高压氧干预对绿色荧光蛋白小鼠骨髓间充质干细胞培养及增殖分化的影响

Effects of hyperbaric oxygen intervention on the culture and proliferation of bone marrow mesenchymal stem cells in mice labeled by green fluorescent protein

目的 探讨骨髓间充质干细胞培养及高压氧对其增殖分化的影响.方法 采用全骨髓细胞贴壁培养法提取绿色荧光蛋白小鼠骨髓间充质干细胞进行培养,取P3代骨髓间充质干细胞,用神经节苷脂和胶质细胞源性神经生长因子诱导液进行成神经元样细胞诱导,并在培养及诱导时进行高压氧干预,1次/d,为高压氧干预组（高压氧组）.采用免疫荧光检测神经元特异性烯醇化酶（neuron specific enolase,NSE）表达,绘制细胞生长曲线.未进行高压氧干预的为对照组.结果 贴壁培养法分离培养骨髓间充质干细胞,经多次换液后可纯化细胞.高压氧组间充质干细胞对数生长期提前,进入平台期后细胞数量多于对照组,高压氧干预后细胞于24 h内出现轴突、树突,48 h后2组对比,高压氧组80％以上细胞出现突起,且细胞突起较长、较粗.对照组突起的细胞比例不足80％,细胞分散,突起短,未连成网状.结论 骨髓间充质干细胞全骨髓贴壁培养法操作简单,细胞繁殖能力强,经高压氧干预后骨髓间充质干细胞生长加快.在细胞诱导时联合高压氧干预,细胞分化数量多,突起长.提示高压氧能促进体外骨髓间充质干细胞向神经元样细胞增殖和分化.

Objective To investigate the effects of hyperbaric oxygen （HBO） on the culture and proliferation of bone marrow mesenchymal stem cells （MSCs）.Methods MSCs were derived from mice labeled by green fluorescent protein,isolated from whole bone marrow cells by adherent culture method.MSCs of the third generation were induced into neuron-like cells by using galactose-cerebroside and gliocyte-derived nerve growth factor coupled with daily HBO intervention.The expression of neuron specific enolase （NSE） was detected with immunofluorescence,and cell growth curve was delineated.Results MSCs could be ideally isolated by adherent culture method after repeated replacement of culture medium.Following HBO intervention,the exponential stage of MSC growth in the HBO group was advanced and the number of MSCs in the plateau stage was greater than that of the control group.Twenty-four hours after HBO intervention,axons and dendrites could be seen in the HBO group.After forty-eight hours,when comparisons were made between the 2 groups,noticeable changes could be found in MSCs.In the HBO group,processes were found in more than 80％ MSCs,with the processes being relatively long and thick.By comparison,in the control group,less than 80％ MSCs were detected to have processes,which were short and dispersed.Conclusions The whole bone marrow mesenchymal stem cell adherent culture method had the advantages of simple manipulation and good cell reproductivity,and HBO intervention could accelerate the growth of MSCs.In the induction of the cells supplemented by HBO intervention,large numbers of cells were differentiated with longer processes,indicating that HBO intervention could enhance the proliferation and differentiation of cells from in vitro bone marrow MSCs to neuron-like cells.