大鼠骨髓来源的平滑肌祖细胞培养及鉴定

Culture and identification of smooth muscle progenitor cells from rat bone marrow

目的培养和鉴定从鼠骨髓中分离的平滑肌祖细胞,观察其在体外增殖、分化过程中相关细胞表型的变化。方法用密度梯度离心法获得鼠骨髓单个核细胞,用条件培养基进行诱导分化,免疫荧光双标技术(α-SMA、CD14)鉴定平滑肌祖细胞(SPC)。Western blot检测α-SMA蛋白,Real-time PCR法检测α-SMA mRNA表达。结果鼠骨髓单个核细胞诱导培养4d时开始贴壁,7d变为梭形,14d呈典型的"峰""谷"样形态;4d时开始出现α-SMA-CD14双荧光阳性细胞;培养1d时无α-SMA蛋白表达,4d后增强,10～14d达高峰,21d仍持续高水平;培养1d的细胞α-SMA mRNA表达低(P<0.01),4d后增强,14d达高峰(P<0.01),21d后仍持续高水平。结论本试验成功地从鼠骨髓单个核细胞中分离培养出SPC,并在体外扩增分化成平滑肌样细胞。

Objective To isolate and identify smooth muscle observe specific expression of smooth muscle progenitor cells progenitor cells （SPCs） from rat bone marrow and to during proliferation and differentiation in vitro. Methods MNCs were isolated by density gradient centrifugation from rat marrow and cultured in conditioned nutrient medium, identification was performed by immunofluorescent staining （α-SMA, CD14）. Smooth muscle cells specific markers （α-SMA） were checked with Western blotting and Real-time PCR at different time. Results During culturing, cells adhered and became spindle shaped with outgrowth at 4 d and 7 d, and showed typical ＂peak＂ ＂valley＂ at 14 d. Both α-SMA and CD14 were positive after 4 d. Expression of α-SMA was not found at 1 d with Western blotting, but it gradually enhanced at 4 d and reached the peak from 10 d to 14 d, then maintaned high-level at 21 d. The results with Real-time PCR indicated that no expression of α-SMA mRNA within non-induced cells was found, but after being induced it gradually enhanced at 4 d and got to peak at 14 d, then kept the high-level at 21 d, low-expression at 1 d was significantly different from the other ones （P 〈 0. 01 ）. Conclusion SPCs may be isolated and cultured from rat marrow monocytes, then proliferated, differentiated into smooth muscle-like cells.