PLZF通过靶向抑制miR-146a促进CXCR4表达调控奶山羊精原干细胞增殖
发布时间：2018-07-20 17:08
【摘要】：精原干细胞(SSCs)的分化与自我更新决定了精子发生的起始,SSCs的增殖是维持大量精子产生的基础。SSCs的分化与自我更新是一个受到严密调控的过程,研究参与SSCs自我更新与增殖过程中的因子对研究精子发生具有重大的意义。近期研究表明早幼粒白血病锌指蛋白(PLZF),趋化因子受体4(CXCR4)以及miR-146a与SSCs的自我更新与增殖相关。本实验以PLZF-miR-146a-CXCR4为研究主线,以分离纯化的奶山羊精原干细胞(gSSCs)为实验材料,通过Western Blot,免疫荧光染色,qRT-PCR,单荧光素酶载体报告系统,双荧光素酶载体报告系统以及流式细胞术研究三者之间的互相作用关系以及对gSSCs增殖的调控作用。研究发现PLZF通过抑制miR-146a的转录来促进CXCR4的表达致使ERK1/2的磷酸化增强,最终促进gSSCs的自我更新和增殖。1.PLZF,miR-146a和CXCR4在雄性奶山羊睾丸中表达规律的研究通过qRT-PCR技术对3月龄,6月龄,9月龄,12月龄以及18月龄奶山羊睾丸进行了转录水平的检测,发现PLZF与CXCR4的转录趋势基本一致,但与miR-146a的转录趋势相反。通过免疫荧光染色技术对PLZF和CXCR4的定位以及CXCR4在3月龄,6月龄,9月龄和18月龄的睾丸组织的表达进行了研究,结果表明PLZF可与CXCR4共同定位于精原干细胞,CXCR4的蛋白检测与mRNA检测结果相近。2.PLZF,miR-146a和CXCR4之间相互作用关系的研究在gSSCs中超表达和干扰PLZF,通过qRT-PCR和Western Blot检测其对mi R-146a和CXCR4转录水平和蛋白水平的调控。发现PLZF的超表达可以促进CXCR4的表达,同时抑制miR-146a的转录,抑制PLZF的表达则会出现相反的结果。随后,通过单荧光素酶载体报告系统在293T细胞中检测PLZF与miR-146a启动子区域的互作关系,证明PLZF靶向miR-146a的启动子区抑制其转录。在gSSCs中转染mi R-146a模拟物、抑制物及无义物,通过qRT-PCR和Western Blot检测其CXCR4的表达变化,发现miR-146a会抑制CXCR4的表达,之后通过双荧光素酶载体报告系统检测miR-146a是否靶向调控CXCR4,结果证实miR-146a间接调控CXCR4的表达。3.PLZF,miR-146a和CXCR4对gSSCs自我更新和增殖的影响通过Western Blot检测PLZF,miR-146a和CXCR4对ERK1/2磷酸化水平的影响,结果表明PLZF和CXCR4表达量上升会促进ERK1/2的磷酸化;而降低PLZF的表达,则会降低ERK1/2的磷酸化。通过流式细胞周期检测技术研究对gSSCs增殖的影响,发现PLZF,CXCR4的表达量上升会促进gSSCs增殖。
[Abstract]:The differentiation and self-renewal of spermatogonial stem cells (SSCs) determine the initiation of spermatogenesis and the proliferation of SSCs is the basis of maintaining a large number of spermatozoa. The differentiation and self-renewal of SSCs is a tightly regulated process. The study of the factors involved in the process of self-renewal and proliferation of SSCs is of great significance to the study of spermatogenesis. Recent studies have shown that zinc finger protein (PLZF), chemokine receptor 4 (CXCR4) and miR-146a are associated with self-renewal and proliferation of SSCs in promyelocytic leukemia. In this experiment, PLZF-miR-146a-CXCR4 was used as the main line of study, and the purified dairy goat spermatogonial stem cells (gSSCs) were used as experimental materials. Western blot, immunofluorescence staining, qRT-PCRand single luciferase carrier report system were used. Double luciferase vector reporting system and flow cytometry were used to study the interaction and regulation of GSSCs proliferation. It was found that PLZF promoted the expression of CXCR4 by inhibiting the transcription of miR-146a, resulting in increased phosphorylation of ERK1 / 2. Finally promote the self-renewal and proliferation of gSSCs. 1. The expression of PLZFN miR-146a and CXCR4 in the testis of male dairy goats was studied. The transcription level of testis was detected by qRT-PCR at the age of 3 months, 6 months, 9 months, 12 months, and 18 months, respectively. It was found that the transcription trend of PLZF and CXCR4 was basically the same, but contrary to that of miR-146a. The localization of PLZF and CXCR4 and the expression of CXCR4 in testis at the age of 3 months, 6 months, 9 months and 18 months were studied by immunofluorescence staining. The results showed that PLZF could co-locate with CXCR4 in spermatogonial stem cell line CXCR4. 2. The interaction between PLZF and CXCR4 was studied in gSSCs by overexpression and interference with PLZF4. The results of qRT-PCR and Western blot were used to detect the expression of MIR-146a and CXCR4 in gSSCs. The regulation of transcription and protein levels. It was found that the overexpression of PLZF could promote the expression of CXCR4, inhibit the transcription of miR-146a, and inhibit the expression of PLZF. Then, the interaction between PLZF and miR-146a promoter region was detected in 293T cells by a single luciferase vector reporting system, which proved that PLZF-targeted promoter region of miR-146a inhibited its transcription. The expression of CXCR4 was detected by qRT-PCR and Western Blot. It was found that miR-146a could inhibit the expression of CXCR4. The effect of miR-146a on the expression of CXCR4. 3. The effects of miR-146a and CXCR4 on the self-renewal and proliferation of gSSCs were detected by Western Blot. The results showed that the increased expression of PLZF and CXCR4 increased the phosphorylation of ERK1 / 2, but decreased the expression of PLZF, decreased the phosphorylation of ERK1 / 2. The effect of flow cytometry on the proliferation of gSSCs was studied. It was found that the increased expression of PLZFG CXCR4 could promote the proliferation of gSSCs.
