本文選題：MicroRNA + 卵泡發(fā)育��； 參考：《南華大學》2014年博士論文

【摘要】：國家衛(wèi)生和計劃生育委員會最新數(shù)據(jù)表明,2013年我國不孕癥發(fā)病率7%~10%,并有逐年上升的趨勢。在不孕癥的諸多病因中,廣泛排卵障礙性不孕是女性不孕癥常見的類型,約占25%~35%[1-3]。卵泡發(fā)育成熟障礙可引起不排卵,進而導致不孕,研究卵泡發(fā)育成熟障礙的調(diào)控機制是目前生殖領域的熱點。卵泡發(fā)育是一個極其復雜的過程,包括不同時期卵泡形態(tài)和功能的變化,受內(nèi)分泌、局部因子、基因和微小RNA(miRNA)等多因素調(diào)控。對卵泡發(fā)育調(diào)控機制已經(jīng)取得一些進展,但還有很多問題亟待解決。本研究用miRNA基因芯片篩選在模擬人體卵巢內(nèi)環(huán)境的條件下,不同發(fā)育時期的人卵母細胞差異表達的mi RNA,選取特異性高表達miRNA作為研究靶標,用生物信息學方法分析靶標miRNA的靶基因;通過研究靶標miRNA對靶基因表達及功能的影響,探討靶標miRNA調(diào)控卵細胞發(fā)育成熟的分子機制,尋找調(diào)控卵細胞發(fā)育成熟的藥物靶點,為防治不孕不育提供提供新的實驗依據(jù)。第一部分不同發(fā)育期人卵母細胞miRNA的差異表達目的:篩選在模擬人體卵巢內(nèi)環(huán)境的條件下,不同發(fā)育時期的人卵母細胞差異表達的miRNA,選取特異性高表達miRNA作為研究靶標,分析靶標miRNA的靶基因。方法:收集人GV期、MI期卵母細胞,用含胰島素樣生長因子-1(IGF-1)的培養(yǎng)液培養(yǎng),以模擬人卵巢內(nèi)環(huán)境,用miRNA微陣列技術(shù)篩選不同發(fā)育時期的卵母細胞差異性表達的miRNAs;用生物信息學技術(shù)預測其靶基因。結(jié)果:用含IGF-1的培養(yǎng)液培養(yǎng),GV期卵母細胞183個miRNA表達上調(diào),117個mi RNA表達下調(diào);MI期卵母細胞145個miRNA表達上調(diào),200個miRNA表達下調(diào)。其中,miR-133b在MI期卵母細胞的表達上調(diào)32倍,在GV期卵母細胞miR-133b表達無差異。靶基因分析軟件顯示miR-133b的一個靶基因是TAGLN2;miR-133b的結(jié)合位點在TAGLN2 3’UTR第215-250位核苷酸。小結(jié):在模擬人體卵巢內(nèi)環(huán)境的條件下,GV期卵母細胞183個mi RNA表達上調(diào),117個miRNA表達下調(diào),MI期卵母細胞145個miRNA表達上調(diào),200個miRNA表達下調(diào)。mi R-133b是MI期卵母細胞特異性高表達的miRNA,其靶基因可能是TAGLN2。第二部分mi R-133b與TAGLN2基因的相互作用目的:分析mi R-133b與轉(zhuǎn)膠蛋白2(TAGLN2)的相互作用,確證TAGLN2是mi R-133b的靶基因。方法:用基因重組技術(shù)構(gòu)建TAGLN2-3’UTR及其突變表達載體(分別命名為psi CHECK-TAGLN2-3’UTR和psi CHECK-TAGLN2-3’UTR-m),用mi R-133b分別與psi CHECK或psi CHECK-TAGLN2-3’UTR或psi CHECK-TAGLN2-3’UTR-m共轉(zhuǎn)染HEK 293T,用雙熒光素酶報告基因檢測系統(tǒng)分析mi R-133b與TAGLN2結(jié)合情況,用免疫印跡和Realtime-PCR分析mi R-133b對TAGLN2表達的影響,免疫熒光分析TAGLN2在MI期卵母細胞的表達及其在亞細胞定位。結(jié)果:成功構(gòu)建psi CHECK-TAGLN2-3’UTR和psi CHECK-TAGLN2-3’UTR-m表達載體,mi R-133b與psi CHECK-TAGLN2-3’UTR共轉(zhuǎn)染HEK 293T細胞后,熒光素酶活性顯著降低(p=0.000.01),mi R-133b與空載體psi CHECK共轉(zhuǎn)染HEK 293T細胞后,熒光素酶活性沒有明顯變化(p=0.590.05)。mi R-133b與psi CHECK-TAGLN2-3’UTR-m共轉(zhuǎn)染HEK 293T細胞后,熒光素酶活性也沒有明顯變化(p=0.620.05)。mi R-133b與TAGLN2 3’UTR第215-250位核苷酸序列結(jié)合。mi R-133b mimics使TAGLN2表達下調(diào);mi R-133b inhibitor可以使TAGLN2表達上調(diào)。TAGLN2定位分布人MI期卵母細胞漿,細胞核較少。結(jié)論:mi R-133b是MI卵母細胞特異性高表達mi RNA,其靶基因是TAGLN2、結(jié)合位點是TAGLN2的3’UTR第215-250位核苷酸序列。mi R-133b下調(diào)TAGLN2 m RNA和蛋白的表達。TAGLN2定位于人MI期卵母細胞漿。第三部分mi R-133b靶向TAGLN2調(diào)控卵泡的發(fā)育成熟目的:探討mi R-133b靶向TAGLN2調(diào)控卵泡發(fā)育成熟的機制。方法:首先,選擇4周齡和8周齡的雌性ICR小鼠,各6只,采用免疫印記方法檢測TAGLN2在4周齡和8周齡小鼠卵巢的表達水平。第二,隨機選擇4周齡雌性ICR小鼠,分4組,每組6只,采用免疫組化方法檢測TAGLN2在竇前卵泡、竇狀卵泡、排卵前卵泡及黃體中的定位和表達。第三,隨機選擇8周齡雌性ICR小鼠,分為3組,每組10只,用免疫熒光染色方法檢測GV期、GVBD期及MII期卵母細胞中TAGLN2的表達水平。第四,隨機選擇8周齡雌性ICR小鼠,分成3組,每組8只,采用TAGLN2 si RNA分析沉默TAGLN2基因前后,卵母細胞直徑、透明帶厚度變化情況。第五,隨機選擇8周齡雌性ICR小鼠,隨機分成5組,每組8只,通過卵巢多點注射法,將mi R-133b模擬物(mi R-133b minic)、mi R-133b模擬物的陰性對照(mi R-133b minic negative control)、mi R-133b抑制物(mi R-133b inhibitor)、mi R-133b抑制物的陰性對照(mi R-133b inhibitor negative control)及上述制劑的溶劑注射入卵巢,分別統(tǒng)計每只小鼠獲卵數(shù),用免疫印跡和實時定量PCR方法檢測卵巢TAGLN2的表達。第六,收集GV期卵母細胞,隨機分成5組通過顯微注射法將mi R-133b minic、mi R-133b minic negative control、mi R-133b inhibitor、mi R-133b inhibitor negative control及上述制劑的溶劑注射至卵母細胞胞漿,觀察各組卵母細胞的成熟情況。