人脐带间充质干细胞生物学特性及体外转化为神经细胞的实验研究

发布时间：2018-03-11 19:44

本文选题：人脐带　切入点：间充质干细胞　出处：《苏州大学》2007年硕士论文　论文类型：学位论文

【摘要】： 第一部分:人脐带间充质干细胞分离纯化及基本生物学特性研究目的探讨人脐带及脐静脉内皮下间充质干细胞(MSC)分离、培养方法,并对其生物学特性进行初步分析。方法无菌条件下收集足月妊娠剖宫产新生儿脐带,PBS洗去血管内的残存瘀血,用两种方法进行培养:1、组织块培养剔出脐带动静脉,组织剪将剩余组织剪碎至1mm3大小组织块。直接将组织块接种于DMEM-LG培养基进行原代培养。2、脐静脉内皮/内皮下分离培养将洗净的脐静脉灌入0.1%Ⅳ型胶原酶,37℃消化20分钟,收集消化液离心后接种培养。观察并记录其形态学变化,细胞达到融合状态时,消化传代进行纯化和扩增。流式细胞仪检测MSC的细胞表面标志。绘制细胞生长曲线,流式细胞仪测定细胞周期。结果两种分离方法均可获得成纤维样MSC。组织块贴壁法1周后于组织块间隙可见散在分布的纺锤型细胞,3周左右达80%融合。脐静脉胶原酶消化法,细胞接种后第二天即可见少量形态各异的贴壁细胞,散在分布。1周左右时,贴壁细胞形成集落,占优势的是成纤维样MSCs及少量铺路石样细胞,3周左右可达80%融合。经传代培养后,可得到较为纯化的成纤维样MSC,细胞约3天即可达到70%融合,需再次传代或冻存,细胞可传代20代以上。分别取第4代、第10代脐带MSC行流式细胞术表面标记检测,结果表明细胞表型无明显差异,高表达CD49e、CD29、CD44,低表达CD106、CD11b,不表达CD34,对第4、10代脐带MSCs生长曲线进行分析,其平均倍增时间为33.1h, 35.2h。流式细胞仪进行细胞周期检测表明,脐带MSCs有60-70%细胞处于G0-G1期。结论组织块贴壁法和脐静脉胶原酶消化法均可从脐带组织分离得到成纤维样MSCs。该细胞具有较强的增殖能力,细胞表面分子检测显示脐带来源的间充质干细胞表面标志与骨髓MSC相同。第二部分:人脐带间充质干细胞向神经细胞定向诱导分化的研究目的探讨bFGF联合N2添加剂对脐带来源MSCs向神经细胞分化的情况,进而为脐带MSC向神经移植提供理论依据。方法取第4代MSC消化后进行细胞爬片,细胞在玻片上生长3天后,即覆盖玻片的60%左右时,进行诱导分化。在诱导剂的培养液中加入30ng/ml bFGF㧏10ml/L N2添加剂。每天计数神经元样细胞分化率;每天取分化后的细胞爬片,用免疫组化的方法检测神经元样细胞特性性标志物Nestin、NSE、NF表达,并计数阳性细胞表达率,比较不同天数bFGF对神经细胞分化能力的差异。结果加入诱导液24小时后即可见部分MSCs胞体有一定的收缩,胞体成球形或不规则,胞体折光性变强,3天后大部分MSCs出现明显形态改变,有的细胞突起进一步伸出二级或三级分支,并可见轴突、树突及胞体的椎样改变,在细胞密集处突起交织成网,4天后,绝大部分细胞形态都已发生了向神经细胞的改变,分化率达87.7%,此后部分细胞脱落、死亡,多数细胞可存活10天以上。诱导分化后的第2天行免疫组化即可见Nestin表达成强阳性,约35.03%,于第4天达到高峰70.88%,NSE和NF第7天染色阳性率分别达到50.1%、47.51%。结论人脐带间充质干细胞具有向神经细胞分化的潜能,神经营养剂N2联合bFGF能提高脐带间充质干细胞分化为神经样细胞的分化率。
[Abstract]:Part 1: isolation and purification of human umbilical cord mesenchymal stem cells and their basic biological characteristics
Objective to investigate the isolation and culture of human umbilical cord and umbilical vein mesenchyme stem cells (MSC), and to analyze its biological characteristics.
Methods collected in sterile condition of full-term pregnancy cesarean section newborn umbilical cord blood PBS, residual wash vessel, by two kinds of methods of culture: 1, tissue culture from umbilical cord vein tissue, cut the remaining tissue cut to size 1mm3 tissue. Direct tissue inoculated on DMEM-LG medium for primary culture.2, human umbilical vein endothelial / endothelial isolation will wash the umbilical vein into 0.1% collagenase digestion, 37 degrees 20 minutes, medium collected after inoculation. The centrifugal digestive fluid to observe and record the change of morphology, the cells reached confluence, digestion and passage were purified and amplified. Cell surface markers by flow cytometry the detection of MSC. Cell growth curve and cell cycle were determined by flow cytometry.
The results can be obtained two kinds of separation methods of fibroblast like MSC. tissue adherent to the tissue after 1 weeks of spindle cells scattered in space distribution, about 3 weeks to 80%. Fusion of umbilical vein collagenase digestion. Cells were inoculated second days after a small amount of different forms of adherent cells scattered in.1 week, adherent cell colony formation, the dominant is fibroblast like MSCs and a small amount of cobblestone like cells, about 3 weeks up to 80% fusion. After subculture, can