CAJ | 학술논문

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.
목적 탐토골수간충질간세포배양급고압양대기증식분화적영향.방법 채용전골수세포첩벽배양법제취록색형광단백소서골수간충질간세포진행배양,취P3대골수간충질간세포,용신경절감지화효질세포원성신경생장인자유도액진행성신경원양세포유도,병재배양급유도시진행고압양간예,1차/d,위고압양간예조(고압양조).채용면역형광검측신경원특이성희순화매(neuron specific enolase,NSE)표체,회제세포생장곡선.미진행고압양간예적위대조조.결과 첩벽배양법분리배양골수간충질간세포,경다차환액후가순화세포.고압양조간충질간세포대수생장기제전,진입평태기후세포수량다우대조조,고압양간예후세포우24 h내출현축돌、수돌,48 h후2조대비,고압양조80％이상세포출현돌기,차세포돌기교장、교조.대조조돌기적세포비례불족80％,세포분산,돌기단,미련성망상.결론 골수간충질간세포전골수첩벽배양법조작간단,세포번식능력강,경고압양간예후골수간충질간세포생장가쾌.재세포유도시연합고압양간예,세포분화수량다,돌기장.제시고압양능촉진체외골수간충질간세포향신경원양세포증식화분화.
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.