【作者】 汲晶；

【导师】 李光来；

【作者基本信息】 山西医科大学 ， 神经病学， 2010， 硕士

【摘要】 目的：脂肪间充质干细胞(Adipose-derived stem cells, ADSCs)和骨髓间充质干细胞(Bone marrowmesenchymal stem cells, BMSCs)均具有分化为神经细胞的潜能。探讨ADSCs与BMSCs的取材、原代及传代培养、干细胞表型的鉴定,比较其分离,培养及扩增的不同点,比较ADSCs与BMSCs在分化中的最佳诱导时间,最佳诱导剂剂量,以及诱导阳性率的不同。探讨脂肪干细胞和骨髓间充质干细胞在体外培养和诱导分化为神经细胞方面的差异,选择最优的种子细胞,为中枢神经统统疾病的治疗提供前提和保障。方法：ADSCs提取自山西医科大学生理教研室提供的vista大鼠皮下脂肪组织,BMSCs提取自同一只大鼠四肢长骨骨髓,体外分离、培养、扩增。流式细胞仪法检测干细胞表面CD34, CD45, CD90,CD105的表达。MTT法检测两种来源的干细胞生长情况,绘制生长曲线图。使用5 mmol/LBME+2% DMSO,5ng/mL、20ng/mL、40ng/mLbFGF的无血清培养基对传至3-5代的干细胞进行诱导分化,无血清的DMEM培养基做空白对照,使用倒置显微镜动态观察诱导前后及诱导过程中细胞的形态学变化。通过免疫细胞化学法检测分化后细胞的NSE、MAP2表达情况,证明ADSCs分化的结果并计算阳性率。结果：原代脂肪干细胞呈单细胞悬浮状态,3h后即开始贴壁,12-24h大量贴壁,24h第一次换液后贴壁细胞形态上和BMSCs类似,8-10d后可1：3传代,此后每2-3天可长满一代,传代细胞逐渐表现为折光性较强的均一梭状细胞并呈漩涡状生长,ADSCs可传20代次以上,增长非常迅速。原代骨髓干细胞接种后可见大量红细胞,48h半量换液后可见少量细胞开始贴壁,72h贴壁细胞开始增多呈纺锤状、圆形或椭圆形,折光性较强,5-6天左右全量换液可见散在的贴壁细胞呈集落状生长,2周左右形成较大的集落,并开始相互融合,达到传代要求,传代细胞生长速度较传代前加快,但仍远低于ADSCs,7-8代以后生长速度逐渐减慢,表现出衰老现象。细胞传代接种后第3天左右进入指数生长期,ADSCs第6天达到生长峰值,约7天后进入平台期。BMSCs第8天达到生长峰值,9天后进入平台期。流式细胞术检测细胞表面标志CD90(+), CD105(+),CD34(-), CD45(-),低浓度bFGF诱导阳性率极低,中,高浓度组成熟神经细胞表达的NSE, MAP2阳性。BMSCs最佳诱导浓度20ng/mL, ADSCs最佳诱导浓度40ng/mL, bFGF优于化学诱导剂,ADSCs诱导阳性率高于BMSCs。结论：1、ADSCs与BMSCs都具有多向分化潜能且表达相似的表面标志,他们都可以定向诱导分化为神经细胞。2、ADSCs比BMSCs来源广泛,原代及传代培养的时间短,增殖能力更强,并且更易培养,不易衰老。3、一定诱导条件下,ADSCs诱导阳性率高于BMSCs,脂肪组织是一种新的间充质干细胞来源。更多还原

【Abstract】 Objective:Both the ADSCs and BMSCs have the potential of differentiating into neural cells. Investigate the draw materials、primary culture and serial subcultivation、the identification of stem cells phenotypes between ADSCs and BMSCs, and compare their difference of isolation cultruration、proliferation and the best induction time、the best induction dose、the induction positive rate in differentiation. To investigate the difference between the ADSCs and BMSCs in vitro cultivation and differentiation into Neural cells. Both ADSCs and BMSCs have the potential of differentiating into neural cells. We will choose the best seed cell, meantime,to apply the guarantee to curing the central nervous system diseases.Methods:This study was carried out in the key lab of biochemistry and molecular biology, Shan xi medical university. ADSCs was extracted from subcutaneous adipose tissue of vista rat provided by physiology department. BMSCs was extracted from the bone marrow of the same rat’s limbs, which was isolated, cultured and proliferation in vitro.the expression of CD34,CD44, CD45, CD90 and CD 105 on the surface of stem cells was detected by flow cytometry. The growth of the two cells was evaluated by MTT assay graphed in growth curve. The stem cells was induced into three or five passage by 5 mmol/LBME and 2%DMSO or different concentrations of bFGF(5ng/mL、20ng/mL、40ng/mL). The changes of the cells before and after induced were observed by inverted microscope and immunocytochemistry. The expression of NSE、MAP2 on the surface of cells after differation were observed by immunocytochemical method. Demonstrate the differentiative result of ADSCs and to calculate their differentiative rate.Results:The growth of ADSCs was faster than BMSCs. their cell surface marker was CD44(+),CD90(+), CD105(+),CD34(-),CD45(-),the induced rate of low concentration bFGF was significant lower. The mature neurocyte with high concentration was NSE、MAP2 positive. The optimum induced concentration of BMSCs was 20ng/ml, while the ADSCs was 40ng/ml. bFGF was superior to the general chemical inducers. The positive induced rate of ADSCs excelled BMSCs.Conclusion: 1. Both ADSCs and BMSCs have multipotentiality. and their surface marker were similar. They can be induced differentiation into neurocyte.2. The source of ADSCs was more extensive than BMSCs, the ADSCs have shorter incubation time、more optimum multiplication capacity. They can be cultured easily and they are not to be aged.3. In the special induced conditions, the positive induced ratio of ADSCs was higher than BMSCs. The adipose tissue was a new source of Mesenchymal Stem Cells更多还原