BMP4调控睾丸支持细胞增殖的初步研究

Preliminary Study on BMP4 Regulating Proliferation of Testicular Sertoli Cells

旨在研究BMP4对睾丸支持细胞增殖的调控。本试验选取0、1、2和3月龄组的健康大足黑山羊公羊各3只,采集睾丸支持细胞,每次试验均设3个生物学重复和3次技术重复,通过体外培养、细胞免疫荧光、基因干扰、过表达、qPCR和Western blotting等技术对BMP4是否通过Id2对山羊睾丸支持细胞进行调控以及它们的调控关系进行验证。结果发现,BMP4在2月龄组大足黑山羊睾丸支持细胞中的表达量极显著高于0、1月龄组(P<0.01);在一定范围内,随着BMP4浓度的增加睾丸支持细胞的增殖活性有所增强,BMP4浓度为200 ng·mL^(-1)时其增殖能力最强(P<0.05);干扰BMP4后,细胞增殖活力显著下降(P<0.05),但在72 h后回到正常水平(P>0.05),PCNA的表达极显著降低(P<0.01),表明干扰BMP4后细胞的增殖能力受到限制,细胞增殖指数测定结果进一步说明,干扰BMP4后细胞的增殖能力受到限制;过表达BMP4后,Id2基因表达水平极显著增加(P<0.01),表明BMP4对Id2具有正向调控作用;相对过表达BMP4再干扰Id2试验组,干扰Id2试验组的PCNA基因的表达水平显著降低(P<0.05),这进一步证明BMP4可以调控该基因和蛋白表达。综上所述,山羊睾丸支持细胞的增殖活力在一定范围内与BMP4的浓度呈正相关;且BMP4能够正向调控Id2的表达,并通过促进Id2的表达进而促进支持细胞的增殖。本研究为阐明BMP4调控山羊睾丸支持细胞的分子机制和生理功能提供了基础。

The study aimed to explore the the regulation of BMP4 on the proliferation of testicular sertoli cells.Three healthy Dazu black goat rams in the 0-,1-,2-and 3-month-old groups were selected respectively,and the testicular sertoli cells were collected.Each experiment set up 3 biological replicates and 3 technical replicates.Whether BMP4 regulated goat testicular sertoli cells through Id2 and their regulatory relationship were verified by in vitro culture,cell immunofluorescence,gene interference,overexpression,qPCR and Western blotting and other technologies.The results showed that the expression level of BMP 4 in testicular sertoli cells of Dazu black goat in 2-month-old group was extremely significantly higher than that in 0-,1-month-old groups(P<0.01);Within a certain range,with the increase of BMP4 concentration,the proliferation activity of testicular sertoli cells increased,and the proliferation ability was the strongest when the concentration of BMP4 was 200 ng·mL^(-1)(P<0.05);After interfering BMP 4 expression,the cell proliferation activity significantly decreased(P<0.05),but returned to the normal level after 72 h(P>0.05),and the expression of PCNA was extremely significantly reduced(P<0.01),indicating that the cell proliferation ability was restricted after interfering BMP 4 expression,and the cell proliferation index measurement results further illustrated that the cell proliferation ability was restricted after interfering BMP 4 expression;After BMP 4 overexpression,the expression levels of Id2 extremely significantly increased(P<0.01),indicating that BMP4 had a positive regulatory effect on Id2;Compared to the overexpressing BMP 4 and then interfering Id 2 group,in the interfering Id 2 group,the expression levels of PCNA gene significantly reduced(P<0.05),which further proved BMP4 could regulate the expression of this gene and protein.In summary,the cell proliferation activity of goat testicular sertoli cells is positively correlated with the concentration of BMP4 within a certain range;BMP4 can positively regulate the expression of Id2,and promote the proliferation of goat testicular sertoli cells through promoting the expression of Id2.This study provides a basis for elucidating the molecular mechanism and physiological functions of BMP4 regulating goat testicular sertoli cells.