永生化山羊睾丸间质细胞系的建立

发布时间：2018-03-03 15:50

本文选题：山羊　切入点：睾丸间质细胞　出处：《西北农林科技大学》2015年硕士论文　论文类型：学位论文

【摘要】：睾丸间质细胞(interstitial cell)又称leydig cell(LCs),分布于曲细精管间的疏松的结缔组织之间,其数量占睾丸中细胞总数的2%~4%,主要功能是合成与分泌雄激素,参与或促进精子发生过程的各种生理功能。很多生理、内分泌、药理或毒理因素等可能干扰睾丸间质细胞的正常功能,导致睾酮分泌异常进而导致雄性哺乳动物生殖能力下降或丧失。虽然可以通过原代睾丸间质细胞研究睾丸间质细胞的调节及睾酮分泌等功能,但其有限的增殖能力限制了很多研究的开展,特别是各种因素对该细胞的长期效应性研究。本实验使用屠宰场健康山羊获得的睾丸组织进行间质细胞分离纯化培养,向细胞内稳定转染含有人端粒酶逆转录酶亚基(human telomerase reverse transcriptase,hTERT)的表达载体,并进行细胞筛选,以期获得永生化的山羊睾丸间质细胞系,为后续的研究奠定基础。获得主要结果如下:1.使用胶原酶消化培养法,2.5 mg/mL胶原酶消化1 h,过滤、低速离心、自然沉降获取单个细胞,培养细胞为纤维状,形态一致,有典型的胞间联系,细胞界限清楚,连接紧密,形态均匀,增殖旺盛,睾丸间质细胞特异的3β-HSD染色成阳性。2.使用脂质体转染法,将质粒pCI-neo-hTERT转入第4代的山羊睾丸间质细胞,经浓度为500μg/mL的G-418筛选后,挑选出耐药细胞克隆扩大培养,所获细胞状态良好,生长增殖旺盛,传至85代。使用RT-PCR和免疫荧光对30代、50代hTERT-GLCs测定,hTERT检测均呈阳性,而对照的原代睾丸间质细胞呈阴性,证明hTERT在hTERT-GLCs中稳定表达,说明外源基因己成功转染到睾丸间质细胞中并表达。3.对转染后第30代、50代的山羊睾丸间质细胞进行RT-PCR鉴定证明hTERT-GLCs可稳定表达其合成睾酮通路上的关键酶和受体:StAR、P450scc、3β-HSD、LH-R,且ELISA检测证明其睾酮分泌能力和原代睾丸间质细胞无明显差异,证明其依然具有正常睾酮分泌能力。4.对该细胞系进行生长曲线测定和细胞周期检测,相比于原代睾丸间质细胞,该细胞系具有更快的生长速率和更长的S期;核型分析检测,其结果呈现山羊染色体正常的核型(2n=60),表明其染色体正常;端粒长度检测,其不同代次间端粒长度稳定,且长度和Hela细胞相比没有明显差异,表明其端粒酶表达强度趋于稳定;软琼脂实验检测,该细胞系软琼脂中不能形成细胞克隆,表明该细胞依然保留贴壁生长和细胞通讯依赖性,没有致瘤性。综上所述,本实验分离纯化山羊睾丸间质细胞,将人端粒酶逆转录酶基因hTERT成功导入细胞基因组中,诱导细胞端粒酶的激活,阻止端粒缩短并维持足够且稳定的长度,最终使细胞实现永生化,成功构建山羊睾丸间质细胞系,为后续研究奠定了基础。
[Abstract]:Interstitial cell of testis, also known as leydig cell, distributes between loose connective tissue between seminiferous tubules. Its number accounts for 2% of the total number of cells in testis. Its main function is to synthesize and secrete androgen. Participate in or promote the various physiological functions of spermatogenesis. Many physiological, endocrine, pharmacological or toxicological factors may interfere with the normal function of testicular interstitial cells, This leads to abnormal testosterone secretion, which leads to the reduction or loss of reproductive capacity in male mammals. Although the regulation of testicular stromal cells and testosterone secretion can be studied through primary testicular stromal cells, However, its limited proliferative ability limits the development of many studies, especially the long-term effects of various factors on the cells. In this study, the mesenchymal cells were isolated and cultured from the testis of slaughterhouse healthy goats. The expression vector containing human telomerase reverse transcriptasehTERT was stably transfected into the cells and screened to obtain immortalized goat testicular stromal cell lines. The main results are as follows: 1. Using collagenase digestion and culture method to digest for 1 hour, filter, centrifuge at low speed, natural sedimentation to obtain a single cell, the cultured cells are fibrous, and the morphology is the same. There is typical intercellular connection, the cell line is clear, the connection is close, the shape is uniform, the proliferation is exuberant, the specific 3 尾 -HSD staining of testicular interstitial cells is positive .2.Using the liposome transfection method, the plasmid pCI-neo-hTERT is transferred into the 4th generation goat testicular interstitial cells. After screening with G-418 at the concentration of 500 渭 g / mL, the drug-resistant cells were selected for extended culture. The cells were in good condition and proliferative, and passed to 85th generation. RT-PCR and immunofluorescence were used to detect hTERT in hTERT-GLCs of 30 ~ 50 generations. In contrast, the primary stromal cells of testis were negative, which proved that hTERT was stably expressed in hTERT-GLCs. The results showed that exogenous gene had been successfully transfected into testicular stromal cells and expressed .3.The RT-PCR analysis of goat testicular stromal cells at passage 30 and 50 showed that hTERT-GLCs could stably express the key enzyme and receptor on the synthesis of testosterone pathway, the receptor: Starn P450 sccf3 尾 -HSDN LH-R. ELISA showed that there was no significant difference between testosterone secretion and primary testicular stromal cells. The results showed that the cell line still had normal testosterone secretion ability. 4. The cell line had faster growth rate and longer S phase than primary testicular stromal cells, and the karyotype analysis was used to detect the growth curve and cell cycle of the cell line, and the results showed that the cell line had higher growth rate and longer S phase than the primary testicular stromal cells. The results showed that the karyotype of goat chromosome was normal, indicating that the chromosome was normal, the telomere length was stable in different generations, and there was no significant difference between the length of telomere and Hela cells, which indicated that the intensity of telomerase expression tended to be stable. Soft Agar assay showed that the cell clone could not be formed in soft Agar, indicating that the cell remained dependent on adherent growth and cell communication and had no tumorigenicity. In conclusion, goat testicular mesenchymal cells were isolated and purified. The human telomerase reverse transcriptase gene (hTERT) was successfully introduced into the cell genome, which induced the activation of telomerase, prevented telomere shortening and maintained sufficient and stable length, finally immortalized the cells and successfully constructed goat testicular stromal cell line. It lays a foundation for further study.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S827

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