不同培养方法和细胞因子对小鼠生精细胞的增殖分化效应

发布时间：2018-01-02 20:45

本文关键词：不同培养方法和细胞因子对小鼠生精细胞的增殖分化效应　出处：《安徽医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的:比较曲细精管片段培养和混合细胞共培养两种不同方法对小鼠生精细胞的增殖分化效应,旨在建立一个高效、稳定的小鼠生精细胞体外培养方法,获得更多接近成熟的单倍体精子细胞。方法:对7~8天龄小鼠生精细胞分别进行曲细精管片段培养和混合细胞共培养,通过细胞形态学观察、存活率和粗线期精母细胞特异性基因P19、单倍体精子细胞特异性基因TP1检测及染色体倍性分析,比较两种培养方法生精细胞的存活、增殖以及分化情况。结果:曲细精管片段培养法第10~12天可见到圆形精子细胞,13~14天出现少量带鞭毛的精子细胞或长形精子,第10天可检测出单倍体峰和单倍体精子细胞特异性基因TP1表达;混合细胞培养法5~7天可见圆形精子细胞,第5天即可检测出单倍体峰和单倍体精子细胞特异性基因TP1表达,8~10天少量精子细胞长出鞭毛或胞体渐变成长形。混合细胞培养法所获细胞数、存活率及单倍体精子细胞分化率均显著高于曲细精管片段培养法(P0.05)。培养过程中,各级生精细胞随培养时间延长,存活率逐渐降低,细胞数减少,曲细精管片段培养和混合细胞培养分别在第3周和第4周出现大部分生精细胞和支持细胞脱落死亡。结论:曲细精管片段培养和生精细胞-支持细胞共培养均可获得单倍体精子细胞,较之曲细精管片段培养法,混合细胞共培养法存活率更高,可更早获得更多单倍体精子细胞。目的:通过在生精细胞体外培养过程中添加不同浓度表皮生长因子(epidermal growth factor,EGF)和干细胞因子(stem cell factor,SCF),探讨两种细胞因子对生精细胞增殖、分化的最佳作用浓度以及它们之间的最佳组合浓度。方法:对7~8天龄小鼠生精细胞进行混合细胞体外培养,在培养基中分别添加不同浓度的EGF和SCF(分别为5 ng/ml、10 ng/ml、20 ng/ml、40 ng/ml、100 ng/ml),并进行EGF和SCF的交互实验,通过细胞形态学观察、存活率和粗线期精母细胞特异性基因P19、单倍体精子细胞特异性基因TP1检测及染色体倍性分析,探讨EGF和SCF对生精细胞的体外增殖分化效应。结果:加入EGF或SCF2-4天,各浓度组中可见不同程度增殖呈团或链状的细胞团紧密连接,以EGF 20ng/ml组和SCF 40ng/ml组较为明显,第5-7天出现圆形精子细胞,8-10天少部分精子细胞长出鞭毛或渐变成长形精子细胞,第4周出现大部分生精细胞和支持细胞脱落死亡。培养第7天,EGF浓度为20ng/ml、SCF浓度为40ng/ml时生精细胞数和存活率显著高于与其它各浓度组(P0.05),且浓度为40ng/ml的SCF可显著提高单倍体峰并降低P19/TP1比值(P0.05)。EGF与SCF配伍时,EGF浓度为12 ng/ml时生精细胞增殖率最高,而SCF的作用效果呈线性递增趋势。结论:在混合生精细胞体外培养体系中,添加一定浓度的EGF和SCF可显著提高生精细胞数和存活率,而且SCF可提高体单倍体精子的形成率,两者交互实验时,对细胞增殖有相互叠加效应。
[Abstract]:Objective: to compare the proliferation and differentiation effects of seminiferous tubule fragment culture and mixed cell co-culture on mouse spermatogenic cells in order to establish an efficient and stable culture method of mouse spermatogenic cells in vitro. More haploid sperm cells were obtained. Methods: the spermatogenic cells of 7-day-old mice were cultured with seminiferous tubules and mixed cells, and the morphology of spermatogenic cells was observed. Survival rate, spermatocyte specific gene P19, haploid spermatocyte-specific gene TP1 and chromosome ploidy analysis were compared. Results: on the 10th and 12th day of seminiferous tubule culture, a small amount of sperm cells with flagellated or long sperm could be found in the round spermatozoa on day 1314. The haploid peak and haploid sperm cell specific gene TP1 expression could be detected on the 10th day. Round spermatocytes could be found in mixed cell culture method for 5 days. Haploid peak and haploid sperm cell specific gene TP1 expression could be detected on the 5th day. After 10 days, a small number of spermatozoa cells grew flagella or the cell body gradually formed. The number of cells obtained by mixed cell culture method. The survival rate and the differentiation rate of haploid sperm cells were significantly higher than those of seminiferous tubule culture (P0.05). In the process of culture, the survival rate and the number of spermatogenic cells decreased with the increase of culture time. The majority of spermatogenic cells and Sertoli cells died in the third and fourth weeks of seminiferous tubule culture and mixed cell culture, respectively. Haploid sperm cells could be obtained in seminiferous tubule fragment culture and spermatogenic cell-Sertoli cell co-culture. The survival rate of mixed cell coculture was higher than that of seminiferous tubule culture, and more haploid sperm cells could be obtained earlier. Objective: to add epidermal growth factor of different concentrations to spermatogenic cells in vitro. To investigate the proliferation of spermatogenic cells by two cytokines: EGF) and stem cell factor (SCF). Methods: mixed spermatogenic cells of 7-day old mice were cultured in vitro. Different concentrations of EGF and SCF were added to the medium (5 ng / ml, 10 ng / ml, 20 ng / ml, 40 ng/ml, respectively). 100ng 路ml ~ (-1) EGF and SCF were used to study the cell morphology, survival rate and spermatocyte specific gene P19 in coarse-line phase. The effects of EGF and SCF on the proliferation and differentiation of spermatogenic cells in vitro were studied by TP1 detection and chromosome ploidy analysis. Results: EGF or SCF2-4 were added to the spermatogenic cells for days. In each concentration group, the cell clusters with different degrees of proliferation were closely connected, especially in the EGF 20ng / ml group and the SCF 40ng / ml group. On the 5th to 7th day, a few of the spermatozoa cells grew out of flagella or maturation. At the 4th week, most of the spermatogenic cells and Sertoli cells died, and on the 7th day of culture, most of the spermatogenic cells and Sertoli cells died. The number and survival rate of spermatogenic cells at EGF concentration of 20 ng / ml were significantly higher than those of other groups (P 0.05). When the concentration of SCF was 40ng / ml, the haploid peak was significantly increased and the ratio of P19 / TP1 was decreased when P0.05N 路EGF was compatible with SCF. When the concentration of EGF was 12 ng/ml, the proliferation rate of spermatogenic cells was the highest, while the effect of SCF showed a linear increasing trend. Conclusion: in the culture system of mixed spermatogenic cells in vitro. Adding a certain concentration of EGF and SCF could significantly increase the number and survival rate of spermatogenic cells, and SCF could increase the formation rate of haploid spermatozoa. In the experiment of interaction between the two groups, there was a superposition effect on the proliferation of spermatogenic cells.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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