分析mi R-133b對小鼠卵母細胞成熟的影響。結(jié)果:免疫印記顯示TAGLN2在4周齡和8周齡小鼠卵巢組織均有表達,8周齡小鼠卵巢TAGLN2的表達水平顯著高于4周齡小鼠卵巢(p=0.0320.05),免疫組化顯示TAGLN2在竇前卵泡、竇狀卵泡、排卵前卵泡、黃體有表達,且定位于顆粒細胞包膜以及胞漿,細胞核幾乎無表達。顆粒細胞TAGLN2的表達水平隨著卵泡發(fā)育逐漸降低,排卵前卵泡達到最低(p=0.0060.01p0.05),至黃體期其表達水平較排卵前卵泡增強。免疫熒光染色顯示TAGLN2在小鼠卵母細胞胞漿表達,GVBD期卵母細胞TAGLN2的表達水平顯著低于GV期卵母細胞的表達水平(p=0.000.01),MⅡ期卵泡卵母細胞中TAGLN2的表達水平最低(p=0.020.05)。與對照組相比,TAGLN2-si RNA處理組卵母細胞直徑顯著增大(p=0.010.05),透明帶厚度變化無顯著差異(p0.05)。分別給小鼠卵巢及卵母細胞注射mi R-133b minic、mi R-133b minic negative control、mi R-133b inhibitor、mi R-133b inhibitor negative control以注射上述制劑的溶劑作為對照,結(jié)果顯示:與對照組比較,mi R-133b mimic處理組GV期和MII期卵母細胞數(shù)增多,但無統(tǒng)計學意義(p0.05),mi R-133b inhibitor處理組小鼠GV期和MII期卵母細胞數(shù)顯著降低(p0.05);mi R-133b mimic顯著誘導卵母細胞的成熟(p0.05),mi R-133b inhibitor顯著抑制卵母細胞的成熟(p0.05)。mi R-133b mimic下調(diào)TAGLN2的表達,mi R-133b inhibitor上調(diào)TAGLN2的表達。結(jié)論TAGLN2定位于卵泡中顆粒細胞膜和細胞質(zhì),其表達與卵母細胞成熟呈負相關(guān)。mi R-133b抑制小鼠卵巢TAGLN2 m RNA及蛋白的表達,促進小鼠卵母細胞成熟。
[Abstract]:The latest data from the National Committee for health and family planning shows that the incidence of infertility in China is 7%~10% in 2013 and has been increasing year by year. Among the many causes of infertility, extensive ovulatory infertility is a common type of female infertility, which accounts for the development of 25%~35%[1-3]. follicles to cause infertile, and then lead to infertility. The regulation mechanism of follicle development disorder is a hot spot in the field of reproductive development. Follicle development is an extremely complex process, including changes in follicle morphology and function at different times, regulated by many factors such as endocrine, local factor, gene and small RNA (miRNA). Many problems need to be solved. This study uses miRNA gene chip to screen mi RNA differentially expressed in human oocytes at different developmental stages, select specific high expression miRNA as research target and analyze target gene of target miRNA by bioinformatics, and study target gene table by target miRNA. To explore the molecular mechanism of the target miRNA regulating the maturation of oocyte, to find the drug targets to regulate the maturation of oocyte, and to provide new experimental basis for the prevention and control of infertility. The first part of the differential expression of miRNA in human oocyte at different developmental stages: screening the conditions in the simulation of human ovarian environment. The differential expression of miRNA in human oocytes at different developmental stages selected specific high expression miRNA as the research target and analyzed target gene of target miRNA. Methods: to collect human GV, MI oocytes, culture medium containing insulin-like growth factor -1 (IGF-1), to simulate the human ovarian environment, and to screen by miRNA microarray technology. The differentially expressed miRNAs in the oocytes of the same developmental period; the target gene was predicted by bioinformatics. Results: the expression of 183 miRNA expressions