骨髓间充质干细胞定向分化心脏瓣膜构成细胞的实验研究

发布时间：2018-02-01 10:24

本文关键词： 骨髓间充质干细胞定向分化内皮细胞骨髓间充质干细胞定向分化平滑肌细胞骨髓间充质干细胞定向分化成纤维细胞　出处：《重庆医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 第一部分骨髓间充质干细胞定向分化内皮细胞的实验研究目的:建立可以在体外稳定地获得大量高纯度内皮细胞的简便方法,并对获得细胞进行形态观察及表面标志鉴定。材料和方法:1.取4周龄SD大鼠,雌雄不拘,无菌条件下提取原代骨髓间充质干细胞,体外通过全髓贴壁法培养、纯化、扩增,将获得的P3-BMSCs采用流式细胞术进行细胞表面抗原鉴定,应用的抗体为CD29和CD34。2.取P3-BMSCs分为2组:实验组和对照组;实验组细胞在L-DMEM(20%FBS+10ng/ml VEGF)条件下培养14d;对照组细胞以未加特殊诱导剂的L-DMEM+10%FBS的培养液培养14d;每日于倒置显微镜下观察细胞生长情况,培养到7d和14d时取实验组和对照组细胞爬片,用Anti-FactorⅧ进行免疫细胞化学鉴定。结果:1.通过全髓贴壁法可获得均质性较好的细胞,获得的细胞培养到第3代时经流式细胞术鉴定发现,细胞表达CD29的阳性率超过96%,而不表达CD34;BMSCs为均一的长梭形,漩涡状、团簇状生长,在体外增殖能力强。2.实验组P3-BMSCs经L-DMEM(20%FBS+10ng/ml VEGF)诱导培养后,细胞逐渐变的粗大, 10d左右倒置显微镜下出现明显的“鹅卵石”样形态,而对照组仍为均一的长梭形,漩涡状、团簇状生长,细胞形态无明显变化;用免疫细胞化学技术鉴定发现,诱导7d和14d的细胞表达Anti-FactorⅧ的阳性率分别为82%和93%,而对照组细胞阴性表达Anti-FactorⅧ。结论:通过全髓贴壁法可获得数量足够的、均质的骨髓间充质干细胞;BMSCs在L-DMEM(20%FBS+10ng/ml VEGF)诱导条件下可以定向分化为内皮细胞,通过此法可在体外获得大量高纯度的内皮细胞。第二部分骨髓间充质干细胞定向分化平滑肌细胞的实验研究目的:建立可以在体外稳定地获得大量高纯度平滑肌细胞的简便方法,并对获得细胞进行形态观察及表面标志鉴定。材料和方法:实验组细胞P3-BMSCs在L-DMEM(20%FBS+2ng/ml PDGF-BB)条件下诱导培养14d;对照组P3-BMSCs以未加特殊诱导剂的L-DMEM+10%FBS培养液培养14d;每日于倒置显微镜下观察细胞生长情况,培养7d和14d时取实验组和对照组细胞爬片,分别用Anti-Actin alpha、Anti-Vimentin进行免疫细胞化学鉴定。结果: P3-BMSCs经L-DMEM(20%FBS+2ng/ml PDGF-BB)诱导培养后,细胞体积逐渐变大,呈不规则梭形,密集与稀疏相交错,形成“峰-谷”状形态,而对照组仍为均一的长梭形,漩涡状、团簇状生长,细胞形态无明显变化;分别应用Anti-Actin alpha、Anti-Vimentin进行免疫细胞化学检测,结果显示:7d和14d时实验组细胞表达Anti-Actin alpha的阳性率分别为78%和87%,而Anti-Vimentin表达为阴性;对照组细胞对Anti-Actin alpha和Anti-Vimentin均呈阴性表达。结论: BMSCs在L-DMEM(20%FBS+2ng/ml PDGF-BB)诱导条件下可以定向分化为平滑肌细胞,通过此法可在体外获得大量高纯度的平滑肌细胞。第三部分骨髓间充质干细胞定向分化成纤维细胞的实验研究目的:建立可以在体外稳定地获得大量高纯度成纤维细胞的简便方法,并对获得细胞进行形态观察及表面标志鉴定。材料和方法:实验组细胞P3-BMSCs在L-DMEM(20%FBS+20ng/ml b-FGF)条件下进行诱导培养14d,对照组P3-BMSCs以未加特殊诱导剂的L-DMEM+10%FBS的培养液培养14d;每日于倒置显微镜下观察细胞生长情况,培养7d和14d时取实验组和对照组细胞爬片,分别用Anti-Actin alpha、Anti-Vimentin进行免疫细胞化学鉴定。结果: P3-BMSCs经L-DMEM(20%FBS+20ng/ml b-FGF)诱导培养后,细胞逐渐变得细长,纺锤形,呈束状、网状排列生长,而对照组仍为均一的长梭形,漩涡状、团簇状生长,细胞形态无明显变化;分别应用Anti-Actin alpha、Anti-Vimentin进行免疫细胞化学鉴定,结果显示:实验组细胞诱导培养7d和14d时,细胞表达Anti-Actin alpha的阳性率分别为72%和82%,表达Anti-Vimentin的阳性率分别为75%和85%;而对照组细胞对Anti-Vimentin和对Anti-Actin alpha均呈阴性表达。结论: BMSCs在L-DMEM(20%FBS+20ng/ml b-FGF)诱导条件下可以定向分化为成纤维细胞,通过此法可在体外获得大量高纯度的成纤维细胞。
[Abstract]:Experimental study on directional differentiation of endothelial cells by bone marrow mesenchymal stem cells
Objective: to establish a simple and convenient method for obtaining a large number of highly purified endothelial cells in vitro, and to observe the morphology of the obtained cells and identify the surface markers.
