脂肪与骨髓来源的间充质干细胞诱导分化为神经细胞的比较

发布时间：2018-01-05 16:00

本文关键词：脂肪与骨髓来源的间充质干细胞诱导分化为神经细胞的比较　出处：《山西医科大学》2010年硕士论文　论文类型：学位论文

【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。
[Abstract]:Objective: adipose derived mesenchymal stem cells (Adipose-derived stem cells, ADSCs) and bone marrow mesenchymal stem cells (Bone marrowmesenchymal stem cells, BMSCs) have the potential to differentiate into nerve cells. ADSCs and BMSCs were discussed, and cultured in vitro, identification of stem cell phenotype, the separation, culture and different points of amplification, the best induction time between ADSCs and BMSCs in the differentiation of the optimal dose of inducer, and different induction rate. To investigate the adipose derived stem cells and bone marrow mesenchymal stem cells cultured in vitro and induced to differentiate into neural cells between the optimal selection of seed cells, to provide the premise and guarantee for the all the treatment of central nervous diseases.
Methods: ADSCs was extracted from Department of physiology of Shanxi Medical University Vista rats subcutaneous adipose tissue, BMSCs extraction from the same rat bone marrow in vitro, isolation, culture, amplification. Flow cytometry method for the detection of stem cell surface CD34, CD45, CD90, CD105 method to detect the expression of.MTT two sources of stem growth the cell growth curves, using 5 mmol/LBME+2%. DMSO, 5ng/mL, 20ng/mL, no radical on the passage to 3-5 induced differentiation of stem cells cultured in serum 40ng/mLbFGF, serum free DMEM medium as blank control, using the inverted microscope to observe morphological changes before and after induction and dynamic process of cells induced by NSE cells. The detection of differentiation by immunocytochemistry, the expression of MAP2, ADSCs proved differentiation results and calculated the positive rate.
Results: the primary adipose derived stem cells in single cell suspension, 3h is adhered to the wall, a lot of adherent 12-24h, 24h first medium change after adherent cells morphologically similar to BMSCs, 8-10d after 1:3 passages, then every 2-3 days can be covered with a generation of cells gradually showed homogeneous spindle cell refraction strong and swirl growth, ADSCs can spread over 20 times, grew very rapidly. The primary bone marrow stem cells after inoculation showed a large number of red blood cells, 48h was half changed after a small amount of cells adhered to the wall, 72h began to increase in adherent cells were fusiform, round or oval. The refractive index of strong, 5-6 days was all changed scattered in adherent cells showed colony like growth, about 2 weeks to form a larger colony, and fusion of the passage, the cells grow faster than before to speed up the passage, but still much lower than that of ADSCs, 7-8 in growth speed gradually Slow down, showing aging phenomenon. About third days after inoculation of cells entered exponential growth stage, ADSCs sixth days to reach the peak of growth, about 7 days into the plateau.BMSCs eighth days to reach the peak of growth, 9 days into the plateau. Flow cytometry to detect the surface markers of CD90 (+), CD105 (+), CD34 (-), CD45 (-), low concentration bFGF induced positive rate is extremely low, and the high concentration group of mature neural cells and expression of NSE, MAP2 positive.BMSCs optimal concentration of 20ng/mL, ADSCs 40ng/mL bFGF is better than the optimal concentration, chemical inducers, ADSCs induced positive rate higher than BMSCs.
Conclusion:
1, both ADSCs and BMSCs have multidirectional differentiation potential and express similar surface markers, and they can be directed to induce differentiation into neural cells.
2, ADSCs is more extensive than BMSCs. The primary and subculture time is short, the proliferation ability is stronger, and the culture is more easy to be cultivated, and it is not easy to senescence.
3, under certain induction conditions, the positive rate of ADSCs is higher than that of BMSCs, and adipose tissue is a new source of mesenchymal stem cells.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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