雄性生殖细胞介导的转基因技术的研究

发布时间：2018-03-01 02:22

  本文关键词： 非免疫缺陷受体 精原干细胞 生殖细胞移植 精子发生 转基因技术　出处：《西北农林科技大学》2016年博士论文　论文类型：学位论文

【摘要】：雄性生殖细胞是指通过有性生殖繁衍后代的雄性生物生殖系统中能将遗传信息传递给后代的一类细胞,其中精原干细胞是整个机体内唯一贯穿一生,既可通过增殖维持自身数目稳定又能定向分化为精子的成体干细胞;同时,精原干细胞在自发或诱导条件下,具有分化为三胚层的潜能,具有类似胚胎干细胞的特征而备受生物医学、转化医学和畜牧业广泛关注。目前对精原干细胞的研究主要集中在分子标记的筛选、体外培养体系建立及维持、经典及表观遗传修饰相关信号通路的研究、基于精原干细胞修饰的转基因动物制作以及睾丸组织块移植等领域,而这些研究中采用的免疫缺陷受体模型大大限制了精原干细胞的科研与应用,特别是实际生产及其在畜牧行业的推广;同时,基于精原干细胞显微移植的转基因动物制作具有一定的技术难度。因此,精原干细胞介导的简单易行的转基因动物制作策略亟需探讨。本研究主要着力于探究睾丸组织及精原干细胞非免疫缺陷移植受体模型制作的方法,探索体内简单易行的雄性生殖细胞介导的转基因技术的新方法。本研究获得如下结果:1,本研究探究了不同浓度的腺嘌呤(200mg/kg,260mg/kg,320mg/kg和380mg/kg)对非免疫缺陷大鼠(Sprague-Dawley,SD大鼠)睾丸的损伤情况,通过相关生理指标和睾丸切片观察,候选得到精原干细胞移植受体在SD大鼠体内的最佳的作用浓度(320mg/kg)。同时作为阳性对照,摸索了白消安(一种广泛使用精原干细胞受体模型制备的药物)在SD大鼠受体制作的合适作用浓度;从存活率、体重、睾丸指数及睾丸切片确定了腺嘌呤处理优于白消安处理,可作为替代方案阻滞内源性精子发生,为精原干细胞移植提供合适的微环境。同时,使用酶消化、差异贴壁及免疫磁珠分选等方法富集高纯度小鼠精原干细胞(Lin28阳性率达到84%,THY-1阳性率达到92%),同时利用体外慢病毒感染修饰的方法标记小鼠的精原干细胞,并移植到受体大鼠曲细精管内,首次检测到非免疫缺陷受体内异种移植完整的精子发生。本实验也使用细胞系和分离的原代生殖细胞及支持细胞作为研究对象,研究腺嘌呤对其体外作用机理;使用TUNEL证实染色体中DNA发生断裂,使用蛋白免疫印迹实验检测到了凋亡早期相关蛋白Caspase3和凋亡执行蛋白PARP浓度依赖性表达,初步探明了腺嘌呤通过诱导细胞凋亡对生殖细胞产生了特异的生殖毒性。2,本研究使用慢病毒体内感染雄性生殖细胞的策略,获得了超过67%的转基因后裔,生殖能力正常,外源转基因可稳定遗传给后代;同时也揭示了该策略与亲本年龄及慢病毒注射部位没有相关性(P0.05),这提示我们可以在大家畜中尝试使用慢病毒注射睾丸间质部位获得转基因后裔,简化大家畜生产及育种中的繁琐环节和技术难题。同时,也比较了不同慢病毒系统对睾丸内精原干细胞感染能力,与慢病毒p CD513B-CMV-MCS-EF1相比,慢病毒p Lenti V-H1-MCS-CMV感染精原干细胞之后获得的转基因后代能正常表达外源转基因片段;进一步研究揭示不同慢病毒整合到子代基因组后,外源基因启动子序列甲基化水平存在显著差异:前者外源转基因片段启动子甲基化水平高达87.8%,而后者仅为45.5%。本研究为以后使用慢病毒载体制作转基因动物特别是对于曲细精管移植难于实现的大家畜提供了新思路。3,本研究探索了使用非免疫缺陷小鼠(成年昆明小鼠)作为睾丸组织块移植的受体,既可以完成同种不同品系间睾丸移植的精子发生,也首次检测到异种睾丸组织块移植后(大鼠睾丸移植到小鼠皮下),具有完整的异源精子发生。本研究同时发现,使用大量的睾丸组织块移植到非免疫缺陷受体小鼠皮下检测到移植组织块经历了破碎、重塑进而完成精子发生的过程;同时也发现在非免疫缺陷小鼠使用免疫抑制剂可提高异源组织块存活和完成精子发生的成功率,将完整精子发生的成功率从2-5%提高到了8.3%,并且重现了移植组织块经历破碎、重塑进入完成精子发生的过程,为精子发生及睾丸组织发育的研究提供了新模型,可为转基因动物制作提供新途径。综上所述,本研究着力探索了非免疫缺陷受体模型下精原干细胞研究的两大策略:其一是基于精原干细胞移植的相关研究,另一个是睾丸组织块移植的研究。探究了腺嘌呤作用于SD大鼠(免疫正常大鼠)睾丸组织生殖毒性,获得一种新型的可用于精原干细胞移植受体模型,经过输出小管移植获得了完整的异种移植精子发生;同时优化了基于慢病毒载体的依赖精原干细胞转基因动物制作的途径,从甲基化水平揭示了不同载体不同启动子在体内表达外源基因差异的原因。同时也对非免疫缺陷受体皮下睾丸组织块移植进行了探索,配合使用免疫抑制剂提高了异源组织块存活率和精子发生率。本研究对于精原干细胞功能、精子发生及睾丸组织发育提供了新见解,对于转基因动物制作提供了简单易行的策略,对于大家畜种和珍稀动物在生物学研究及实际应用具有重要意义。
[Abstract]:Male germ cells refers to the male reproductive system by sexual reproduction biological offspring can transmit genetic information to a class of cell progeny, which spermatogonial stem cells are the body only throughout life, can maintain its stability and the number of proliferation to differentiate into sperm of adult stem cells; at the same time, fine the original stem cells in spontaneous or induced conditions, can differentiate into three germ layers with similar potential of embryonic stem cells has attracted extensive attention in biomedical characteristics, translational medicine and animal husbandry. The spermatogonial stem cell research mainly concentrated in the screening of molecular markers, system establishment and maintenance of in vitro epigenetic research modification of related signaling pathways and classic spermatogonial stem cells modified by transgenic animal production and testicular tissue transplantation field based on immune defects in these studies