【作者】 于雪；

【导师】 杨利国；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2014， 博士

【摘要】 通过研究培养条件(包括血清浓度、饲养层细胞及三种生长因子)对水牛精原干细胞(SSCs)增殖的影响,最终建立水牛SSCs体外增殖的培养体系。通过介导生长抑素RNA干扰的PB转座子转染SSCs,得到含目的基因片段的SSCs;在移植转基因SSCs至昆明鼠睾丸后,分析转基因SSCs的增殖与分化功能。该研究为克隆转基因水牛提供新途径；为SSCs的生物学特性、多潜能性及定向分化的表观遗传学和精子发生机理等研究提供技术平台；结合SSCs的冷冻保存,为水牛的种质资源保存提供新手段；为其他家畜和人的SSCs的研究提供理论参考。1.水牛精原干细胞的体外培养与分子标志探索水牛SSCs特异性分子标志的发展,体外培养条件的优化以及其多能性的保持,在水牛的选择性遗传修饰中具有重要的意义。本研究取4～5月龄杂交水牛(摩拉×沼泽,49条染色体)睾丸,经Western blot发现水牛睾丸中存在两种蛋白异构体(135kDa大片段和90kDa小片段),CDH1为水牛精原干细胞的特异性分子标志；免疫组织化学分析发现水牛睾丸精原细胞上存在CDH1的表达,且在体细胞中无表达。纯化后的精原干细胞(流式分析纯化富集效率达到53%)体外培养7天后,CDH1在细胞克隆中也检测到表达,。而且在精原干细胞体外培养过程中,不同生长因子组合对增殖影响的研究发现,与对照或仅添加单一生长因子组相比,共同添加20ng/mL GDNF、10ng/mL FGF2和1000U/mL LIF能显著提高水牛精原干细胞的克隆数(约提高2倍)和增殖率(约提高3倍),还能显著上调精原细胞特异性和多能性相关标志物(BCL6B, GFRA1和POU5F1)的mRNA表达水平,同时下调受体酪氨酸激酶(KIT)。本研究结果证实水牛存在CDHl特异性分子标志,可用于建立体外培养体系,从而有利于水牛的遗传修饰。2.生长抑素干扰载体转染BHK-21细胞及水牛SSCs克隆生长抑素(SS)在体内的主要作用是抑制生长激素(GH)的分泌,而抑制SS的分泌则可以促进动物的生长。如果干扰SSCs的SS基因后进行移植,可获得长期整合的转基因后代,从而得到生长状况良好的优异个体。但是,由于SSCs本身没有SS的分泌,所以对于干扰效果的验证需在其他SS分泌型细胞上实施。本研究设计并构建了3条针对SS基因序列的干扰片段,以PB载体介导经脂质体转染到BHK-21细胞(仓鼠肾成纤维细胞,经鉴定具备分泌SS的细胞)以沉默SS基因的表达,经过放射性免疫(RIA)和荧光定量PCR结果得知其中一个干扰载体在细胞水平的干扰效果十分明显,可达到50%以上。另外发现,在干扰生长抑素的表达后,BHK-21细胞的早期凋亡显著降低,细胞更多地进入S期。在体外干扰细胞的生长抑素表达是否影响生长抑素受体的变化目前未见报道,因此,我们检测了生长抑素基因五个受体表达量的变化。结果表明,生长抑素受体SSTR3的表达都下调。随后,应用RT-PCR和Western Blot方法检测与凋亡和周期相关的基因,发现Caspase-3、p21的表达水平都显著下调,而Bcl-2的表达水平则显著上调。这些结果表明,生长抑素可能通生长抑素受体2和3来调控Caspase-3与Bcl-2等基因的活动,从而促进BHK-21细胞的凋亡。选择干扰效果最佳的pshRNA-2质粒并优化转染方法后,成功转染SSCs,转染率达90%以上。3.转基因SSCs克隆的异种移植为了确认转染后SSCs干细胞的潜能,通过DBA染色鉴定证实异种移植小鼠睾丸曲细精管的基底膜上存在供体水牛精原细胞。为了探讨水牛精原干细胞移植到昆明小鼠后的增殖情况,首先对受体动物(昆明小鼠)注射不同剂量(30,35,40mg/kg体重)的白消安,以消除内源性精原细胞的干扰,依据体重、睾丸组织形态学和受体小鼠的睾酮激素水平综合评判消除效果,发现以40mg/kg体重的剂量为最优。之后将体外转染的水牛SSCs克隆(>58%纯度)打散重悬后,经显微注射到小鼠曲细精管内,在注射后1天、4天、1周、2周、3周、1个月和2个月,收集各实验组和对照组小鼠睾丸,应用扁豆凝集素(DBA)免疫组化和PCR方法检测供体精原细胞的存在,发现水牛睾丸细胞作为新鲜的供体,在受体鼠的睾丸曲细精管中可见形成圆形的细胞克隆,这表明精原干细胞在受体睾丸中成功增殖和克隆。但是2个月后,受体睾丸中克隆数减少,并且这些细胞表现为纤维组织细胞形态并失去了生精能力。以上结果说明,用白消安处理的昆明小鼠睾丸能够实现水牛精原干细胞的克隆和增殖,从而使其成为合适的动物模型进行评估水牛精原干细胞在受体小鼠中的增殖。水牛精原干细胞能够在受体小鼠睾丸中存活并增殖2个月。更多还原

