人血管内皮细胞生长因子165基因转染大鼠骨髓基质细胞的实验研究

目的探讨对大鼠骨髓基质细胞进行重组人血管内皮细胞生长因子(VEGF)165基因修饰的可行性。方法采用全骨髓细胞贴壁培养法获得稳定的大鼠骨髓基质细胞,分别在脂质体LipofectamineTM2000和新型的阳离子聚合物Sofast介导下行pcDNA3.1-VEGF165转染骨髓基质细胞,在倒置显微镜下观察转染后细胞形态和生长情况的变化,通过免疫细胞化学鉴定VEGF在细胞中的表达情况。结果脂质体LipofectaminTM和新型阳离子聚合物Sofast介导转染的阳性细胞率分别为11.34%和11.42%,两者无明显差异。而细胞存活率分别为74.60%和85.88%,后者稍高于前者(P<0.05)。结论真核表达载体pcDNA3.1-VEGF165可以成功地在骨髓基质细胞中表达,新型阳离子聚合物Sofast的转染效率与传统的脂质体无明显差异,而细胞毒性略小于传统的脂质体。

Expression of Human VEGF165 Gene in Rat Bone Marrow Stromal Cells in vitro

Objective The purpose of this study was to evaluate the feasibility of VEGF165 gene transfection into bone marrow stromal cells and make the groundwork of VEGF gene therapy for the revascularization of bone tissue engineering. Methods Bone marrow stromal cells(BMSC) were cultured in vitro, and were tested for the dexamethasone-induced potential to form mineralized-like tissue in culture in the presence of ascorbic acid and beta-glycerophosphate. The vector pcDNA3.1-VEGF165 was transfected into bone marrow stroma cells with the method of liposome mediated or Sofast transfectam reagent. The expression level of human VEGF165 in the transformed cells was detected by immunocytochemistry. Results The percent of the positive cells after VEGF165 gene transfection mediated by liposome and cationic polymer was (11.34) and (11.42) respectively. There was no significant difference in gene transfer efficacy between liposome and cationic polymer mediated gene delivery. While the percent of the number of survival cells after gene transfection was 74.60 and 85.88 respectively, the cytoxity of liposome was a little higher than that of cationic polymer. Conclusion The experiment demonstrated that rhVEGF165 was successfully expressed in BMSC. This result could serve as a basis for the next step of revascularization of bone tissue engineering.