FHL2在小鼠卵巢中的表达和定位及对卵泡颗粒细胞的调控作用研究

发布时间：2018-02-28 05:37

本文关键词： FHL2 表达定位颗粒细胞细胞凋亡与周期类固醇激素　出处：《华中农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：FHL2(four and a half LIM domains protein 2)属于LIM-Only蛋白家族中的一员,可以与不同的蛋白因子互作,作为转录共调节子参与调控基因表达、细胞的增殖分化、细胞迁移和凋亡等诸多生理调节过程。FHL2在卵巢组织中表达,诱导性腺向卵巢分化,并参与调控抑制素基因的表达。但FHL2在小鼠卵巢中的表达定位模式,及对卵泡颗粒细胞的增殖、凋亡及类固醇激素分泌调控作用尚不清楚。为了阐明FHL2在卵巢中的表达定位规律及对卵泡颗粒细胞的调控作用,本研究从基因表达、组织表达、亚细胞定位、功能研究等几方面研究了FHL2对小鼠卵泡颗粒细胞的调控作用。结果如下:1、FHL2在小鼠卵巢中的表达与定位(1)在不同日龄(7d、14d、21d、28d)的小鼠卵巢中,应用RT-PCR的方法证明了FHL2在小鼠卵巢中表达,且在不同日龄小鼠卵巢中表达量没有差异;(2)免疫组化结果表明,FHL2在小鼠卵巢中主要定位于卵泡颗粒细胞和基质细胞中,在各个时期卵泡中表达量差异不大。(3)细胞免疫荧光结果表明,FHL2在小鼠卵泡颗粒细胞亚细胞定位于细胞核。2、FHL2干扰对卵泡颗粒细胞生长和凋亡及分泌功能的影响(1)卵泡颗粒细胞转染干扰质粒72h后,应用流式细胞仪检测细胞凋亡情况,发现转染组中正常细胞比例显著高于对照组(91.52±0.78 vs 85.78±0.14,P0.05),细胞晚期凋亡率也显著低于对照组(4.18±0.16 vs 10.18±1.81,P0.05),凋亡相关蛋白Bcl-2显著上调,Bax和Caspase-3表达水平显著下调,说明干扰后颗粒细胞的存活能力提高。(2)利用流式细胞术分析细胞周期,发现FHL2干扰组处于S期颗粒细胞比例显著多于对照组(36.83±0.40 vs 24.02±0.33,P0.05),处于G1期的细胞比例显著低于对照组(48.31±1.02 vs 62.96±0.72,P0.05),周期相关蛋白Cyclin E和Cyclin B表达水平显著提高,P21表达水平显著下调,干扰后影响了颗粒细胞的有丝分裂过程;(3)应用MTT检测转染干扰质粒后的小鼠卵巢颗粒细胞增殖情况,结果表明,相对于对照组,FHL2干扰组细胞活力显著增加(53.60%vs 15.43%,48h,P0.05;109.86%vs 33.33%,72h,P0.05),干扰后显著促进了颗粒细胞的增殖;(4)应用ELISA方法检测转染后颗粒细胞类固醇分泌水平,结果表明,相对于对照组,FHL2干扰组雌二醇(8.19±0.19 vs 2.53±0.41,ng/L,P0.01)和孕酮水平(13.96±1.07 vs 1.64±0.08,ng/m L,P0.01)显著升高。本课题研究了FHL2在小鼠卵巢组织及卵泡颗粒细胞亚细胞中的表达定位,发现FHL2在不同日龄的小鼠卵巢中表达量没有差异,且主要定位于卵泡颗粒细胞的细胞核中,在不同发育阶段卵泡中表达量差异不大。进一步研究FHL2对颗粒细胞调控作用,结果表明干扰FHL2后能够抑制卵泡颗粒细胞凋亡,促进细胞增殖和分泌类固醇激素,并对细胞的分裂周期有明显的影响。这些结果说明FHL2对小鼠卵泡发育有重要的调控作用,为进一步研究FHL2对卵泡发育调控机制和信号通路研究奠定了基础。
[Abstract]:FHL2(four and a half LIM domains protein 2 (FHL2(four and a half LIM domains protein 2) is a member of the LIM-Only protein family, which can interact with different protein factors and play a role in the regulation of gene expression and cell proliferation and differentiation as a transcriptional comodulator. Many physiological regulation processes such as cell migration and apoptosis. FHL2 is expressed in ovarian tissue, induces gonad differentiation into ovary, and participates in regulating the expression of inhibin gene. In order to elucidate the expression and localization of FHL2 in ovary and the regulation of follicular granulosa cells, we studied the gene expression and tissue expression in order to elucidate the expression and localization of FHL2 in follicular granulosa cells. The regulatory effects of FHL2 on mouse follicular granulosa cells were studied in subcellular localization and functional study. The results were as follows: 1) the expression and localization of FHL2 in mouse ovaries were as follows: (1) in the ovaries of mice with different ages of 7 days, 14 days, 21 days and 28 days, the expression and localization of FHL2 in mouse ovaries were as follows. The expression of FHL2 in mouse ovary was proved by RT-PCR method, and there was no difference in the expression of FHL2 in ovary of different day old mice. The immunohistochemical results showed that FHL2 was mainly localized in follicular granulosa cells and stromal cells in mouse ovary. The results of