【Abstract】 By studying the different conditions (including serum concentrations, feeder cells and several types of growth factors) on the proliferation of SSCs buffalo, we eventually establish in vitro culture system for proliferation of SSCs. By PB transposon vector, SSCs has been transfected with the SS gene fragment. After transplantation of genetically modified SSCs to the recipient’s testicles, we analysed the proliferation and differentiation of transgenic SSCs function on different time after transplatation. The study will provide new opportunities and pathways for clone and production of transgenic buffalo; provide a platform for research on other potential mechanisms of epigenetic biological characteristics of SSCs, directed differentiation and spermatogenesis; provide new tools for buffalo germplasm conservation by combination of cryopreserved SSCs; provide a theoretical reference for the study of other livestock and human SSCs.1. Buffalo spermatogonial stem cells in vitro culture and molecular markers explorationDevelopment of suitable selective marker for buffalo spermatogonial stem cells (SSCs), optimization of long term in-vitro culturing conditions and their pluripotent retention capacity in buffaloes can be of prime importance in selective genetic modifications of this species. In the present study, we identified CDH1as a specific marker for buffalo SSCs and revealed that it exists in two protein isoforms (large (135kDa) and small (90kDa) subunits) in the three-month-old cross-bred (Murrah×swamp,49chromosomes) buffalo testis. Furthermore, immunohistochemical analysis revealed that CDH1expression was present in spermatogonia but absent from somatic cells in testis. After7days of enrichment, expressions of CDH1were also detectable in in-vitro culture colonies (-53%enrichment efficiency by FACS). For long term culture of SSCs, proliferation studies with different factors showed that combination of20ng/mL GDNF,10ng/mL FGF2and1000U/mL LIF can significantly promote number of colonies (-2folds) and proliferation of buffalo SSCs (-3folds) compared to those of control or single treatment groups, furthermore, addition of these combination growth factors significantly up regulated the mRNA level of spermatogonial specific and pluripotency related markers (BCL6B, GFRA1, and POU5F1) while down regulated receptor tyrosine kinase (KIT). These findings indicate the identification of a new buffalo SSCs marker, furthermore, it may help in establishing culture that would help in genetic modifications of these buffaloes. 2. Transfection of BHK-21and SSCs colonies with somatostatin RNAi vectorSomatostatin (SS) play a major role in inhibiting the secretion of growth hormone (GH), so animal growth character can be promoted by inhibiting the secretion of SS. If we knock down SS gene out of SSCs, we can get long-term integration transgenic offspring through SSCs transplantion, and then furtherly get excellent individual in good growth condition. But previous studies in our laboratory have confirmed SSTR is not present in buffalo SSCs, so the moderating effects of need to be verified on other SS secreting cells. In this study, three PB delivered interference vector was designed and constructed for SS gene sequences, and lipo-mediated transfected into BHK-21cells (hamster kidney fibroblasts, identified by SS secreting cells). Result of radioimmunoassay (RIA) and fluorescence quantitative PCR (qPCR) showed that interference effects from one of the interfering carrier can reach more than50%at the cellular level. In addition, we found that the expression of somatostatin after RNAi, the early phase apoptotic of BHK-21cells significantly reduced, and showed more cells into S phase. The effects of Somatostatin in vitro interference on cell somatostatin receptor expression have not been reported, so we examined the changes in the expression of the five somatostatin receptors. The results showed that all somatostatin receptor expression was down regulated. Apoptosis and cell cycle-related genes were detected by RT-PCR and Western Blot, showing Caspase-3, p21and Bax expression levels were significantly reduced, while the expression of Bcl-2levels were significantly upregulated. These results suggest that somatostatin may regulate Caspase-3activity and Bcl-2/Bax genes through somatostatin receptor2and3to promote apoptosis in BHK-21cells. Through these results we have chosen the best interference pshRNA-2plasmid. After optimizing transfection methods coupled with SSCs in vitro conditions, we conducted successful SSCs transfection, which has laid a solid foundation for the production of transgenic animals.3. Transgenic SSCs colonies xenotranplantationFor confirmation of their stem cell potential, DBA-stained cells were identified in the basal membrane of seminiferous tubules of xenotransplanted mice testis. In order to examine the propagation of buffalo spermatogonial stem cells following transplantation into Kunming mice. For preparation of recipient animals, we also optimized the dosage of busulfan in Kunming mice for successful depletion of endogenous spermatogenesis. This was followed by microinjection of enriched donor buffalo germ cells (58%purified) into mouse seminiferous tubules. After treatment with different doses of busulfan (30,35,40 mg/kg b.w.), we found that a dose of40mg kg-1is optimal for the ablative treatment. This optimization was based on body weight, testicular histomorphology, and testosterone level of recipient mice. After transplantation, mouse testes were analyzed at day1d,4d,1wk,2wk,3wk,1mo and2mo after transplantation with Dolichos biflorus agglutinin (DBA) immunohistochemistry and PCR for the presence of donor germ cells. As fresh explants, buffalo testes cells were observed as small colonies of round cells within mouse seminiferous tubules, indicating successful proliferation and colonization of germ cells, however, at2months, the number of colonies decreased although, these cells appeared as fibrous tissues and devoid of spermatogenic capacity. In conclusion, busulfan-treated Kunming mouse testis are capable of colonizing the buffalo germ cells, thus making it suitable model for evaluation and further development of buffalo germ cells transplantation in mice. The buffalo spermatogonial stem cells are able to be alive and propagated for2months in mouse testis.更多还原