大鼠骨髓源神经干细胞向血管内皮细胞样细胞分化的实验研究

发布时间：2018-11-29 12:49

【摘要】：目的:观察SD大鼠骨髓源神经干细胞(bone marrow mesenchymal stem cells-derived neural stem cells,BMSCs-NSCs)在血管内皮细胞(vascular endothelial cells,VEC)培养条件下是否分化为内皮样细胞(endothelial-like cells,ELCs)。方法:本实验采用全骨髓培养法分离培养大鼠双侧股骨、胫骨以及肱骨的骨髓,利用骨髓源基质干细胞(bone marrow mesenchymal stem cells,BMSCs)的贴壁生长特性和多次传代培养使其纯化。研究BMSCs的外形改变并绘制P3(Passage 3)代细胞的生长曲线,探讨BMSCs的最适接种密度,利用流式细胞仪检测BMSCs标志物CD34、CD44、CD45、CD90的表达情况。将所得P3代BMSCs消化形成单细胞悬液,调整细胞密度后置于神经干细胞(neural stem cells,NSCs)诱导培养基(20ng/mL bFGF、20ng/mL EGF、B27)中进行悬浮培养,通过免疫细胞化学染色观察神经球表面标志物CD133、Nestin的表达情况。培养所得神经球消化形成单细胞,并进行免疫细胞化学染色检测VEC表面标志物CD31、vWF的表达情况。将所得NSCs悬液置于Matrigel胶质培养基中培养,观察是否形成血管腔样结构。结果:1.BMSCs早期呈簇状生长,细胞呈梭形。P3 BMSCs流式细胞仪检测显示CD44阳性细胞比率为91.4%,CD90阳性细胞比率为93.7%;CD34、CD45的表达均呈阴性反应。2.P3代细胞生长曲线呈“S”型,其接种最佳密度区间在5×104~1×105/mL。3.BMSCs培养1-2天时细胞聚集呈团块状,随后呈“桑葚状”球形结构,免疫细胞化学染色显示CD133、Nestin的表达均呈阳性反应。4.骨髓源神经球在VEC培养基中培养7天时,细胞呈扁平状,三角形或多角形,外观似“铺路石”状。在Matrigel胶质培养基中可形成管腔样结构。结论:1.体外利用无血清悬浮培养法,可将大鼠BMSCs诱导分化为NSCs。2.BMSCs-NSCs体外诱导可定向分化为ELCs,并且在Matrigel胶中可形成管腔样结构。
[Abstract]:Aim: to investigate the differentiation of SD rat bone marrow derived neural stem cells (bone marrow mesenchymal stem cells-derived neural stem cells,BMSCs-NSCs) into endothelioid cells (endothelial-like cells,ELCs) in cultured vascular endothelial cells (vascular endothelial cells,VEC). Methods: the bone marrow of bilateral femur, tibia and humerus were isolated and cultured by whole bone marrow culture method. The adherent growth characteristics of bone marrow derived stromal cells (bone marrow mesenchymal stem cells,BMSCs) and the purification of the bone marrow derived stem cells (bone marrow mesenchymal stem cells,BMSCs) were obtained. The shape change of BMSCs was studied and the growth curve of P3 (Passage 3) generation cells was plotted. The optimal inoculation density of BMSCs was discussed. The expression of BMSCs marker CD34,CD44,CD45,CD90 was detected by flow cytometry. The P3 passage BMSCs was digested to form a single cell suspension, and the cell density was adjusted and then cultured in a neural stem cell (neural stem cells,NSCs (20ng/mL bFGF,20ng/mL EGF,B27) induction medium. The expression of CD133,Nestin was observed by immunocytochemical staining. The cultured neurospheres were digested to form single cells and the expression of CD31,vWF on the surface of VEC was detected by immunocytochemical staining. The obtained NSCs suspension was cultured in Matrigel glial medium to observe whether the vascular lumen structure was formed. Results: in the early stage of 1.BMSCs, the cells grew in clusters and the cells were fusiform. P3 BMSCs flow cytometry showed that the ratio of CD44 positive cells was 91.4 and that of CD90 positive cells was 93.7. The expression of CD34,CD45 was negative. The growth curve of 2.P3 cells was "S" type, and the best density of 2.P3 was "mulberry" globular structure after 1-2 days of incubation with 5 脳 10 ~ 4 ~ 1 脳 105/mL.3.BMSCs. Immunocytochemical staining showed that the expression of CD133,Nestin was positive. 4. 4. When cultured in VEC medium for 7 days, the cells were flattened, triangular or polygonal, and looked like paving stones. The lumen like structure can be formed in Matrigel colloid medium. Conclusion: 1. Rat BMSCs could be induced to differentiate into NSCs.2.BMSCs-NSCs by serum-free suspension culture in vitro, and could be induced to differentiate into ELCs, in vitro. The tubular structure could be formed in Matrigel gel.
【学位授予单位】：青海大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R741

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