【作者】 王继东；

【导师】 姚珍薇；

【作者基本信息】 重庆医科大学 ， 妇产科学， 2013， 博士

【摘要】 多囊卵巢综合征（polycystic ovary syndrome, PCOS）是临床妇科最常见的内分泌紊乱症候群，也是生育期女性不孕的主要原因。卵泡发育异常是PCOS的病理生理特征之一，目前发病机制不清。有文献报道PCOS患者血清激活素（activins）表达水平降低，而activins可能具有促进颗粒细胞增殖与分化、调控卵泡发育等作用，因此我们推测activins可能在PCOS卵泡发育障碍中发挥重要作用。为明确activins在卵泡发育中的作用及可能的分子机制，我们设计本实验。第一部分：慢病毒载体（LV-eGFP）转染大鼠卵巢颗粒细胞的体内外实验研究目的：探讨LV-eGFP体外转染卵巢颗粒细胞的有效性及体内转染大鼠卵巢组织的最佳剂量及时效性。方法：大鼠卵巢颗粒细胞的原代培养并传代，LV-eGFP病毒颗粒体外转染颗粒细胞，观察绿色荧光蛋白（eGFP）的表达情况。大鼠卵巢显微注射的方法以不同转染剂量在体转染大鼠卵巢组织。于转染第3天观察各剂量组卵巢组织切片的eGFP表达情况。确定最佳转染剂量后，分别以转染5天、15天、30天、45天、60天、75天为观察时间点，观察eGFP持续表达及变化情况；并同时检测大鼠全身其他组织器官是否有eGFP的表达。结果：体外转染实验中，在转染第3天可以观察到颗粒细胞内有eGFP的表达，并随着转染时间延长其表达量增多。体内转染大鼠卵巢第5天，不同剂量组大鼠卵巢均有明显eGFP表达；剂量1组（2×106TU病毒颗粒）与剂量2组（10×106TU病毒颗粒）卵巢组织切片荧光半定量分别为0.2311±0.0203及0.2307±0.0199；两组间差异无统计学意义（P=0.976）。随转染时间的延长，绿色荧光蛋白的表达量增加，于转染30天时表达量达到峰值并可以持续高效表达至75天（不同观察时间点卵巢组织荧光强度值分别为0.2307±0.0199，0.3119±0.0213，0.3462±0.0264，0.3568±0.0127，0.3496±0.0133及0.3513±0.0172）。同时，在大鼠全身其它组织器官均有绿色荧光蛋白的高效持续表达。结论：LV-eGFP可以在体外有效转染颗粒细胞，并且可以在活体大鼠卵巢及其他组织器官高效持续地表达。第二部分：lenti-eGFP-inhba过表达载体的构建及鉴定目的：构建inhba基因过表达的慢病毒载体（lenti-eGFP-inhba）并观察转染大鼠卵巢颗粒细胞的有效性。方法：pLVX-EGFP-3FLAG质粒系统经EcoR I酶切使之线性化。inhba基因（NM₀08380）PCR扩增后同源重组入线性化的表达质粒系统。经转化、菌落阳性克隆鉴定，western blotting检测表达质粒内目的基因的表达情况。构建成功的慢病毒载体（lenti-eGFP-inhba）转染大鼠卵巢颗粒细胞（MOI=20），观察目的基因表达。结果：PCR扩增产物与目的基因大小吻合，重组质粒系统经转化后获得阳性克隆，转染293T细胞可见eGFP-inhba融合蛋白的表达。western blotting获得76kDa分子量大小阳性条带，与融合蛋白分子量大小一致。lenti-eGFP-inhba体外转染大鼠卵巢颗粒细胞，倒置荧光显微镜下可见eGFP的表达。结论：lenti-eGFP-inhba表达载体成功构建，并可以有效转染大鼠颗粒细胞。第三部分：activinA对大鼠卵泡发育的影响研究目的：观察慢病毒载体介导inhba基因体内转染大鼠卵巢组织，大鼠生殖内分泌功能的变化。方法：30只SD大鼠随机分为三组：对照组（CON）、inhba基因过表达组（INH）及空病毒载体组（GFP）。Inhba过表达慢病毒载体（LV-eGFP-inhba）通过显微注射转染大鼠卵巢组织，CON组给予相同体积的PBS溶液，GFP组给予相同剂量的空病毒载体（LV-eGFP）。结果：inhba在基因及蛋白水平均成功过表达于INH组大鼠卵巢。窦状卵泡数量在三组间无显著差异（P>0.05）；INH组大鼠窦状卵泡直径大于CON组及GFP组（P<0.05）。ELISA法及放射免疫法检测activin A、雌二醇（E2）、孕激素（P）、卵泡刺激素（FSH）的血清水平在INH组大鼠高于CON组及GFP组大鼠（P<0.05）；INH组黄体生成素（LH）的血清水平低于CON组及GFP组（P>0.05）。雌激素受体（ER-α、ER-β）及FSH受体（FSHR）在mRNA和蛋白的表达水平，INH组大鼠均高于另外两个对照组（P<0.05）；孕激素受体（PR）在三组大鼠的基因及蛋白表达水平无显著差异（P>0.05）；黄体生成素受体（LHR）基因、蛋白表达水平，在INH组低于其他两组（P<0.05）。结论：inhba基因在大鼠卵巢过表达，影响生殖内分泌功能并促进窦状卵泡的发育。第四部分：activinA促进卵泡发育的分子机制研究目的：探讨activin A促进颗粒细胞增殖与调控卵泡发育的分子机制。方法：携带inhba基因的慢病毒过表达载体（lenti-eGFP-inhba）体外体内转染大鼠卵巢颗粒细胞及卵巢组织。绘制生长曲线并计算细胞倍增时间；流式细胞技术检测细胞周期及细胞凋亡；免疫组织化学染色检测SCF及c-Kit蛋白在卵巢组织的表达；western blotting检测信号通路蛋白smad2、MERK5、nur77及SCF的表达水平。结果：inhba基因过表达组（INH组）颗粒细胞在体外增殖速率明显高于另外两个对照组（CON组及GFP组），差异具有统计学意义（P<0.05）；INH组细胞倍增时间短于CON组及GFP组（P<0.05）。免疫组化提示大鼠卵巢SCF主要表达于颗粒细胞及黄体细胞，c-Kit主要表达于黄体细胞及各级卵泡的卵母细胞，二者在INH组的表达量高于CON组及GFP组（P<0.05）。western blotting结果提示smad2、MERK5、nur77及SCF蛋白的表达水平在INH组高于CON组及GFP组，差异具有统计学意义（P<0.05）。结论：activin A通过smad2、MERK5、nur77介导的信号通路促进颗粒细胞增殖，颗粒细胞又分泌SCF促进卵泡发育。更多还原

【Abstract】 Polycystic ovary syndrome （PCOS） is the most common clinicalgynecological endocrine disorder syndrome, and main causes of infertilityfor reproductive age women. Follicular dysplasia is one of thepathophysiological features of PCOS, but the specific pathogenesis is stillunclear. Several researches proved activins hormone can promote cellproliferation and differentiation, regulate possible follicular development.Combined with PCOS patients serum activin hormone expression leveldecreased, we speculated that activins may play an important role in thePCOS follicular developmental disorders. We designed this experiment toclear activins functions in follicular development and