ADSCs培养观察及rhBMP-2转染对其生长增殖的影响

In vitro growth pattern of ADSCs and effects of rhBMP-2 transfection on growth and proliferation of ADSCs

目的观察脂肪源性干细胞(ADSCs)体外生长规律,并探讨重组人骨形态发生蛋白(rhBMP-2)转染后ADSCs增殖变化情况。方法用酶消化法从1岁龄贵州小香猪腹壁脂肪组织中获取ADSCs,进行连续传代培养,倒置显微镜下观察其相关生长特性。用细胞消化计数法和四唑盐比色法(MTT)对rhBMP-2转染ADSCs前后生长增殖情况进行比较,计算倍增时间,绘制生长曲线。结果传代后ADSCs增殖速度比原代有所增快,于第4～6代趋于稳定。细胞形态由多角形、圆形和不规则形逐渐向成纤维细胞样和梭形过渡。rhBMP-2转染后ADSCs群体倍增时间增加8～10h,但对ADSCs生长增殖无明显影响(P<0.05)。结论ADSCs在体外培养至4～6代可获得较稳定状态。并且ADSCs作为宿主细胞被转染rhBMP-2基因后未发生畸变及生长增殖明显改变,说明rhBMP-2基因修饰的ADSCs可以作为携带目的基因的种子细胞应用于再生医学研究。

Objective To observe in vitro growth pattern of adipose-derived stem cells （ADSCs） and effects of rhBMP-2 transfection on growth and proliferation of ADSCs. Methods Enzyme digestion method was applied to obtaining ADSCs from the abdominal adipose tissues of the yearling Guizhou minipigs, and the ADSCs were cultivated consecutively, their related growth pattern was observed under the inverted microscope. Cell counting and MTT assay were adopted to compare the status of ADSCs growth and proliferation before and after rhBMP-2 transfection,and then the colony doubling time was calculated and the growth curve was drawn. Results The ADSCs proliferation was accelerating after descendant culture in vitro,and tended to be stable in the 4--6th generation. The cell shape was becoming fibroid and fusiform shape from polygon, rotundity and abnormity. The colony doubling time increased 8-10 hours after transfection,but there was no difference in the growth proliferation （P〈0.05）. Conclusion ADSCs can attain steady state when cultivated to the 4--6th generation in vitro;furthermore, no aberration or apparent alteration of growth and proliferation of ADSCs occurred after transfection with rhBMP-2. The study indicats that rhBMP-2 modified ADSCs may be used in study and research in the regeneration medicine as the seed cells with the targeted genes.