immunofluorescence showed that FHL2 was located in the nucleus of mouse follicular granulosa cells and the effect of FHL2 interference on the growth, apoptosis and secretory function of follicular granulosa cells. Granulosa cells were transfected with interference plasmids for 72 hours. Flow cytometry was used to detect apoptosis. It was found that the percentage of normal cells in the transfected group was significantly higher than that in the control group (91.52 卤0.78 vs 85.78 卤0.14 P0.05), and the late apoptosis rate was significantly lower than that in the control group (4.18 卤0.16 vs 10.18 卤1.81 P0.05G). The apoptosis-related protein Bcl-2 significantly up-regulated the expression of Bax and Caspase-3. The results showed that the survival ability of granulosa cells increased after interference. 2) flow cytometry was used to analyze the cell cycle. It was found that the percentage of granulosa cells in S phase in FHL2 interference group was significantly higher than that in control group (36.83 卤0.40 vs 24.02 卤0.33 P0.05G), and the percentage of cells in G1 phase was significantly lower than that in control group (48.31 卤1.02 vs 62.96 卤0.72). The expression level of cyclin Cyclin E and Cyclin B increased significantly down-regulation of P21 expression. The mitotic process of granulosa cells was affected by interference. MTT was used to detect the proliferation of granulosa cells after transfection of interfering plasmids. Compared with the control group, the cell viability of FHL2 interference group was significantly increased by 53.60 vs 15.4348 h, P0.05 109.86 vs 33.3372 h P0.05, and significantly promoted the proliferation of granulosa cells. The ELISA assay was used to detect the steroid secretion level of transfected granulosa cells. Compared with the control group, the levels of estradiol and progesterone were significantly increased in FHL2 interference group (8.19 卤0.19 vs 2.53 卤0.41 ng 路L ~ (-1)) and progesterone level 13.96 卤1.07 vs 1.64 卤0.08 ng / m ~ (-1) P ~ (0.01). The expression of FHL2 in mouse ovarian tissue and follicular granulosa cells was studied in this study. It was found that there was no difference in the expression of FHL2 in the ovary of different day old mice, and it was mainly located in the nucleus of follicular granulosa cells, but there was no difference in the expression of FHL2 in follicles at different stages of development. Further study on the regulatory effect of FHL2 on granulosa cells was carried out. The results showed that interfering with FHL2 could inhibit the apoptosis of follicular granulosa cells, promote cell proliferation and secretion of steroid hormones, and have a significant effect on cell division cycle. These results suggest that FHL2 plays an important role in the regulation of follicular development in mice. It lays a foundation for further study on the regulation mechanism and signal pathway of follicle development by FHL2.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S814.1

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