本文關(guān)鍵詞： FHL2 表達(dá)定位 顆粒細(xì)胞 細(xì)胞凋亡與周期 類固醇激素　出處：《華中農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：FHL2(four and a half LIM domains protein 2)屬于LIM-Only蛋白家族中的一員,可以與不同的蛋白因子互作,作為轉(zhuǎn)錄共調(diào)節(jié)子參與調(diào)控基因表達(dá)、細(xì)胞的增殖分化、細(xì)胞遷移和凋亡等諸多生理調(diào)節(jié)過程。FHL2在卵巢組織中表達(dá),誘導(dǎo)性腺向卵巢分化,并參與調(diào)控抑制素基因的表達(dá)。但FHL2在小鼠卵巢中的表達(dá)定位模式,及對卵泡顆粒細(xì)胞的增殖、凋亡及類固醇激素分泌調(diào)控作用尚不清楚。為了闡明FHL2在卵巢中的表達(dá)定位規(guī)律及對卵泡顆粒細(xì)胞的調(diào)控作用,本研究從基因表達(dá)、組織表達(dá)、亞細(xì)胞定位、功能研究等幾方面研究了FHL2對小鼠卵泡顆粒細(xì)胞的調(diào)控作用。結(jié)果如下:1、FHL2在小鼠卵巢中的表達(dá)與定位(1)在不同日齡(7d、14d、21d、28d)的小鼠卵巢中,應(yīng)用RT-PCR的方法證明了FHL2在小鼠卵巢中表達(dá),且在不同日齡小鼠卵巢中表達(dá)量沒有差異;(2)免疫組化結(jié)果表明,FHL2在小鼠卵巢中主要定位于卵泡顆粒細(xì)胞和基質(zhì)細(xì)胞中,在各個時期卵泡中表達(dá)量差異不大。(3)細(xì)胞免疫熒光結(jié)果表明,FHL2在小鼠卵泡顆粒細(xì)胞亞細(xì)胞定位于細(xì)胞核。2、FHL2干擾對卵泡顆粒細(xì)胞生長和凋亡及分泌功能的影響(1)卵泡顆粒細(xì)胞轉(zhuǎn)染干擾質(zhì)粒72h后,應(yīng)用流式細(xì)胞儀檢測細(xì)胞凋亡情況,發(fā)現(xiàn)轉(zhuǎn)染組中正常細(xì)胞比例顯著高于對照組(91.52±0.78 vs 85.78±0.14,P0.05),細(xì)胞晚期凋亡率也顯著低于對照組(4.18±0.16 vs 10.18±1.81,P0.05),凋亡相關(guān)蛋白Bcl-2顯著上調(diào),Bax和Caspase-3表達(dá)水平顯著下調(diào),說明干擾后顆粒細(xì)胞的存活能力提高。(2)利用流式細(xì)胞術(shù)分析細(xì)胞周期,發(fā)現(xiàn)FHL2干擾組處于S期顆粒細(xì)胞比例顯著多于對照組(36.83±0.40 vs 24.02±0.33,P0.05),處于G1期的細(xì)胞比例顯著低于對照組(48.31±1.02 vs 62.96±0.72,P0.05),周期相關(guān)蛋白Cyclin E和Cyclin B表達(dá)水平顯著提高,P21表達(dá)水平顯著下調(diào),干擾后影響了顆粒細(xì)胞的有絲分裂過程;(3)應(yīng)用MTT檢測轉(zhuǎn)染干擾質(zhì)粒后的小鼠卵巢顆粒細(xì)胞增殖情況,結(jié)果表明,相對于對照組,FHL2干擾組細(xì)胞活力顯著增加(53.60%vs 15.43%,48h,P0.05;109.86%vs 33.33%,72h,P0.05),干擾后顯著促進(jìn)了顆粒細(xì)胞的增殖;(4)應(yīng)用ELISA方法檢測轉(zhuǎn)染后顆粒細(xì)胞類固醇分泌水平,結(jié)果表明,相對于對照組,FHL2干擾組雌二醇(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)顯著升高。本課題研究了FHL2在小鼠卵巢組織及卵泡顆粒細(xì)胞亞細(xì)胞中的表達(dá)定位,發(fā)現(xiàn)FHL2在不同日齡的小鼠卵巢中表達(dá)量沒有差異,且主要定位于卵泡顆粒細(xì)胞的細(xì)胞核中,在不同發(fā)育階段卵泡中表達(dá)量差異不大。進(jìn)一步研究FHL2對顆粒細(xì)胞調(diào)控作用,結(jié)果表明干擾FHL2后能夠抑制卵泡顆粒細(xì)胞凋亡,促進(jìn)細(xì)胞增殖和分泌類固醇激素,并對細(xì)胞的分裂周期有明顯的影響。這些結(jié)果說明FHL2對小鼠卵泡發(fā)育有重要的調(diào)控作用,為進(jìn)一步研究FHL2對卵泡發(fā)育調(diào)控機(jī)制和信號通路研究奠定了基礎(chǔ)。
[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 鹵0.78 vs 85.78 鹵0.14 P0.05), and the late apoptosis rate was significantly lower than that in the control group (4.18 鹵0.16 vs 10.18 鹵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 鹵0.40 vs 24.02 鹵0.33 P0.05G), and the percentage of cells in G1 phase was significantly lower than that in control group (48.31 鹵1.02 vs 62.96 鹵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 鹵0.19 vs 2.53 鹵0.41 ng 路L ~ (-1)) and progesterone level 13.96 鹵1.07 vs 1.64 鹵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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S814.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 丁麗華,焦園園,劉雨飛,方言,周蕾,王曉輝,黃翠芬,葉棋濃;FHL2與IGFBP-7存在直接相互作用[J];軍事醫(yī)學(xué)科學(xué)院院刊;2005年04期

2 熊志紅;孫啟鴻;葉棋濃;;FHL2蛋白的研究進(jìn)展[J];軍事醫(yī)學(xué)科學(xué)院院刊;2007年04期

3 譚曉虹;徐恒;韋長元;曹驥;崔凌飛;劉斐;;RNA干擾p21對肝癌細(xì)胞SMMC-7721增殖的影響[J];世界華人消化雜志;2011年08期

，

本文編號：1545984