稳定表达角质生长因子与缺氧诱导因子的大鼠骨髓间充质干细胞的制备

Preparation of Bone Marrow Mesenchymal Stem Cells Stably Modulated by KGF-HIF in Rats

目的制备角质生长因子（KGF）与缺氧诱导因子（HIF）修饰的大鼠骨髓间充质干细胞（MSCs）,评价细胞因子表达效率及其活性。方法骨髓全细胞贴壁培养法分离培养大鼠骨髓MSCs,经免疫荧光法检测细胞表面标志物后,以KGF、HIF重组腺病毒感染,酶联免疫吸附试验（ELISA）检测KGF、HIF的表达水平,成骨细胞增殖实验评价KGF、HIF的活性。结果获得了可在体外连续传代、高表达CD44、CD133表面分子的骨髓MSCs;KGF、HIF细胞因子获得稳定表达,且在感染24 h即出现表达,随培养时间的延长表达量逐渐上升,至120 h开始下降;骨髓MSCs培养上清含量为50%~100%时成骨细胞增殖水平明显提高。结论成功制备可稳定表达KGF、HIF的大鼠骨髓MSCs,其表达的KGF、HIF因子含量高,可促进成骨细胞的增殖,具有良好的生物活性。

Objective To prepare bone marrow mesenchymal stem cells（ MSCs） modulated by kinetics gain factor（ KGF） and hypoxia-inducible factor（ HIF） in rats,and to evaluate the expression efficiencies and activities of KGF and HIF. Methods Bone marrow mesenchymal stem cells of rats were isolated and cultured using whole bone marrow cells adherence method,and cell surface markers were identified using immunofluorescence testing,and then adenovirus infections were restructured by KGF and HIF. The expression levels of KGF and HIF were detected using enzyme linked immunosorbent assay（ ELISA）,and the activities of KGF and HIF were evaluated using proliferation assay of osteoblasts. Results Serial passage and high expressions in vitro of surface markers CD44 and CD133 were obtained in bone marrow mesenchymal stem cells,and the expressions of CD44 and CD133 were stable. The CD44 and CD133 were expressed at the 24 thh infection,and the expression levels were increasing with prolonged time,and the levels began to decrease at the 120 thh infection. The osteoblasts proliferation was obvious when supernatant content was 50%-100% by bone marrow MSCs culture. Conclusion KGF / HIF modulated rat bone marrow MSCs can be successfully achieved in rats. KGF and HIF can be highly expressed in these cells,which may promote osteoblastic multiplication with good biological activity.