the treatment group(P＜0.05)[Conclutions]①MSCs which was transplanted into VD rats’ hippocampus can live and migrate in VD rats’cerebral,and improve cognition of VD rats at 4w after translpantation.②The transplanted MSCs could enhance the expression of the BDNF.It suggests that the improvement of cognition of VD rats is probably due to the increased BDNF.更多还原