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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。方法取体重100 g左右雄性Sprague-Dawley(SD)大鼠后肢股骨和胫骨骨髓,采用原代全骨髓培养法,多次传代纯化,体外扩增后,观察细胞形态,并用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分为实验组和对照组: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;对照组采用普通的DMEM-LG培养。观察诱导后细胞形态,检测诱导后第5代细胞平滑肌α-肌动蛋白(SM--αactin)、钙结合蛋白(calponin)、平滑肌肌球蛋白重链(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。实验组经诱导后的BMSCs生长较缓慢,略呈椭圆形,检测SM--αactin、calponin、SMMHC显著表达;对照组的细胞形态及生长速度和实验组BMSCs相似,但不表达SM--αactin、calponin和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,可为组织工程构建小口径血管提供平滑肌种子细胞。
Objective To investigate the feasibility and mechanism of bone marrow mesenchymal stem cells (BMSCs) as a small-caliber vascular seed cells. Methods The bone marrow of the hindlimb femur and tibia of male Sprague-Dawley (SD) rats weighing about 100 g was obtained. The cells were subcultured and purified repeatedly in primary bone marrow culture. After being expanded in vitro, the cell morphology was observed. Immunofluorescence and flow cytometry Instrument detection of CD34, CD90, CD105 cytokines, to identify whether the BMSCs. BMSCs were divided into experimental group and control group. The experimental group was cultured in low glucose medium (DMEM-LG) containing all-trans retinoic acid and db-cAMP. The control group was cultured with ordinary DMEM-LG to cultivate. The morphological changes of the induced cells were observed. The expression of SM - αactin, calponin and SMMHC in the 5th passage cells were detected. Results The cells obtained from primary culture showed long fusiform shape after many subcultures and showed characteristic swirling growth. The positive expression of CD34, CD90 and CD105 were detected. The induced BMSCs in the experimental group grew slowly, slightly oval, and the expressions of SM - αactin, calponin and SMMHC were significant. The cell morphology and growth rate in the control group were similar to those in the experimental group, but not in the experimental group. calponin and SMMHC. Conclusion BMSCs can differentiate into vascular smooth muscle cells under the induction of all-trans retinoic acid and provide smooth muscle seed cells for tissue engineering of small-caliber blood vessels.