大鼠骨髓间充质干细胞体外诱导分化为血管平滑肌样细胞的实验研究

发布时间：2019-04-23 20:41

【摘要】：目的探讨骨髓间充质干细胞(BMSCs)体外分离培养以及扩增的方法并鉴定细胞性质。方法取100 g左右雄性SD大鼠后肢股骨、胫骨骨髓,原代全骨髓培养法,多次传代纯化,体外扩增后,观察细胞形态,绘制细胞生长曲线,并利用免疫组化及流式细胞学检测培养细胞中CD34、CD90、CD105细胞标志性因子,鉴定原代培养获取的细胞是否为BMSCs。结果原代培养所获取的细胞形态呈长梭形,生长呈现特征性的漩涡状,生长曲线为典型的S型,免疫组化结果显示CD34阴性,CD44阳性,CD54阳性,流式细胞学检测结果显示CD34阴性,CD90、CD105阳性。结论利用全骨髓培养法可成功分离骨髓间充质干细胞,全骨髓培养较为简便、易行。经传代后细胞纯度增高,10代以内的细胞纯度高,活性好。目的探讨骨髓间充质干细胞(BMSCs)作为构建小口径血管种子细胞的可行性,及其诱导机制。方法取100g左右雄性SD大鼠后肢股骨、胫骨骨髓,原代全骨髓培养法,多次传代纯化,体外扩增后,观察细胞形态,并免疫荧光及流式细胞仪检测CD34、CD90、CD105细胞因子,鉴定是否为BMSCs。将BMSCs分为实验组及对照组:实验组使用含全反式维甲酸及二丁酰环磷酸腺苷(db-cAMP)的低糖基本培养基(DMEM-LG)培养基培养;对照组采用普通的DMEM-LG培养基培养。观察诱导后的细胞形态并利用免疫荧光技术及流式细胞仪检测诱导后第5代细胞平滑肌α-肌动蛋白(SM-α-actin)、钙结合蛋白(calponin)、肌球蛋白重链(SMMHC)的表达情况。结果原代培养所获取的细胞经过多次传代培养后呈长梭形,呈现特征性的漩涡状生长,检测CD34阴性,CD90、CD105阳性。经诱导后的BMSCs生长较缓慢,略呈椭圆形,检测SM-α-actin、calponin、SMMHC显著表达;对照组细胞的形态及生长速度和BMSCs相似,不表达SM-α-actin、calponin、SMMHC。结论BMSCs在全反式维甲酸的诱导下可向血管平滑肌样细胞表型分化,为组织工程构建小口径血管提供平滑肌种子细胞。目的探讨血管平滑肌样细胞在体外体外静态培养的可能性以及观察细胞生长状态。方法取成年雄性比格犬,处死后,取胸主动脉,长约6cm,并对所取血管行脱细胞实验,获取脱细胞基质支架,并行快速冰冻染色检查,检测脱细胞效果。将获取的脱细胞基质支架材料剪成2x2cm大小的薄片,将诱导完成的血管平滑肌样细胞种植在薄片上,隔日换液,种植15d后将支架薄片行快速冰冻检查、电子扫描电镜检查,观察细胞生长状态。结果快速冰冻染色检查可见诱导完成的血管平滑肌样细胞可种植在血管脱细胞基质支架表面生长,电子扫描电镜可见细胞复杂在支架材料上。结论诱导完成的血管平滑肌样细胞可静态种植在血管脱细胞基质材料上。
[Abstract]:Objective to investigate the methods of isolation, culture and amplification of bone marrow mesenchymal stem cells (BMSCs) in vitro and to identify the cell properties. Methods the femoral and tibial bone marrow of the male SD rats with 100g or so were cultured and purified for many times. After in vitro amplification, the morphology of the cells was observed and the growth curve of the cells was drawn. Immunohistochemistry and flow cytometry were used to detect the marker factors of CD34,CD90,CD105 cells in order to identify whether the cells obtained in primary culture were BMSCs. or not. Results the morphology of the cells was spindle-shaped and the growth curve was typical S-type. The results of immunohistochemistry showed that CD34 was negative, CD44 was positive, CD54 was positive, and CD34 was negative by flow cytometry. CD90,CD105 positive. Conclusion Bone marrow mesenchymal stem cells can be successfully isolated by whole bone marrow culture, and the whole bone marrow culture is simple and easy. After passage, the cell purity increased, the cell purity within 10 generations was high, and the cell activity was good. Objective to investigate the feasibility and induction mechanism of bone marrow mesenchymal stem cells (BMSCs) as seed cells of small diameter blood vessels. Methods the femoral and tibial bone marrow of 100 g male SD rats were cultured and purified for many times. After in vitro amplification, the morphology of the cells was observed, and the CD34,CD90,CD105 cytokines were detected by immunofluorescence and flow cytometry. Identify BMSCs. BMSCs was divided into experimental group and control group: the experimental group was cultured in low glucose basic medium (DMEM-LG) containing all-trans retinoic acid and dibutyryl cyclic adenosine monophosphate (db-cAMP), and the control group was cultured in common DMEM-LG medium. The morphology of induced cells was observed and the expression of SM- 伪-actin), calcium binding protein (calponin), myosin heavy chain (SMMHC) was detected by immunofluorescence and flow cytometry. Results the cells obtained from primary culture showed long fusiform shape and characteristic swirl-like growth after repeated subculture. CD34-negative and CD90,CD105-positive cells were detected. After induction, BMSCs grew slowly and showed a slightly oval shape, and the expression of SM- 伪-actin,calponin,SMMHC was detected. In the control group, the morphology and growth rate of the cells were similar to those of BMSCs, but no expression of SM- 伪-actin,calponin,SMMHC. was observed in the cells of the control group. Conclusion BMSCs can differentiate into vascular smooth muscle-like cells induced by all-trans retinoic acid and provide smooth muscle seed cells for tissue engineering. Objective to investigate the possibility of static culture of vascular smooth muscle-like cells (VSMCs) in vitro and to observe the state of cell growth. Methods the adult male Beagle dogs were killed and the thoracic aorta was taken for about 6 cm. The acellular matrix scaffolds were obtained and the acellular effect was detected by rapid frozen staining. The obtained acellular matrix scaffold material was cut into 2x2cm-size thin slices, and the induced vascular smooth muscle-like cells were implanted on the thin slices, then the solution was exchanged every other day. After 15 days of implantation, the scaffolds were subjected to rapid freezing examination and electron scanning electron microscopy (SEM), and the scaffolds were subjected to rapid freezing examination and electron scanning electron microscopy (SEM). The state of cell growth was observed. Results Rapid frozen staining showed that the induced vascular smooth muscle-like cells could grow on the surface of the acellular matrix scaffold, and the cells were complicated on the scaffold material by electron scanning electron microscopy (SEM). Conclusion the induced vascular smooth muscle-like cells can be statically implanted on the acellular matrix material.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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