in GV oocytes was up-regulated by IGF-1 culture medium, and the expression of 117 mi RNA was down regulated, the expression of 145 miRNA in MI oocytes was up and 200 miRNA expressions downregulated. Among them, miR-133b was in MI phase oocyte. The expression of the cell was up to 32 times, and there was no difference in the expression of miR-133b in GV oocyte. The target gene analysis software showed that a target gene of miR-133b was TAGLN2, and the binding site of miR-133b was in TAGLN2 3 'UTR 215-250 nucleotide. Down regulation, the expression of 145 miRNA in MI oocytes was up, and 200 miRNA expression down regulated.Mi R-133b was the specific and high expression of miRNA in MI oocytes. The target gene may be the interaction between TAGLN2. second mi R-133b and TAGLN2 gene. Methods: TAGLN2-3 'UTR and its mutant expression vectors were constructed by gene recombination technology (named psi CHECK-TAGLN2-3' UTR and psi CHECK-TAGLN2-3 'UTR-m, respectively). Analysis of the combination of MI R-133b with TAGLN2, the effects of MI R-133b on the expression of TAGLN2 by immunoblotting and Realtime-PCR, and the expression of TAGLN2 in MI oocyte and its subcellular location by immunofluorescence. After N2-3 'UTR co transfected HEK 293T cells, luciferase activity decreased significantly (p=0.000.01). After MI R-133b and psi CHECK co transfected HEK 293T cells, luciferase activity was not significantly changed. 05).Mi R-133b and TAGLN2 3 'UTR nucleotide sequence combined with.Mi R-133b mimics to reduce the expression of TAGLN2, MI R-133b inhibitor can make TAGLN2 expression up-regulated and distributes human oocyte pulp with fewer nuclei. The point is TAGLN2 3 'UTR nucleotide sequence 215-250 nucleotide sequence.Mi R-133b downregulates TAGLN2 m RNA and protein expression.TAGLN2 located in human MI oocyte pulp. Third mi R-133b targeting TAGLN2 regulates the maturation of follicles. The expression level of TAGLN2 in the ovary of 4 week and 8 week old mice was detected by immuno imprinting. Second, 4 weeks old female ICR mice were randomly selected, and 6 rats in each group were divided into 4 groups. The location and expression of TAGLN2 in preantral follicles, antral follicles, preovulal follicles and corpus luteum were detected by immunohistochemistry. Third, random. 8 weeks old female ICR mice were selected to be divided into 3 groups, 10 rats in each group. The expression level of TAGLN2 in phase GV, GVBD and MII oocytes was detected by immunofluorescence. Fourth, 8 weeks old female ICR mice were randomly selected and divided into 3 groups, 8 in each group. The diameter of oocyte and the thickness of the zona pellucida were changed by TAGLN2 Si RNA analysis. Fifth, randomly selected 8 weeks old female ICR mice, randomly divided into 5 groups, 8 rats in each group. The MI R-133b analogue (MI R-133b minic), the negative control of MI R-133b analog (MI R-133b minic negative), and the negative control inhibitor of MI R-133b, were randomly assigned to each group. Ibitor