饰胶蛋白聚糖对猪卵泡颗粒细胞表型和功能的影响  

Effects of decorin on the phenotype and function of porcine follicular granulosa cells

作　　者：杨菲菲[1] 杨飞[2,3] 孙义姗 黄涛 YANG Feifei;YANG Fei;SUN Yishan;HUANG Tao(Jinhua Vocational and Technical College,Jinhua 321000,China;College of Animal Science and Technolongy,Shihezi Uniersity,Shihezi 832000,China;Tiankang Biological Co.,Ltd.,Urumqi 830001,China)

机构地区：[1]金华职业技术学院,浙江金华321000 [2]石河子大学动物科技学院,新疆石河子832000 [3]天康生物股份有限公司,乌鲁木齐830001

出　　处：《黑龙江畜牧兽医》2021年第4期35-42,153,共9页Heilongjiang Animal Science And veterinary Medicine

基　　金：教育部留学归国人员科研启动基金项目(20131092)。

摘　　要：为了研究转化生长因子-β(TGF-β)信号通路中的饰胶蛋白聚糖(decorin, DCN)基因对猪卵泡颗粒细胞的调控作用,试验构建DCN基因干扰和过表达载体,并将构建好的干扰和过表达重组质粒转染猪卵泡颗粒细胞,通过实时荧光定量PCR技术进行有效载体的筛选以及检测其对TGF-β信号通路下游基因(TGF-βRⅠ、TGF-βRⅡ、SMAD1、SMAD2、SMAD3、SMAD4、SMAD5)和细胞凋亡基因(Bax、Bcl-2)mRNA的表达水平影响,然后用MTT法和流式细胞仪检测DCN基因干扰和过表达对卵泡颗粒细胞增殖及凋亡的影响。结果表明:DCN干扰(pSIREN-DCN01、pSIREN-DCN02、pSIREN-DCN03)和过表达[pcDNA3.1(+)-DCN]载体构建成功,其中pSIREN-DCN01抑制DCN基因表达的效果最好,干扰效率最高。过表达DCN基因时,TGF-βRⅠ基因mRNA相对表达量下降,使得SMAD2、SMAD3基因mRNA相对表达量也下降,进而同时抑制卵巢颗粒细胞增殖和促进其凋亡;干扰DCN基因时,TGF-βRⅠ基因mRNA相对表达量显著上升(P<0.05),使得SMAD2、SMAD3基因mRNA相对表达量也上升(P<0.05),进而促进颗粒细胞增殖和凋亡。说明DCN基因可通过调节TGF-β信号通路促进猪卵泡颗粒细胞的凋亡,间接影响猪的繁殖性能。To investigate the regulation of decorin(DCN) gene in the transforming growth factor-β(TGF-β) signaling pathway on porcine follicular granulosa cells, in this study, DCN interference vector and overexpression vector were designed and constructed. Porcine ovarian granulosa cells were transfected with the constructed interference and overexpression recombinant plasmids, and used real-time quantitative PCR technology to screen effective vector and detect mRNA expression levels of TGF-β signaling pathway downstream genes(TGF-βRⅠ, TGF-βRⅡ, SMAD1, SMAD2, SMAD3, SMAD4, SMAD5) and apoptosis genes(Bax, Bcl-2). Finally, the effects of DCN interference and overexpression on the proliferation and apoptosis of follicular granulosa cells were detected by MTT method and flow cytometry. The results showed that DCN interference(pSIREN-DCN01、pSIREN-DCN02、pSIREN-DCN03)and overexpression vectors(pcDNA3.1(+)-DCN)were successfully constructed, among which pSIREN-DCN01 had the best effect of inhibiting DCN expression and the highest interference efficiency. When overexpression of DCN gene, the mRNA expression of TGF-βRⅠ gene decreased, so that the mRNA expression of SMAD2 and SMAD3 decreased, at the same time, it inhibited the proliferation and apoptosis of granulosa cells. However, interfering with DCN gene, the mRNA expression of TGF-βRⅠ increased significantly(P<0.05), so that the mRNA expression of SMAD2 and SMAD3 also increased(P<0.05), and promoted the proliferation and apoptosis of granulosa cells. The results suggested that the DCN gene significantly promoted apoptosis of ovarian granulosa cells by regulating the TGF-β signaling pathway, and indirectly affect the reproductive performance of pigs.

关 键 词：猪 TGF-Β信号通路 饰胶蛋白聚糖 颗粒细胞 干扰 过表达 

分 类 号：Q782[生物学—分子生物学]