候选基因LBC在鸡精原干细胞形成过程中的作用研究

发布时间：2019-02-28 11:04

【摘要】：精原干细胞(Spermatogonial stem cells,SSCs)是雄性成体干细胞中唯一一种能够将遗传物质传递给下一代的干细胞。SSCs作为生殖干细胞,是精子发生的基础,在研究精子发生机制、阐述男性不育病因和治疗男性不育症等领域有着不可替代的作用。精原干细胞的形成受到相关基因的调控,但是其具体分子机制现在仍然未知。本研究针对精原干细胞上特异表达候选基因LBC进行功能验证,以如皋黄鸡作为试验动物,在体外和体内通过敲除和过表达LBC基因的方法,研究LBC基因在鸡精原干细胞形成过程中的作用,为阐明精原干细胞发生及分化机制提供参考。本文主要研究如下:1.如皋黄鸡LBC基因的克隆根据NCBI提供的LBC基因序列,设计引物克隆LBC基因的CDS区,并将其连接至pMD19-T载体中进行测序。测序结果表明pMD19-T-LBC构建正确,与公布的原鸡LBC基因CDS区同源性达99.43%,编码氨基酸有两个发生改变,成功克隆出如皋黄鸡中LBC基因,可用于后续研究。2.LBC基因多克隆抗体制备及亚细胞定位研究制备LBC基因的抗原多肽,注射小鼠进行免疫,收集血清后通过间接免疫荧光技术(IFA)进行抗体效价检测;构建真核表达载体pEGFP-N1-LBC,转染DF-1细胞48h后于荧光倒置显微镜下观察转染结果,并利用RT-PCR和间接免疫荧光试验进一步鉴定LBC-eGFPmRNA和蛋白水平的表达状况。结果发现,RT-PCR能检测到融合蛋白转录水平的表达,荧光显微镜观察和IFA检测结果都显示LBC-eGFP融合蛋白在细胞核和细胞质中都有表达,与生物信息学预测结果相一致。3.LBC基因对精原干细胞形成的影响构建LBC敲除载体CRISPR/Cas9-LBC和过表达载体pcDNA3.0-LBC,转染DF-1细胞后利用测序、T7E1酶切和Western Blot技术验证载体活性;分别转染ESC,在RA诱导培养基中培养,采用形态学观察、免疫化学检测和QRT-PCR等方法在体外检测LBC缺失与过表达对ESC向SSC分化的影响,同时鸡胚注射CRISPR/Cas9-LBC和pcDNA3.O-LBC载体,采用石蜡切片和流式分析的方法在体内检测LBC缺失与过表达对PGCs和SSCs形成的影响。结果表明CRISPR/Cas9-LBC和pcDNA3.0-LBC载体均构建成功且具有活性;体外实验中,与RA诱导组相比,LBC过表达显著地促进了ESC向SSC的分化,且integrin α6、integrinβ1等SSCs标记基因的表达量也有显著的增加,在第10天,相对于诱导组中integrinα6的相对表达量(1.96),在过表达组中达到2.34;相反的,LBC敲除,ESCs向SSCs的分化受到一定程度的抑制,第6天克隆形成之后,不能形成精原样细胞,且全能性标记基因Sox2的表达量的降低受到抑制,相对于诱导组中相对表达量(0.96),在敲除组中达到1.03,integrinα6、integrinβ1表达量也相对减少;体内试验中,与正常发育的鸡胚相比,LBC过表达显著的促进了PGC和SSC细胞的生成,而LBC敲除相对减少了 PGC和SSC细胞的生成。
[Abstract]:Spermatogonial stem cells (Spermatogonial stem cells,SSCs) are the only male adult stem cells that can transfer genetic material to the next generation of stem cells as reproductive stem cells, which is the basis of spermatogenesis, in the study of the mechanism of spermatogenesis. Expounding the causes of male infertility and the treatment of male infertility and other fields has an irreplaceable role. The formation of spermatogonial stem cells is regulated by related genes, but the molecular mechanism of spermatogonial stem cells is still unknown. In this study, the specific expression of candidate gene LBC on spermatogonial stem cells was verified by knockout and over-expression of LBC gene in vitro and in vivo, using Rugao yellow chicken as experimental animal. To study the role of LBC gene in the formation of chicken spermatogonial stem cells and to provide a reference for elucidating the mechanism of spermatogonial stem cell development and differentiation. The main research in this paper is as follows: 1. The cloning of LBC gene of Rugao yellow chicken was based on the sequence of LBC gene provided by NCBI. The primers were designed to clone the CDS region of LBC gene and cloned into pMD19-T vector for sequencing. The sequencing results showed that the pMD19-T-LBC was constructed correctly and had 99.43% homology with the published CDS region of the original chicken LBC gene. Two amino acids were changed and the LBC gene in Rugao yellow chicken was cloned successfully. 2. Preparation of polyclonal antibody against LBC gene and subcellular localization study to prepare antigen polypeptide of LBC gene, immunize mice, collect serum and detect antibody titer by indirect immunofluorescence technique (IFA); The eukaryotic expression vector pEGFP-N1-LBC, was constructed and transfected into DF-1 cells for 48 hours. The transfection results were observed under fluorescence inversion microscope, and the expression of LBC-eGFPmRNA and protein were further identified by RT-PCR and indirect immunofluorescence assay. The results showed that RT-PCR could detect the expression of fusion protein at transcriptional level. The results of fluorescence microscopy and IFA showed that LBC-eGFP fusion protein was expressed in both nucleus and cytoplasm. 3. The effect of LBC gene on the formation of spermatogonial stem cells is consistent with the predicted results of bioinformatics. 3. LBC knockout vector CRISPR/Cas9-LBC and over-expression vector pcDNA3.0-LBC, were transfected into DF-1 cells and sequenced. The activity of the vector was verified by T7E1 digestion and Western Blot. The transfected ESC, were cultured in RA-induced medium. The effects of LBC deletion and over-expression on the differentiation of ESC into SSC were detected by morphological observation, immunochemical assay and QRT-PCR in vitro. At the same time, CRISPR/Cas9-LBC and pcDNA3.O-LBC vectors were injected into chicken embryos. Paraffin sections and flow cytometry were used to detect the effect of LBC deletion and overexpression on the formation of PGCs and SSCs in vivo. The results showed that both CRISPR/Cas9-LBC and pcDNA3.0-LBC vectors were constructed successfully and had activity. In vitro, compared with RA-induced group, the over-expression of LBC significantly promoted the differentiation of ESC into SSC, and the expression of integrin 伪 6, integrin 尾 1 and other SSCs-labeled genes also increased significantly on the 10th day, and the expression of integrin 伪 6, integrin 尾 1 and other SSCs-labeled genes were significantly increased on the 10th day. The relative expression of integrin 伪 6 in the overexpressing group was 2.34 compared with that in the induced group (1.96). On the contrary, LBC knockout and ESCs differentiation into SSCs were inhibited to a certain extent. After 6 days of cloning, spermatogonia-like cells could not be formed, and the decrease of the expression of totipotency marker gene Sox2 was inhibited. Compared with the induction group (0.96), the expression of integrin 伪 6 and integrin 尾 1 in the knockout group was 1.03, and the expression level of integrin 尾 1 was also decreased in the knockout group. In vivo, compared with normal chicken embryos, over-expression of LBC significantly promoted the production of PGC and SSC cells, while LBC knockout decreased the production of PGC and SSC cells.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S831

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