Materials and methods: 1. 4 week old SD rats of both sexes under sterile conditions to extract primary bone marrow mesenchymal stem cells in vitro by whole marrow adherent culture, purification, amplification, the P3-BMSCs obtained by cell surface antigens by flow cytometry, using antibodies to CD29 and CD34.2. P3-BMSCs were divided into 2 groups: experimental group and control group; experimental group of cells in the L-DMEM (20%FBS+10ng/ml VEGF) under the condition of 14d culture medium group; control cells without special inducer in the culture of L-DMEM+10%FBS 14d; daily on cell growth was observed under inverted microscope, the experimental group and control group cell culturing to 7d and 14d, identified by immunocytochemistry with Anti-Factor VIII.
Results: 1. through the whole marrow adherence method can obtain better homogeneity of cells, the cells were cultured to the third generation by flow cytometry showed that the cells, the positive expression rate of CD29 is more than 96%, while the expression of CD34; BMSCs long spindle shape, uniform swirling, cluster growth by L-DMEM in the in vitro proliferation of.2. experimental group P3-BMSCs (20%FBS+10ng/ml VEGF) after induction, the cells gradually become coarse and the apparent "cobblestone" form about 10d under the microscope, while the control group is still long spindle shape, uniform swirling, cluster growth, cell morphology did not change significantly; found immunocytochemistry identification, induction of 7D and 14d positive expression rate of Anti-Factor VIII respectively 82% and 93%, while the control group were negative expression of Anti-Factor VIII.
Conclusion: sufficient quantity of homogeneous bone marrow mesenchymal stem cells can be obtained by full pulp adherence method. BMSCs can be differentiated into endothelial cells under L-DMEM (20%FBS+10ng/ml VEGF) induction condition, and a large number of high-purity endothelial cells can be obtained in vitro.
Experimental study on directional differentiation of smooth muscle cells by bone marrow mesenchymal stem cells in the second part
Objective: to establish a simple and convenient method for obtaining a large number of high purity smooth muscle cells in vitro, and to observe the morphology of the obtained cells and identify the surface markers.
Materials and methods: the experimental group of P3-BMSCs cells in L-DMEM (20%FBS+2ng/ml PDGF-BB) cultured under the condition of 14d; control group P3-BMSCs without special inducer L-DMEM+10%FBS medium 14d; daily on cell growth was observed under inverted microscope, cultured 7d and 14d from the experimental group and the control group were treated with Anti-Actin cell climbing slices alpha, identified by immunocytochemistry Anti-Vimentin.
Results: P3-BMSCs by L-DMEM (20%FBS+2ng/ml PDGF-BB) after induction, cell volume became larger, irregular fusiform, dense and sparse staggered, the formation of "peak valley" shape, while the control group is still long spindle shape, uniform swirling, cluster growth, cell morphology did not change significantly the application of Anti-Actin alpha Anti-Vimentin; respectively, were detected by immunocytochemistry. The results showed that: 7d and 14d positive cells in the experimental group the expression of Anti-Actin alpha were 78% and 87%, while the expression of Anti-Vimentin was negative; the control group of Anti-Actin alpha and Anti-Vimentin cells were negative.
Conclusion: BMSCs can be differentiated into smooth muscle cells under the induction of L-DMEM (20%FBS+2ng/ml PDGF-BB). By this method, a lot of high purity smooth muscle cells can be obtained in vitro.
Experimental study on directional differentiation of bone marrow mesenchymal stem cells into fibroblasts in the third part
Objective: to establish a simple and convenient method for obtaining a large number of highly purified fibroblasts in vitro, and to observe the morphology of the obtained cells and identify the surface markers.
Materials and methods: the experimental group of P3-BMSCs cells in L-DMEM (20%FBS+20ng/ml b-FGF) were induced and cultured under the condition of 14d, 14d P3-BMSCs in cultured control group without special inducer of L-DMEM+10%FBS; daily on cell growth was observed under inverted microscope, cultured 7d and 14d from the experimental group and the control group cell climbing films, respectively with Anti-Actin alpha, identified by immunocytochemistry Anti-Vimentin.
Results: P3-BMSCs by L-DMEM (20%FBS+20ng/ml b-FGF) after induction, cells became elongated, spindle, a bundle, mesh arrangement growth, while the control group is still long spindle shape, uniform swirling, cluster growth, cell morphology did not change significantly; respectively using Anti-Actin alpha, identified by immunocytochemistry. The results showed that: the experimental group of Anti-Vimentin cells induced by 7d and 14d when cultured cells, the positive expression rate of Anti-Actin alpha were 72% and 82%, the expression of Anti-Vimentin positive rates were 75% and 85%; while the control group of Anti-Actin cells on the expression of Anti-Vimentin and alpha were negative.
Conclusion: BMSCs can be differentiated into fibroblasts under L-DMEM (20%FBS+20ng/ml b-FGF) induction. By this method, a large number of high purity fibroblasts can be obtained in vitro.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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