negative control) and the solvent of the above preparation were injected into the ovary. The number of eggs obtained in each mouse was measured respectively. The expression of TAGLN2 in the ovary was detected by immunoblotting and real-time quantitative PCR. Sixth, the GV oocyte was collected, and the MI R-133b minic was randomly divided into 5 groups by microinjection, MI R-133b minic. Bitor, MI R-133b inhibitor negative control and the solvent of the aforementioned preparation were injected into the oocyte cytoplasm to observe the maturation of oocyte in each group. The effect of MI R-133b on the maturation of oocyte in mice was analyzed. Results: the immune imprint showed that TAGLN2 was expressed at 4 weeks and 8 weeks of age in mouse ovary tissues, and the table of TAGLN2 in the ovary of 8 weeks old mice The level was significantly higher than the 4 week old mouse ovary (p=0.0320.05). The immunohistochemical staining showed that TAGLN2 was in preantral follicles, sinus like follicles, pre ovulation follicles, and the corpus luteum was expressed in the granulosa cell envelope and cytoplasm, and the nucleus was almost no expression. The expression of TAGLN2 in granular cells decreased gradually with the follicle development, and the follicles reached the lowest level before ovulation. The expression level of p=0.0060.01p0.05 in the luteal phase was stronger than that before ovulation. Immunofluorescence staining showed that TAGLN2 was expressed in the cytoplasm of mouse oocyte, and the expression level of TAGLN2 in GVBD oocyte was significantly lower than that of GV oocyte (p=0.000.01), and the expression level of TAGLN2 in M II oocyte was the lowest (p=0.020.05). Compared with the control group, the oocyte diameter of the TAGLN2-si RNA treatment group increased significantly (p=0.010.05), and there was no significant difference in the thickness of the zona pellucida (P0.05). The mice ovary and oocyte were injected with MI R-133b minic, and MI R-133b minic negative control was injected. As compared with the control group, the results showed that compared with the control group, the number of GV and MII oocytes increased in MI R-133b mimic treatment group, but there was no statistical significance (P0.05). The number of oocytes in GV and MII phase of MI R-133b inhibitor treated mice decreased significantly (P0.05). The maturation of oocyte (P0.05).Mi R-133b mimic down regulated the expression of TAGLN2, and MI R-133b inhibitor up-regulated the expression of TAGLN2. Conclusion TAGLN2 is located in the membrane and cytoplasm of granulosa cells in the follicle, and the expression is negatively correlated with the maturation of oocyte. The cell matures.
【學位授予單位】：南華大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R711.6

【共引文獻】

相關(guān)期刊論文 前2條

1 聶明月;楊曉葵;;miR-23a和miR-27a在卵巢中的生理與病理作用[J];國際生殖健康/計劃生育雜志;2014年05期

2 巴林林;劉冬娥;;微小RNA在卵泡發(fā)育中的作用[J];國際生殖健康/計劃生育雜志;2014年05期

相關(guān)博士學位論文 前1條

1 張曉東;山羊卵巢microRNA的鑒定及生物學功能分析[D];安徽農(nóng)業(yè)大學;2013年

，

本文編號：2012551