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【目的】探讨作为组织工程种子细胞的骨髓间充质干细胞(BMSCs),在体外培养条件下bFGF基因转染对其生物学特性的影响。【方法】穿刺抽取新西兰大白兔胫骨骨髓,采用密度梯度离心法分离BMSCs,在采用贴壁筛选法对分离出的BMSCs进行纯化。将DH-5α菌(含牛bFGF-pcDNA3真核表达载体)扩增,用UNIQ-10柱式质粒小量抽提试剂盒抽提质粒,并对所提取的bFGF-pcDNA3重组表达质粒酶切鉴定。利用脂质体转染bFGF-pcDNA3到BMSCs,G418筛选获得抗性克隆。采用免疫组化、Western blot检测、MTT法检测和流式细胞仪检测转染bFGF的骨髓基质干细胞表达bFGF蛋白、细胞的增殖情况和细胞周期。【结果】密度梯度离心法和贴壁筛选法,获得大量形态均一的BMSCs,部分细胞经成骨诱导培养后,Vonkossa染色可见黑色结节。脂质体介导bFGF-pcDNA3重组表达质粒转染BMSCs,经免疫组化和Western blot检测,证实转染细胞确实表达bFGF,并且表达部位在胞浆。转染细胞增值活力加强,生长曲线上移,处于增殖周期的细胞比例加大(P<0.05)。【结论】用脂质体转染法能将bFGF-pcDNA3重组真核表达载体成功导入体外培养的BMSCs,筛选得到稳定表达bFGF的BMSCs细胞株,自身表达的bFGF可以促进BMSCs增殖。
【Objective】 To investigate the effect of bFGF gene transfection on the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) as seed cells of tissue engineering under in vitro culture conditions. 【Method】 The bone marrow of New Zealand white rabbits was taken by puncture. BMSCs were isolated by density gradient centrifugation and the isolated BMSCs were purified by adherence screening. The DH-5α strain (containing the bFGF-pcDNA3 eukaryotic expression vector) was amplified, and the plasmid was extracted by UNIQ-10 column plasmid extraction kit and identified by bFGF-pcDNA3 recombinant expression plasmid . Lipofectamine was used to transfect bFGF-pcDNA3 into BMSCs, and G418 was screened to obtain resistant clones. Immunohistochemistry, Western blot, MTT assay and flow cytometry were used to detect the expression of bFGF protein in bFGF transfected bone marrow stromal cells, proliferation and cell cycle. 【Result】 A large number of BMSCs were obtained by density gradient centrifugation and adherence screening. Some of the cells were cultured by osteogenic induction. Vonkossa staining showed black nodules. Liposome-mediated bFGF-pcDNA3 recombinant plasmid was transfected into BMSCs, confirmed by immunohistochemistry and Western blot, the transfected cells did express bFGF, and the expression site in the cytoplasm. The viability of transfected cells increased, the growth curve shifted up, and the proportion of cells in the proliferative cycle increased (P <0.05). 【Conclusion】 The bFGF-pcDNA3 recombinant eukaryotic expression vector can be successfully introduced into BMSCs cultured in vitro by lipofection method. The BMSCs stably expressing bFGF can be selected and screened. The bFGF itself can promote the proliferation of BMSCs.