支持细胞对体外培养精原干细胞的作用途径研究

发布时间：2018-01-05 07:16

本文关键词：支持细胞对体外培养精原干细胞的作用途径研究　出处：《重庆医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的通过不同培养条件对精原干细胞(Spermatogonial Stem Cells,SSCs)进行体外培养,观察每种培养条件下SSCs24h贴壁率,增殖特性并绘制其增殖曲线,比较上述指标在不同培养条件下的差异,从而探讨支持细胞对体外培养精原干细胞的作用途径。方法选用7d龄雄性昆明种小鼠,两步酶消化法获得睾丸组织细胞悬液,差异时间贴壁法分离精原干细胞和支持细胞,免疫荧光法和油红O染色法分别对其进行生物学鉴定,流式细胞仪对精原干细胞进行纯度分析。按培养条件的不同将实验分为3组:精原干细胞与支持细胞共培养组(A组),条件培养基组(B组),常规培养基组(C组)。其中B组所用条件培养基按单纯支持细胞培养上清液:双倍浓缩的DMEM/F12:胎牛血清=4.5:4.5:1的比例配置;A、C组所用常规培养基即含体积分数10%胎牛血清的DMEM/F12。台盼兰法测定各组贴壁率,四甲基偶氮唑盐(MTT)法测定各组精原干细胞的吸光度并绘制增值曲线。倒置显微镜下观察各组精原干细胞增殖特点及集落形成情况。比较各组精原干细胞24h贴壁率、增值曲线及存活时间。结果 A组精原干细胞的24h贴壁率大于B组及C组(P0.05),而B、C组间无差异(P0.05);A组精原干细胞接种起即稳定增殖,于(7-10d)形成稳定集落并维持约30d的特性。B组和C组均表现为精原干细胞经短暂的增殖后呈现快速减少的趋势,培养一周后,精原干细胞数量明显减少。结论支持细胞对体外培养精原干细胞的作用是依靠两者之间的直接联系和支持细胞的旁分泌两种途径;仅依靠支持细胞的旁分泌作用不能促进精原干细胞的贴壁和增殖。
[Abstract]:Purpose Spermatogonial Stem cells (SSCs) were cultured in vitro under different culture conditions. The adherent rate and proliferation characteristics of SSCs24h were observed under each culture condition and the proliferation curve was plotted to compare the differences of the above indexes under different culture conditions. In order to explore the role of Sertoli cells on cultured spermatogonial stem cells in vitro. Method Spermatogonial stem cells and Sertoli cells were isolated from male Kunming mice by two-step enzyme digestion. They were identified by immunofluorescence and oil red O staining. The purity of spermatogonial stem cells was analyzed by flow cytometry. According to the different culture conditions, the experiment was divided into three groups: spermatogonial stem cells and Sertoli cells co-culture group (group A), conditioned medium group (group B). The conditioned medium used in group B was as follows: supernatant of Sertoli cell culture: double concentration of DMEM / F12: fetal bovine serum of 4.5: 4.5: 1; DMEM / F12 containing 10% fetal bovine serum was used in the conventional medium used in group A (C). The adherent rate of each group was determined by Trypan blue method. Tetramethylazolium (MTT). The proliferation characteristics and colony formation of spermatogonial stem cells in each group were observed under inverted microscope. 24 h adherent rate of spermatogonial stem cells was compared. Increment curve and survival time. Results The 24 h adherent rate of spermatogonial stem cells in group A was higher than that in group B and group C (P 0.05), but there was no difference between group B and C (P 0.05). In group A, spermatogonial stem cells proliferated stably as soon as they were inoculated. Group B and group C showed a rapid decrease of spermatogonial stem cells after a short period of proliferation, and cultured for one week. The number of spermatogonial stem cells decreased significantly. Conclusion The effect of Sertoli cells on the culture of spermatogonial stem cells in vitro depends on the direct connection between them and the paracrine of Sertoli cells. Paracrine function of Sertoli cells alone can not promote adherent and proliferation of spermatogonial stem cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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