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PLZF对奶山羊雄性生殖干细胞生物学特性的影响

发布时间:2019-01-17 09:57
【摘要】:雄性生殖干细胞(male Germline Stem Cells,mGSCs)是定位于雄性动物睾丸中的曲细精管基底膜上的成体干细胞。mGSCs不仅能够通过自我更新来维持自身群体数量的稳定,还可以通过定向分化产生精子,是唯一一种能够通过自然生殖过程将雄性动物的遗传信息传递到下一代的成体干细胞。PLZF是一种高度保守的多功能转录调控因子,其表达具有严格的组织特异性和时序特异性,参与机体的发育、分化等多种重要的生物学过程。近年来,PLZF作为非常重要的转录因子,已引起人们的广泛关注。PLZF表达于未分化的mGSCs,对其多能性的维持、自我更新和分化的平衡以及雄性个体的生育能力维持等具有重要的调控作用,是被广泛认可的mGSCs的标志基因。目前,虽然对PLZF调控mGSCs自我更新和分化的作用及作用机制的探索已经取得了一些进展,但这些研究都是在小鼠等模式动物中取得的。PLZF对山羊等家畜大动物mGSCs的作用及作用机制却知之甚少。本研究拟在已有基础上,探索PLZF对奶山羊mGSCs生物学特性的的影响,以进一步了解其对精子发生过程的调节机制。本实验通过构建PLZF超表达和干扰慢病毒载体,转导奶山羊mGSCs,并筛选得到了稳定超表达和干扰PLZF的奶山羊mGSCs细胞株。并通过免疫荧光染色,qPCR,Western blot,EdU染色,流式细胞检测等技术手段检测了PLZF对奶山羊mGSCs多能性、自我更新、增殖、分化和凋亡等生物学特性的影响。1.PLZF超表达载体及干扰载体的构建和稳转细胞株的筛选我们构建得到了PLZF慢病毒超表达载体pPLZF-CDH和PLZF慢病毒干扰载体pshPLZF。然后将慢病毒载体包装病毒并转导奶山羊mGSC,通过筛选、鉴定得到了超表达PLZF的稳转细胞株mGSCs-oePLZF及其对照细胞株mGSCs-pCDH,干扰PLZF的稳转细胞株mGSCs-pshPLZF及其对照细胞株mGSCs-pSIH。2.PLZF促进奶山羊mGSCs维持其自我更新和多能性并抑制其分化通过免疫荧光染色、qPCR和Western blot检测,研究发现GFRα1、Vasa、CD49f、CD90、Oct4、Etv5等与mGSCs自我更新和多能性相关的基因表达显著上调,而mGSCs的分化标志基因c-Kit表达水平显著下调。另外我们通过qPCR检测发现Tet1表达水平显著下调,同时Dnmt3a表达水平显著上调,说明PLZF可能促进奶山羊mGSCs的DNA甲基化修饰。3.PLZF抑制奶山羊mGSCs增殖,促进其凋亡我们通过测定细胞生长曲线、CCK8检测、EdU染色、流式细胞周期检测、免疫荧光染色、qPCR、Western blot等实验方法,发现超表达PLZF的奶山羊mGSCs细胞增殖速度变慢、细胞周期在G0/G1期发生阻滞,细胞凋亡相关基因表达上调。通过细胞移植实验发现,移植PLZF超表达稳转细胞的白消安模型小鼠睾丸要明显比对侧移植对照细胞的睾丸小,且曲细精管内形成的细胞克隆也明显比对照组差;相应的,移植PLZF干扰稳转细胞的睾丸明显比对照组大,曲细精管内形成的细胞克隆也比对照组好。
[Abstract]:Male reproductive stem cells (male Germline Stem Cells,mGSCs) are adult stem cells located on the basal membrane of the seminiferous tubules located in the testicles of male animals. MGSCs can not only maintain the stability of its population through self-renewal. It is the only adult stem cell that can transmit genetic information from male animals to the next generation through natural reproduction. PLZF is a highly conserved multifunctional transcriptional regulator. Its expression has strict tissue specificity and timing specificity, and participates in many important biological processes, such as development and differentiation. In recent years, PLZF, as a very important transcription factor, has attracted much attention. PLZF is expressed in undifferentiated mGSCs, to maintain its pluripotency. The balance of self-renewal and differentiation and the maintenance of male fertility play an important role in the regulation of mGSCs, which is widely recognized as a marker gene. At present, although some progress has been made in exploring the role and mechanism of PLZF in regulating the self-renewal and differentiation of mGSCs, However, these studies were obtained in mice and other model animals. The effect and mechanism of PLZF on mGSCs in large domestic animals such as goats are not well understood. This study is to explore the effect of PLZF on the biological characteristics of mGSCs in dairy goats on the basis of previous studies in order to further understand its regulatory mechanism on spermatogenesis. In this study, we constructed PLZF overexpression vector and interfered lentivirus vector, then transduced mGSCs, into dairy goat mGSCs cell line which was stable and interfered with PLZF. Immunofluorescence staining, qPCR,Western blot,EdU staining and flow cytometry were used to detect the pluripotency, self-renewal and proliferation of PLZF to dairy goat mGSCs. Effects of biological characteristics such as differentiation and apoptosis. Construction of 1.PLZF superexpression vector and interference vector and screening of stable cell line we constructed PLZF lentivirus superexpression vector pPLZF-CDH and PLZF lentivirus interference vector pshPLZF. Then the lentivirus vector was packaged and transduced into dairy goat mGSC,. The stable cell line mGSCs-oePLZF and the control cell line mGSCs-pCDH, were identified by screening. The stable transformed cell line mGSCs-pshPLZF which interferes with PLZF and its control cell line mGSCs-pSIH.2.PLZF promote the maintenance of self-renewal and pluripotency and inhibit the differentiation of dairy goat mGSCs by immunofluorescence staining, qPCR and Western blot detection. Oct4,Etv5 and other genes related to mGSCs self-renewal and pluripotency were significantly up-regulated, while mGSCs differentiation marker gene c-Kit expression was significantly down-regulated. In addition, we found that the expression of Tet1 was significantly down-regulated and the expression of Dnmt3a was up-regulated by qPCR assay, which suggested that PLZF might promote DNA methylation of mGSCs in dairy goats. 3.PLZF inhibited mGSCs proliferation of dairy goats. By measuring cell growth curve, CCK8 detection, EdU staining, flow cytometry, immunofluorescence staining, qPCR,Western blot and other experimental methods, we found that the proliferation rate of mGSCs cells with overexpression of PLZF became slower. The cell cycle was blocked at G0/G1 stage and the expression of apoptosis-related genes was up-regulated. The results of cell transplantation showed that the testis of the mice transplanted with stable PLZF cells were smaller than those of the contralateral control cells, and the cell clones formed in the seminiferous tubules were significantly worse than those in the control group. Accordingly, the testis of the transplanted PLZF interfering stable cells were significantly larger than that of the control group, and the cell clones formed in the seminiferous tubule were also better than those in the control group.
【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】:2017
【分类号】:S827

【参考文献】

相关期刊论文 前2条

1 翟源心;郑丽明;华进联;;E-cadherin对奶山羊雄性生殖干细胞自我更新与增殖的影响[J];农业生物技术学报;2016年09期

2 李明昭;刘维帅;华进联;;哺乳动物精子发生中减数分裂的起始[J];中国生物化学与分子生物学报;2013年10期



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