possible molecularmechanism in it.The first part: Lentivirus vectors mediated enhanced greenfluorescence protein gene （LV-eGFP） transfection on rat ovaryObjective: to investigate the transfected efficiency of lentivirus vectorsmediated enhanced green fluorescent protein （eGFP） gene in vitro transfect to the rats’ granular cells and the optimum dosage and the time-effectrelationship on the ovary tissues in vivo. Methods: primary culture of ratovary granular cells was finished, the passage cells were transfected bylentivirus vectors mediated eGFP gene （LV-eGFP） with MOI=20. LV-eGFPwas injected into the rats’ ovary tissues by microinjection. At3days oftransfection, we examine the eGFP expression both in cell in vitro and ovarytissues in vivo. The green fluorescence density of every dosage group in theovaries was calculated. After the rats’ ovaries were microinjected with theoptimum dosage of LV-eGFP, we observed the expression of eGFP in ovariesand others tissues and organs at days5,15,30,45,60and75aftertransfection. Results: after5days of transfection, the eGFP were seen in thegranular cells. For the animal experiment, the expression of eGFP wasobserved at days5of transfection in different dosage group. The greenfluorescence semi-quantitative values for the two groups （2×106TU and10×106TU virosome） were0.2311±0.0203and0.2307±0.0199respectivelyand there was no significance between the two groups （P=0.976）. Theexpression of eGFP increased as the transfection time prolongation and wasat its peak at days30of transfection and can last high-level expression to75days （the green fluorescence density of every time point is0.2307±0.0199，0.3119±0.0213，0.3462±0.0264，0.3568±0.0127，0.3496±0.0133and0.3513±0.0172）. Furthermore, there were efficient and durable expressionof eGFP at other tissues and organs in rats. Conclusion: LV-eGFP may transfect successfully on rats’ ovary granular cells in vitro and ovary tissuesand other organs in vivo simultaneously, the expression of eGFP ishighly-efficient and durable.The second part: The construction and identification oflentivirus vectors mediated inhba gene overexpressionObjective: To construct lentivirus overexpression vectors of inhbagene （lenti-eGFP-inhba） and observe the effectiveness of it transfectedovarian granulosa cells of rats in vitro. Methods: pLVX-EGFP-3FLAGplasmid system linearization was carried out via EcoR I enzyme. Inhba gene（NM₀08380） was amplified by PCR and recombinated homologously intothe linearized plasmid system. The plasmid was constructed viatransformation and colony positive clone identification. Western blottingwas used to test expression in protein level that the target gene in plasmid.Lenti-eGFP-inhba transfected rat ovarian granulosa cells （MOI=20） in vitro,the eGFP and inhba gene expression was observed by inverted fluorescencemicroscope. Results: The PCR amplification product was consistant withthe size of target gene. Recombinant plasmid system obtained positiveclones after transformation; the expression of eGFP-inhba fusion proteinwas visible in which transfected293T cells.76kda positive stripe Westernblotting acquired was accordance with the molecular weight of the fusionprotein. Lenti-eGFP-inhba transfection into rat ovarian granulosa cells invitro, the expression of eGFP was observed by inverted fluorescence microscope. Conclusion: