多种细胞饲养层的制备及其资源建立

发布时间：2018-04-01 14:21

本文选题：睾丸支持细胞　切入点：骨髓基质细胞　出处：《广西医科大学》2008年硕士论文

【摘要】： 目的: 分离培养睾丸支持细胞、骨髓基质细胞、胚胎成纤维细胞和胰腺成纤维细胞,优化培养条件,建立饲养细胞系,为干细胞体外共培养体系的建立提空充足的饲养细胞。方法: 1.使用胶原酶消化法和低渗处理分离睾丸支持细胞(Sertoli cell,SC);全骨髓离心后贴壁培养分离骨髓基质细胞(Bone marrow stromalcell,BMSC),多种培养液对比培养;组织消化法分离培养小鼠胚胎成纤维细胞(Mouse embryonic fibroblast,MEF)和胰腺成纤维细胞(PancreaticFibroblast,PF)。 2.MTF法测定第三代胚胎成纤维细胞和同代胰腺成纤维细胞的生长活力,使用DMSO低温保存细胞;分别取第三代的胚胎成纤维细胞和胰腺成纤维细胞,加入10μg/ml的丝裂霉素C制备饲养层。结果: 低渗处理可以获得富集度较高的SC;含15%FBS的DMEM(L)培养液更适合BMSC体外培养;经组织块贴壁培养法从胰腺结缔组织中分离获得成纤维样细胞,此类细胞具有成纤维细胞生长活性;使用DMSO作为细胞保护剂冻存的MEF和PF,复苏后可继续传代培养;制备了MEF和PF饲养层。结论: 1.低渗处理可以有效地提高睾丸支持细胞的富集度; 2.含15%FBS的DMEM(L)培养液更适合BMSC体外培养; 3.从胰腺结缔组织中分离PF,此细胞和MEF具有相似生物学活性; 4.初步建立了小鼠胚胎成纤维细胞系和胰腺成纤维细胞系。 5.制备了MEF和PF两种饲养层。
[Abstract]:Objective:. Sertoli cells, bone marrow stromal cells, embryonic fibroblasts and pancreatic fibroblasts were isolated and cultured. Methods:. 1. Sertoli cells were isolated by collagenase digestion and hypoosmotic treatment, bone marrow stromal cells were isolated by adherent culture after whole bone marrow centrifugation. Mouse embryonic fibroblast (MEF) and pancreatic fibroblast (Pancreatic fibroblast) were isolated and cultured by tissue digestion. 2.MTF method was used to determine the growth activity of the third generation of embryonic fibroblasts and the pancreatic fibroblasts of the same generation. The cells were cryopreserved by DMSO, and the third generation of embryonic fibroblasts and pancreatic fibroblasts were obtained, respectively. The feeder layer was prepared by adding 10 渭 g/ml mitomycin C. Results:. Hypotonic treatment could obtain high concentration of SCS; the culture medium containing 15s was more suitable for BMSC culture in vitro; fibroblasts were isolated from pancreatic connective tissue by tissue mass adherent culture, and fibroblast-like cells were obtained from pancreatic connective tissue by tissue mass adherent culture. The cells had fibroblast growth activity, MEF and PFL, which were frozen by DMSO as cell protectant, could be further cultured after resuscitation. MEF and PF feeder layers were prepared. Conclusion:. 1. Hypotonic treatment can effectively increase the concentration of Sertoli cells in testis. 2. The culture medium containing 15s was more suitable for BMSC culture in vitro. 3.PFs isolated from connective tissue of pancreas showed similar biological activity to MEF. 4. Mouse embryonic fibroblasts and pancreatic fibroblasts were established. 5. Two feeding layers, MEF and PF, were prepared.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

【引证文献】