PCSK9在多囊卵巢综合征卵巢颗粒细胞中的表达研究

Study on the expression of PCSK9 in ovarian granulosa cells with polycystic ovary syndrome

目的研究前蛋白转化酶枯草溶菌素9(PCSK9)在多囊卵巢综合征(PCOS)卵巢颗粒细胞中的表达情况,并探讨PCSK9表达与PCOS卵巢组织以及睾酮处理的KGN细胞中脂质状况的关系。方法通过来曲唑联合高脂饲料喂养构建PCOS大鼠模型,油红O染色观察PCOS大鼠卵巢组织中脂质分布情况;生物信息学分析筛选PCOS卵巢与正常卵巢转录组数据中参与脂质代谢通路的差异表达基因;采用免疫组织化学和Western blot技术检测PCSK9蛋白在PCOS卵巢组织中的表达情况;采用免疫荧光、qRT-PCR和Western blot技术检测不同浓度睾酮处理的KGN细胞中PCSK9和低密度脂蛋白受体(LDLR)的表达差异;免疫荧光显微镜观察KGN细胞对Dil-LDL的摄取能力。结果油红O染色结果显示PCOS卵巢组织中颗粒细胞上的脂滴分布较正常卵巢组织明显减少;生物信息学分析结果显示PCOS大鼠卵巢组织中上调的PCSK9主要参与胆固醇代谢通路;免疫组织化学和Western blot结果显示PCSK9蛋白主要定位于颗粒细胞且在PCOS大鼠颗粒细胞中表达增加;免疫荧光、qRT-PCR和Western blot结果显示KGN细胞中PCSK9的表达随睾酮浓度的增加而增加,LDLR的表达随睾酮浓度的增加而减少;睾酮(100 nmol/L)可减弱KGN细胞对Dil-LDL的摄取能力。结论PCSK9在PCOS卵巢颗粒细胞中高表达,其可导致LDLR蛋白表达降低,使颗粒细胞脂质摄取不足。

Aim To explore the expression of proprotein convertase subtilisin kexin 9(PCSK9)in polycystic ovary syndrome(PCOS)ovarian granulosa cells,and the relationship between PCSK9 expression and the lipid status of PCOS ovarian tissue and testosterone-treated KGN cells.Methods A rat model of PCOS was constructed by letrozole combined with high-fat diet.Oil red O staining was used to observe the lipid distribution in the ovarian tissue of PCOS rats.Transcriptome data from PCOS ovarian tissue and normal ovarian tissue was analyzed by bioinformatics,and the differentially expressed genes involved in lipid metabolism pathway were screened.Western blot and immunohistochemical techniques were used to detect the protein expression of PCSK9 in PCOS rat tissues.Immunofluorescence,qRT-PCR and Western blot techniques were used to detect the expression differences of PCSK9 and low density lipoprotein receptor(L DLR)in KGN cells treated with different concentrations of testosterone.Immunofluorescence microscope was used to observe the ability of KGN cells uptake Dil-LDL.Results The results of Oil Red O staining showed that the distribution of lipid droplets in granular cells of PCOS ovarian tissue was significantly less than that of normal ovarian tissue.The results of bioinformatics analysis showed that the up-regulated expression of PCSK9 in the ovarian tissue of PCOS rats was mainly involved in the cholesterol metabolism pathway.The results of immunohistochemistry and Western blot showed that the PCSK9 protein was mainly localized in granulosa cells and its expression increased in the granulosa cells of PCOS rats.The results of immunofluorescence,qRT-PCR and Western blot showed that PCSK9 expression was up-regulated with the increased concentration of testosterone,and LDLR expression was decreased with the increased concentration of testosterone in KGN cells.Testosterone(100 nmol/L)could reduce the uptake of Dil-LDL by KGN cells.Conclusion PCSK9 was highly expressed in PCOS ovarian granulosa cells,which could reduce the expression of LDLR protein and lead to insufficient lipid uptake of granulosa cells.