obtain more purified fibroblast like MSC cells, about 3 days can reach 70% confluence, again passage or cryopreservation. Cells can be passaged for more than 20 generations respectively. The fourth generation, cytometry to detect surface markers of umbilical cord MSC tenth generation line flow, results showed no significant difference of cell phenotype, expression of CD49e, CD29, CD44, low expression of CD106 and CD11b, the expression of CD34 on the 4,10 MSCs growth curve of umbilical cord The average doubling time was 33.1h, and the cell cycle detection of 35.2h. flow cytometry showed that there were 60-70% cells in the umbilical cord MSCs in G0-G1 stage.
Conclusion tissue block adherence and umbilical vein collagenase digestion can get fibroblast like MSCs. from umbilical cord tissue. The cell has strong proliferative ability. Cell surface molecular detection shows that umbilical cord derived mesenchymal stem cells surface markers are the same as bone marrow MSC.
The second part: A Study on the directed differentiation of human umbilical cord mesenchymal stem cells into neural cells
Objective to investigate the differentiation of umbilical cord derived MSCs into neural cells by bFGF combined with N2 additive, and to provide a theoretical basis for the transplantation of umbilical cord MSC to the nerve.
Methods fourth MSC after digestion of cell smear, cell growth after 3 days in the slides, which covered the 60% coverslips, differentiation of 30ng/ml bFGF in the culture medium. Adding inducer? 10ml/L N2 additive. Differentiation of neuron like cells count rate per day; every day take differentiated cell climbing slices immunohistochemical method was used to detect characteristics of neuron like cell markers Nestin, NSE, NF expression, and the expression rate of positive cells count, the difference of different days of bFGF on the differentiation of neural cells.
The addition of inducing medium after 24 hours of the visible part of MSCs cell bodies have a certain degree of contraction, cell body into spherical or irregular, cell body index becomes strong, 3 days after the most MSCs appear obvious morphological changes, some cell protrusions further extend two or three branches, and visible axons, dendrites and somata of vertebra like change, interwoven into a network in the cell dense protrusions 4 days later, most cells have been changed into neural cells, the differentiation rate reached 87.7%, then part of cell loss, death, most cells can survive for 10 days. Second days after induction of differentiation of immune group showed that Nestin expressed strong positive, about 35.03% to fourth days to reach the peak of 70.88%, NSE and NF for seventh days the positive staining rate reached 50.1%, 47.51%.
Conclusion human umbilical cord mesenchymal stem cells have the potential to differentiate into neurons. Neurotrophic agent N2 combined with bFGF can improve the differentiation rate of umbilical cord mesenchymal stem cells into neuron like cells.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329.2

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