used by Model greatly limits the spermatogonial stem cell research and application, especially the actual production and promotion in the livestock industry; at the same time, there is a certain difficulty in transgenic animal spermatogonial stem cell transplantation based on production. Therefore, to explore the strategies to transgenic animal spermatogonial stem cells mediated by simple production. The research mainly focuses on exploring testis and spermatogonial stem cells transplantation model making method of non immune deficiency, a new method of transgenic technology of male germ cells mediated in vivo to explore simple. This study as follows: 1, the research results of different concentrations of adenine (200mg/kg, 260mg/kg, 320mg/kg and 380mg/kg) the non immunodeficient rat (Sprague-Dawley SD rats) injury of testis, the related physiological indexes and testicular sections were obtained, the candidate spermatogonial stem cell transplantation in the receptor The optimal concentration of SD rats (320mg/kg). At the same time as a positive control, groping busulfan (a widely used spermatogonial stem cell drug receptor model preparation) in the appropriate concentration of production of receptor SD in rats; from the survival rate, weight, testis index and testis were determined adenine treatment is superior to busulfan treatment, can be used as alternatives to block endogenous spermatogenesis, provide suitable microenvironment for spermatogonial stem cell transplantation. At the same time, the use of enzyme digestion and differential attachment and immunomagnetic separation methods such as enrichment of high purity of mouse spermatogonial stem cells (Lin28 positive rate reached 84%, the positive rate of THY-1 reached 92% at the same time), in vitro using lentivirus infection modified method labeling of mouse spermatogonial stem cells, and transplanted into recipient rat seminiferous tube, first detected by immunocompetent xenograft in vivo intact sperm. This experiment also makes Primary germ cells and Sertoli cells and isolated cell lines as the research object, the research on the mechanism of adenine in vitro; TUNEL chromosome DNA confirmed fracture, using Western blot to detect early apoptosis and apoptosis related protein Caspase3. PARP protein concentration dependent expression, preliminarily adenine produced the reproductive toxicity of specific.2 on germ cells by inducing apoptosis, this study strategy using lentiviral infection of male germ cells, transgenic descendants of more than 67% of the normal reproductive capacity, transgene can be stably inherited; at the same time also revealed no correlation between the strategies and parental age and lentivirus injection site (P0.05), suggesting that we can try to use in all animal lentivirus injected testicular interstitial site to obtain transgenic offspring, we simplify. The tedious aspects and technical problems in production and breeding. At the same time, also compared the different lentiviral system on testicular spermatogonial stem cells to infection, compared with CD513B-CMV-MCS-EF1 lentiviral P, lentivirus