【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】：S814
本文编号：2134185
[Abstract]:The differentiation and self-renewal of spermatogonial stem cells (SSCs) determine the initiation of spermatogenesis and the proliferation of SSCs is the basis of maintaining a large number of spermatozoa. The differentiation and self-renewal of SSCs is a tightly regulated process. The study of the factors involved in the process of self-renewal and proliferation of SSCs is of great significance to the study of spermatogenesis. Recent studies have shown that zinc finger protein (PLZF), chemokine receptor 4 (CXCR4) and miR-146a are associated with self-renewal and proliferation of SSCs in promyelocytic leukemia. In this experiment, PLZF-miR-146a-CXCR4 was used as the main line of study, and the purified dairy goat spermatogonial stem cells (gSSCs) were used as experimental materials. Western blot, immunofluorescence staining, qRT-PCRand single luciferase carrier report system were used. Double luciferase vector reporting system and flow cytometry were used to study the interaction and regulation of GSSCs proliferation. It was found that PLZF promoted the expression of CXCR4 by inhibiting the transcription of miR-146a, resulting in increased phosphorylation of ERK1 / 2. Finally promote the self-renewal and proliferation of gSSCs. 1. The expression of PLZFN miR-146a and CXCR4 in the testis of male dairy goats was studied. The transcription level of testis was detected by qRT-PCR at the age of 3 months, 6 months, 9 months, 12 months, and 18 months, respectively. It was found that the transcription trend of PLZF and CXCR4 was basically the same, but contrary to that of miR-146a. The localization of PLZF and CXCR4 and the expression of CXCR4 in testis at the age of 3 months, 6 months, 9 months and 18 months were studied by immunofluorescence staining. The results showed that PLZF could co-locate with CXCR4 in spermatogonial stem cell line CXCR4. 2. The interaction between PLZF and CXCR4 was studied in gSSCs by overexpression and interference with PLZF4. The results of qRT-PCR and Western blot were used to detect the expression of MIR-146a and CXCR4 in gSSCs. The regulation of transcription and protein levels. It was found that the overexpression of PLZF could promote the expression of CXCR4, inhibit the transcription of miR-146a, and inhibit the expression of PLZF. Then, the interaction between PLZF and miR-146a promoter region was detected in 293T cells by a single luciferase vector reporting system, which proved that PLZF-targeted promoter region of miR-146a inhibited its transcription. The expression of CXCR4 was detected by qRT-PCR and Western Blot. It was found that miR-146a could inhibit the expression of CXCR4. The effect of miR-146a on the expression of CXCR4. 3. The effects of miR-146a and CXCR4 on the self-renewal and proliferation of gSSCs were detected by Western Blot. The results showed that the increased expression of PLZF and CXCR4 increased the phosphorylation of ERK1 / 2, but decreased the expression of PLZF, decreased the phosphorylation of ERK1 / 2. The effect of flow cytometry on the proliferation of gSSCs was studied. It was found that the increased expression of PLZFG CXCR4 could promote the proliferation of gSSCs.
【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】：S814
【参考文献】
相关期刊论文 前1条
1 李冬梅;秦晓东;;精原干细胞自我更新与分化的调控[J];中华男科学杂志;2013年11期
，本文编号：2134185
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