BMP-7诱导脂肪干细胞微团培养下成软骨能力的实验研究

发布时间：2018-06-21 15:41

  本文选题：干细胞 + 微团培养　； 参考：《中国医科大学》2009年硕士论文

【摘要】： 目的 比较脂肪干细胞在BMP-7存在时平面培养和微团培养两种方式下诱导分化成软骨细胞的情况,探讨不同培养方式对脂肪干细胞成软骨能力的影响。寻求一种更好的促进脂肪干细胞成软骨分化的培养方式。 方法 细胞的分离和扩增,选用6周龄健康Wistar雄性大鼠,无菌条件下取大鼠双侧腹股沟脂肪组织,眼科剪剪碎,0.1%的胶原酶37度震荡消化20分钟,含10%胎牛血清的DMEM终止消化,1500g离心10min,含10%胎牛血清的DMEM重悬,接种于培养瓶中培养。每3天换液一次,待细胞生长至接近融合时胰酶/EDTA消化传代。将培养至第3代细胞胰酶/EDTA消化后计数,调整密度为1×10~5/ml,在BMP-7存在条件下进行平面和微团培养。指标检测(1)光镜观察原代脂肪干细胞及诱导7,21天后的生长情况,流式细胞学检测脂肪间充质干细胞的表面标志;(2)MTT法检测不同培养方式下脂肪干细胞的增殖能力;(3)免疫组织化学染色检测培养三周后两组Ⅱ型胶原表达,RT-PCR检测诱导14,21天后平面和微团培养组软骨特异蛋白聚糖和Ⅱ型胶原mRNA的表达及肥大软骨细胞的标志X型胶原mRNA的表达。 结果 脂肪干细胞的形态特征原代脂肪干细胞接种24小时后光镜下可见有少量的细胞贴壁,形态为短梭形或多角形,48小时后贴壁细胞明显增多,约9天左右细胞融合超过90%。诱导7天后平面培养组细胞呈长梭形,微团培养组呈短梭形。诱导21天后两组细胞形态均为圆形。流式细胞仪检测表明,脂肪间充质干细胞高表达CD29和CD44,而CD34,CD45的表达均不足2%。 MTT检测结果表明平面培养组细胞增殖趋势平缓,10天左右进入平台期。而微团培养组各检测点MTT值均显著高于平面培养组,二者统计学有显著性(P＜0.05),表示微团组细胞的增殖要明显快于平面培养组,且微团培养组从生长曲线上看未见明显的平台期。 免疫组化结果显示,培养三周后微团组Ⅱ型胶原表达强阳性而平面组表达弱阳性,RT-PCR结果显示培养14和21天后两组均有软骨特异蛋白聚糖和Ⅱ型胶原mRNA的表达,但微团组表达较强。而微团组培养21天时其X型胶原mRNA的表达弱于平面组。 结论 在BMP-7诱导下脂肪干细胞可以向软骨细胞分化,同时微团培养比平面培养更能促进脂肪干细胞的增殖、分化,并能更好的维持软骨细胞的表型,可以作为组织工程构建软骨的理想选择。
[Abstract]:Objective to compare the differentiation of adipose stem cells into chondrocytes in the presence of BMP-7 and to explore the effects of different culture methods on the chondrogenic ability of adipose stem cells. To seek a better way to promote the differentiation of adipose stem cells into cartilage. Methods the cells were isolated and expanded. Healthy male Wistar rats aged 6 weeks were selected and bilateral inguinal adipose tissue was harvested under aseptic condition. The tissue was digested by 37 degree shock of 0.1% collagenase in ophthalmic shears for 20 minutes. DMEM containing 10% fetal bovine serum was digested by 1500g and centrifuged for 10 min. The DMEM containing 10% fetal bovine serum was suspended and inoculated in culture flask. Once every 3 days, trypsin / EDTA digested and subcultured when the cells grew close to fusion. After digesting trypsin / EDTA at the third passage, the cell density was adjusted to 1 脳 10 ~ (5) / ml, and the flat and microclusters were cultured in the presence of BMP-7. The primary adipose stem cells were observed under light microscope and the growth of primary adipose stem cells was observed 21 days after induction. Proliferation of adipose Mesenchymal Stem cells detected by flow Cytometry and MTT method) Immunohistochemical staining was used to detect the expression of type 鈪，

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