lenti-eGFP-inhba overexpression vector was toconstructed successfully, and can effective transfection granulosa cells inrats.The third part: Changes in reproductive endocrine in ratfollowing intraovary microinjection of inhba lentivirus vectorsObjective: To investigate the reproductive endocrine changes afterinhba overexpression into rat ovary. Methods: Thirty Sprague-Dawley ratswere randomly assigned to three groups. Inhba overexpression lentivirusvectors （LV-eGFP-inhba） were microinjected into rat ovary （INH group）;Control animals received the same amount lentivirus vector empty （CONgroup） or LV-eGFP （GFP group）. Antral follicle amount and diameter werecounted and serum level of activin A, E2, P, FSH, and LH and the expressionof ER-α, ER-β, PR, FSHR, and LHR were measured. Results: There was nosignificant difference among three groups in antral follicle amount （P>0.05）;antral follicle diameter was increased in INH group rats compared with theother group rats （P<0.05）. Serum levels of activin A, E2, P, and FSHhormones were increased and LH was decreased in INH group ratscompared with the other group rats （P<0.05）. The mRNA and proteinexpression of ER-α, ER-β, FSHR was higher in INH group rats than that inthe other group rats （P<0.05）. There was no significant difference in mRNAand protein expression of PR among the three group rats （P>0.05）, LHRexpression was decreased in INH group rats compared with the other two group rats （P<0.05）. Conclusion: inhba overexpression in rat ovary in vivomay change reproductive endocrine function.The forth part: Molecular mechanism of activin A regulationfollicle development in ratObjective: To study the molecular mechanism of activin A hormonepromotes follicular granulosa cell proliferation and regulates follicledevelopment. Methods: Inhba gene overexpression lentivirus vectors（lenti-eGFP-inhba） were transfected into rat ovarian granulosa cells in vitroand ovarian tissue in vivo respectively. We draw the cell growth curve andcalculate the cell doubling time; detect the cell cycle and cell apoptosis byFlow cytometry technology; observe SCF and c-Kit protein expression inovarian tissue through Immunohistochemical staining; estimate expressionlevel of signal pathway proteins smad2, MERK5, nur77and SCF byWestern blotting technology. Results: Granulosa cells proliferation rate ininhba gene overexpression group （INH） was higher than that in the other twocontrol groups （CON group and GFP group） in vitro, the difference wasstatistically significant （P<0.05）; cell doubling time in INH group wasshorter than that in both the CON group and GFP group （P<0.05）.Immunohistochemical staining suggested SCF protein is mainly expressedin ovarian granulosa cells and corpus luteum cells; c-Kit is mainly expressedin luteal cells and the follicular oocytes of different level follicles. Both ofthe two proteins expression in INH group was higher than that in CON group and GFP group （P <0.05）. Western blotting results indicated Smad2,MERK5, nur77and SCF protein expression level in INH group was higherthan that in CON group and GFP group, the statistical difference issignificance （P <0.05）. Conclusions: activin A could promote granulosacell proliferation through smad2, MERK5, nur77mediated signalingpathway and secrete SCF protein to promote follicle development.更多还原