P Lenti V-H1-MCS-CMV infection of spermatogonial stem cells of transgenic progeny obtained after the normal expression of transgene fragments; further study revealed different slow the virus integrated into the genome of offspring, exogenous gene promoter sequence there are significant differences in methylation level: the former transgene fragment promoter methylation levels up to 87.8%, while the latter is only 45.5%. in this study for later use lentiviral vector for making transgenic animal especially provides a new idea for.3 seminiferous tube transplantation is difficult to achieve all animals, this study explored the use of non immunodeficient mice (adult Kunming mice) as testis tissue transplantation receptor, can complete the same Different strains of the testicular transplantation spermatogenesis, also first detected in testicular tissue block transplantation (xenogeneic transplantation of rat testis to mice), with complete heterologous sperm occurrence. The study also found that the use of a large number of testicular tissue transplanted to immunodeficient mice subcutaneous non receptor detected tissue after transplantation broken, and then complete the remodeling process of spermatogenesis; also found that the use of immunosuppressive agents in immunodeficient mice can improve the heterologous tissue survived and complete spermatogenesis success rate, the success rate of complete spermatogenesis increased from 2-5% to 8.3%, and re transplantation tissue through crushing, complete the process of spermatogenesis in remodeling that provides a new model for the study of the development and organization of spermatogenesis, which can provide a new way for the production of transgenic animal. In summary, this study explores the non Immunodeficiency receptor model two strategies under the spermatogonial stem cell research: one is the related research of spermatogonial stem cell transplantation based on a study of testis tissue transplantation. To explore the role of adenine in SD rats (Immune normal rats) reproductive toxicity in testis, a model can be used to spermatogonial stem cell transplantation receptor model, after efferent duct transplantation has achieved complete xenograft sperm; optimize the sperm dependent lentiviral vector based on the original way of making stem cells of transgenic animal, the level of methylation reveals why different carriers from different promoters of exogenous gene expression in vivo. The difference is also carried out exploration on the testis non receptor block with immunodeficiency subcutaneous transplantation, immunosuppression increases the incidence of heterologous tissue and sperm survival rate. This study for spermatogonial stem cell function Organization, and spermatogenesis provides new insights into the development, provides a simple strategy for the production of transgenic animal, for all livestock and rare animal plays an important role in biological research and practical application.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78;Q954.4
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本文编号：1549993