BMP4诱导的Smad9在小鼠卵泡发育中的作用及其机制的初步研究

本研究旨在探讨Smad9在小鼠卵泡发育中的作用，为卵泡生长发育及其调控机制的研究提供新思路。将6周龄的雌性昆明小鼠随机分成3组，分别腹腔注射生理盐水、骨形态发生蛋白-4（BMP4）和Smad9抑制剂（LDN-193189），依次作为对照组、BMP4组和LDN组。48 h后收集卵巢制作石蜡切片，HE染色后观察卵泡发育情况并对各级卵泡进行计数；采用Western blotting和实时荧光定量PCR技术检测Smad9蛋白和mRNA水平。同时通过ELISA试验测定各组小鼠血清中雌二醇（E₂）、孕酮（P₄）、黄体生成素（LH）、卵泡刺激素（FSH）和芳香化酶等的浓度，并检测了CYP19a1、LHR、PRLR和FSHR基因的表达情况，进一步探究Smad9可能的作用机制。结果显示，与对照组相比，BMP4组Smad9蛋白和mRNA水平较高，而LDN组较低；BMP4组有腔卵泡显著增加（P<0.05），而LDN组显著减少（P<0.05）。此外，ELISA试验结果显示，BMP4组血清E₂、FSH和芳香化酶含量增加，而P₄和LH含量降低；LDN组中E₂和芳香化酶的含量降低。实时荧光定量PCR结果显示，BMP4组FSHR和CYP19a1基因的mRNA水平升高，而LHR和PRLR基因的mRNA水平降低；LDN组PRLR基因的mRNA水平升高，而CYP19a1基因的mRNA水平降低。由以上试验结果可以得出，BMP4诱导的Smad9能促进小鼠有腔卵泡的发育，而其可能主要通过影响E₂、PRL和芳香化酶等的产生而起作用。

Preliminary Study on the Role of BMP4-induced Smad9 in Mouse Follicular Development and Its Mechanism

This study was conducted to explore the role of Smad9 on follicular development in mice and provide new ideas for the study of follicular growth and its regulation mechanism.Six-week-old female Kunming mice were randomly divided into 3 groups and injected intraperitoneally with physiological saline,bone morphogenetic protein-4 (BMP4) and Smad9 inhibitor (LDN-193189),respectively,which were used as the control group,BMP4 group and LDN group.After 48 hours,the ovaries were collected to make paraffin sections.Follicles were observed after HE staining and the follicles at different stages of development were counted.Western blotting and Real-time PCR were used to detect Smad9 protein and mRNA levels.At the same time,the concentrations of estradiol (E₂),progesterone (P₄),luteinizing hormone (LH),follicle stimulating hormone (FSH) and aromatase in the serum of each group of mice were measured by ELISA,and the expression of genes such as CYP19a1,LHR,PRLR and FSHR were detected to further explore possible mechanisms of Smad9.The results showed that compared with control group,the Smad9 protein and mRNA levels were higher in BMP4 group and lower in LDN group;The number of antral follicles in BMP4 group was significantly increased (P < 0.05),while LDN group was significantly reduced (P < 0.05).In addition,ELISA results showed that the levels of serum E₂,FSH and aromatase increased,while the levels of P₄ and LH decreased in BMP4 group;The levels of E₂ and aromatase decreased in LDN group.The results of Real-time PCR showed that the mRNA levels of FSHR and CYP19a1 genes increased,while the mRNA levels of LHR and PRLR genes decreased in BMP4 group;The mRNA level of PRLR gene increased,while the mRNA level of CYP19a1 gene decreased in LDN group.From the above experimental results,it could be concluded that BMP4-induced Smad9 promoted antral follicles development,and it might work via affecting the production of E₂,PRL and aromatase.