基础医学与临床 ›› 2008, Vol. 28 ›› Issue (11): 1197-1202.

• 技术与方法 • 上一篇    下一篇

大鼠骨髓来源的平滑肌祖细胞培养及鉴定

步雪峰 严玉兰 刘洋 张志坚 王穆斌   

  1. 江苏大学附属人民医院 江苏大学附属人民医院 江苏大学附属人民医院 江苏大学医学院 江苏大学医学院
  • 收稿日期:2008-04-23 修回日期:2008-07-17 出版日期:2008-11-25 发布日期:2008-11-25
  • 通讯作者: 严玉兰

Culture and Identification of smooth muscle progenitor cells from rat marrow

Xue-feng BU, Yu-lan YAN, Yang LIU, Zhi-jiang ZHANG, Mu-bin WANG   

  1. Affiliated Renmin Hospital Affiliated Renmin Hospital Affiliated Renmin Hospital Medical College. Jiangsu University Medical College. Jiangsu University
  • Received:2008-04-23 Revised:2008-07-17 Online:2008-11-25 Published:2008-11-25
  • Contact: Yu-lan YAN,

摘要: 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4 d时开始贴壁,7 d变为梭形,14 d呈典型的"峰""谷"样形态;4 d时细胞开始出现α-SMA-CD14双荧光阳性;培养1 d 时无α-SMA蛋白表达,4 d后增强, 10 ~14 d达高峰,21 d仍持续高水平;培养1 d 的细胞α-SMA mRNA表达低(P<0.01),4 d后增强,14 d 达高峰(P<0.01),21 d 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。

Abstract: AIM To isolate and identify smooth muscle progenitor cells ( SPCs) from rat bone marrow and observe specific expression profile of the smooth muscle progenitor cells during proliferation and differentiation in vitro. Methods MNCs were isolated by ficoll density gradient centrifugation from rat marrow and cultured in conditioned nutrient chemical, identification was performed by immunofluorescent staining (α-SMA,CD14).And smooth muscle cells specific markers (α-SMA) were determined 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 presented typical "peak" "valley" at 14 d (3 generation). Both α-SMA and CD14 were positive after 4 d. Expression for α-SMA was not found at 1 d with Western blotting,but it gradually enhanced at 4 d and reached to the top from 10d to 14 d ,and still remained high-level at 21 d .The results with Real-time PCR indicated that the expression of α-SMA mRNA within non-induced cells was not found ,but after being induced it gradually enhanced at 4 d and got to peak at 14 d, and still remained high-level at 21 d , low-expression at 1 d was significantly different from the other ones (P<0.01). Conclusion SPCs could be isolated and cultured from rat marrow monocytes, and proliferated, differentiated into smooth muscle -like cells.