山羊脂肪间充质干细胞的体外分离培养与定向分化

发布时间：2018-01-15 10:44

本文关键词：山羊脂肪间充质干细胞的体外分离培养与定向分化　出处：《内蒙古大学》2011年硕士论文　论文类型：学位论文

【摘要】：脂肪间充质干细胞(adipose-derived stromal cells, ADSCs)在不同种类的动物细胞中越来越受到人们的关注。虽然从实验动物(如大鼠)脂肪组织中已经分离出大量脂肪间充质干细胞并进行了相关的研究,但对于家畜的脂肪间充质干细胞(如山羊)却研究的很少。为了分离和培养山羊脂肪间充质干细胞,本实验借鉴分离大鼠脂肪间充质干细胞的方法体外分离培养山羊脂肪间充质干细胞、进行定向诱导培养并与大鼠脂肪间充质干细胞进行比较。结果本实验分离得到的山羊脂肪间充质干细胞在体外增殖稳定,经过多次传代后染色体仍保持正常的二倍性,但在相同接种密度和培养条件下,山羊脂肪间充质干细胞比大鼠脂肪间充质干细胞分裂增殖速度更快。山羊和大鼠脂肪间充质干细胞经CD49d和CD13染色呈阳性,CD34、CD106呈阴性,RT-PCR显示CD44表达呈阳性；成骨诱导后经茜素红染色后均可见明显的骨结节、ALP染色后细胞均呈阳性且表达Osteocalcin;不同的是山羊脂肪间充质干细胞经成骨诱导后形成的骨化结比大鼠脂肪间充质干细胞经诱导后形成的骨化结大且ALP染色更深。两种细胞成脂诱导后经O-油红染色后均有红色脂滴出现在细胞周围且表达PPARy2,不同的是山羊脂肪间充质干细胞诱导后形成的脂滴比大鼠脂肪间充质干细胞诱导后形成的脂滴多且大。应用贴壁培养法、三维培养法和悬滴培养法对两种细胞进行成软骨诱导,诱导后经阿新蓝染色均有软骨陷窝形成且表达COL2A1,不同的是在大鼠脂肪间充质干细胞软骨分化诱导实验中,悬滴培养法诱导后形成的软骨陷窝数量较多、形态更好,在山羊脂肪间充质干细胞软骨分化诱导实验中,贴壁诱导培养法和悬滴诱导培养法效果均好于三维诱导培养法,且形成的软骨陷窝数量多、形态好,但贴壁诱导培养法和悬滴诱导培养法两者相比较并无明显差异。研究结果表明本实验中已经得到纯度较高的山羊和大鼠脂肪间充质干细胞。
[Abstract]:Adipose-derived stromal cells. ADSCs have attracted more and more attention in different animal cells. Although from experimental animals (such as rats). A large number of adipose mesenchymal stem cells have been isolated and studied in adipose tissue. But little research has been done on adipose mesenchymal stem cells (such as goats) in domestic animals. In order to isolate and culture goat adipose mesenchymal stem cells, we used the method of rat adipose mesenchymal stem cells to isolate and culture goat adipose mesenchymal stem cells in vitro. Results the goat adipose mesenchymal stem cells isolated in this experiment proliferate stably in vitro. After several generations, the chromosomes remained normal diploidy, but under the same inoculation density and culture conditions. Goat adipose mesenchymal stem cells divide and proliferate faster than rat adipose mesenchymal stem cells. Goat and rat adipose mesenchymal stem cells are positive for CD34 by CD49d and CD13 staining. The expression of CD44 was positive by CD106 negative RT-PCR. After osteogenesis induced by alizarin red staining, all the cells were positive and expressed Osteocalcinin after ALP staining. The difference is that the ossification of goat adipose mesenchymal stem cells induced by osteogenesis is larger than that of rat adipose mesenchymal stem cells induced by ossification and ALP staining is deeper. After red staining, red lipid droplets appeared around the cells and expressed PPARy2. The difference was that the fat droplets of goat adipose mesenchymal stem cells were more and larger than that of rat adipose mesenchymal stem cells induced by adipose mesenchymal stem cells. Three dimensional culture method and suspension culture method were used to induce the cartilage of the two kinds of cells. After induction, cartilage lacunae were formed and COL2A1 was expressed. The difference is that in the differentiation of adipose mesenchymal stem cells, the number of cartilage lacunae induced by suspension culture is more and the shape is better. In the experiment of differentiation of goat adipose mesenchymal stem cells, the effect of adherent culture method and suspension culture method were better than that of three-dimensional culture method, and the number of cartilage lacunae was more and the shape was good. But there was no significant difference between the two methods. The results show that high purity goat and rat adipose mesenchymal stem cells have been obtained in this experiment.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

【相似文献】