睾丸支持细胞促进骨髓间充质干细胞分化的培养特性研究

发布时间：2018-06-09 03:43

  本文选题：睾丸支持细胞 + 骨髓间充质干细胞　； 参考：《华东理工大学》2012年硕士论文

【摘要】：睾丸支持细胞(SCs)具有分泌多种细胞因子与营养物质的功能,能滋养或促进共培养细胞的增殖或分化,且可形成局部免疫豁免的功能。骨髓间充质干细胞(bmMSCs)作为源自于骨髓的间充质干细胞,bmMSCs在不同的诱导条件下可以分化为多种跨胚层细胞,在组织工程领域具有广泛的研究价值。本论文主要对SCs与bmMSCs分离鉴定、SCs促进bmMSCs定向分化及分化的特性进行初步研究,并建立了利用搅拌式生物反应器实现bmMSCs在微载体上大量增殖的方法,并对扩增后的bmMSCs进行生理特性的研究。具体结果如下： (1)SCs被从10d大的小鼠睾丸中成功分离与鉴定。经苏木精伊红染色与电子扫描显微镜鉴定为支持细胞,具有明显的双核仁特征；通过对培养基与转瓶微载体培养体系的工艺优化,成功实现SCs大量扩增,支持细胞由初始2.25×105cells/ml的接种密度增殖至2.37×106cells/ml。 (2)利用密度梯度离心的方法从骨髓中成功分离出bmMSCs。体外培养时呈现典型的鱼群或漩涡状排列,经流式细胞术检测表面抗原CD29. CD90与CD34鉴定为bmMSCs。通过检测不同血清浓度对bmMSCs体外增殖的影响,发现高浓度血清培养可能会引起bmMSCs分化,影响bmMSCs的生理特性,因此确定含1%FBS的培养基更适宜bmMSCs的体外培养。 (3)采用两种细胞共培养与条件培养基(CM)的培养模式,进行SCs促进bmMSCs定向分化为软骨细胞与成骨细胞的特性研究。通过免疫细胞化学、细胞化学、RT-PCR与、Vestern-blot等方法进行检测,结果表明SCs能够显著促进bmMSCs定向分化。 (4)建立1.5L搅拌式生物反应器的bmMSCs体外大规模扩增的微载体培养方法。在细胞与微载体接种密度分别为2.5×105cells/ml与4mg/ml,搅拌速率实时调控的条件下,利用Cytodex3微载体与低血清(1%)培养基以及1.5L搅拌式反应器,成功地实现了bmMSCs体外大规模扩增。通过每24h更换50%培养基的策略,bmMSCs的扩增倍数达到了10.4倍,最大细胞密度达到2.6x106cells/ml。通过流式细胞术与定向分化检测,用胰酶从微载体上消化下的bmMSCs仍能保留其表型特征以及分化为软骨与成骨细胞的潜能。
[Abstract]:Testicular Sertoli cells (SCS) can secrete many cytokines and nutrients, can nourish or promote the proliferation or differentiation of co-cultured cells, and form the function of local immunity. Bone marrow mesenchymal stem cells (BMSCs), as mesenchymal stem cells derived from bone marrow, can differentiate into many kinds of transdermal cells under different induction conditions. In this paper, we mainly studied the characteristics of promoting the directional differentiation and differentiation of BmMSCs by isolation and identification of SCs from bmMSCs, and established a method to realize the proliferation of bmMSCs on microcarriers by stirred bioreactor. The physiological characteristics of amplified bmMSCs were studied. The results are as follows: 1) the stem cells were isolated and identified successfully from the testis of 10 d old mice. Sertoli cells were identified by hematoxylin eosin staining and electron scanning microscope. Sertoli cells proliferated from the initial inoculation density of 2.25 脳 105cells/ml to 2.37 脳 106 cells / ml 路ml. 2) bmMSCs were isolated from bone marrow by density gradient centrifugation. In vitro culture showed a typical fish colony or whirlpool arrangement, and the surface antigen CD29 was detected by flow cytometry. CD90 and CD34 were identified as bmMSCs. By detecting the effects of different serum concentrations on the proliferation of bmMSCs in vitro, it was found that the high concentration of serum culture might induce the differentiation of bmMSCs and affect the physiological characteristics of bmMSCs. Therefore, the culture medium containing 1s was more suitable for the culture of bmMSCs in vitro. (3) the characteristics of SCs promoting the differentiation of bmMSCs into chondrocytes and osteoblasts were studied by using two culture modes of co-culture and conditioned medium (CMM). The results of immunocytochemistry RT-PCR and Vestern-blot showed that SCs could significantly promote the directional differentiation of bmMSCs. Under the condition that the cell and microcarrier inoculation densities were 2.5 脳 105cells/ml and 4 mg / ml, respectively, and the stirring rate was adjusted in real time, using Cytodex3 microcarrier and low serum 1 layer) medium and 1.5L stirred reactor, BmMSCs were successfully amplified in vitro on a large scale. By replacing 50% culture medium every 24 hours, the expansion times of BmMSCs reached 10.4 times, and the maximum cell density reached 2.6x106 cells / ml. By flow cytometry and directional differentiation detection, bmMSCs digested with trypsin from microcarriers could still retain their phenotypic characteristics and the potential to differentiate into cartilage and osteoblasts.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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