IL-6、GATA6在大鼠肺动脉平滑肌细胞中的表达及对细胞增殖的影响

Expressions of IL-6 and GATA6 in rat pulmonary artery smooth muscle cells and their effects on cell proliferation

目的探讨白细胞介素(IL)-6、转录因子GATA6在血小板源性生长因子-BB(PDGF-BB)诱导大鼠肺动脉平滑肌细胞(PASMCs)增殖中的表达及作用。方法从8周龄SD大鼠中分离肺动脉,采用Ⅰ型胶原酶消化法原代培养PASMCs;采用倒置显微镜观察和免疫荧光染色鉴定细胞;采用CCK-8检测不同剂量(0、15、30、45、60、75μg/L)PDGF-BB分别作用12、24、48 h对PASMCs增殖能力的影响;采用流式细胞术检测30μg/L PDGF-BB诱导细胞增殖48 h的细胞周期;应用qPCR和Western blot技术检测30μg/L PDGF-BB诱导细胞增殖12、24、48 h后IL-6和GATA6 mRNA及蛋白的表达水平。结果15~60μg/L的PDGF-BB诱导24 h、48 h后PASMCs增殖显著,其中以30μg/L诱导48 h效果最佳(P<0.05);流式周期结果表明,PDGF-BB组G1期的百分比较对照组降低,而S期和G2期百分比增高(P<0.01)。PDGF-BB能够诱导细胞增加IL-6 mRNA和蛋白的分泌并降低GATA6 mRNA和蛋白表达水平。相关性分析提示PASMCs的增殖与IL-6 mRNA表达呈正相关,与GATA6 mRNA表达呈负相关(r分别为0.538和-0.647,均P<0.05);IL-6与GATA6的表达呈负相关(r=-0.705,P<0.01)。结论PDGF-BB诱导体外培养的PASMCs增殖;IL-6、GATA6参与了PDGF-BB诱导PASMCs增殖的发生和发展;PASMC增殖与IL-6 mRNA表达上调和GATA6 mRNA表达下调有关。

Objective To explore the expressions and roles of interleukin(IL)-6 and transcription factor GATA6 in the proliferation of rat pulmonary artery smooth muscle cells(PASMCs)induced by platelet-derived growth factor-BB(PDGFBB).Methods Pulmonary arteries were isolated from 8-week-old SD rats,and PASMCs were cultured by typeⅠcollagenase digestion method.The cells were observed by inverted microscope and identified by immunofluorescence staining.The effects of different concentrations of PDGF-BB(0,15,30,45,60 and 75μg/L)on the proliferation of PASMCs at 12 h,24 h and 48 h were detected by CCK-8.Flow cytometry was used to detect the cell cycle of 30μg/L PDGF-BB induced cell proliferation for 48 h.The qPCR and Western blot assay were used to detect the expression levels of IL-6 mRNA and GATA6 mRNA and protein after 30μg/L PDGF-BB induced cell proliferation for 12 h,24 h and 48 h.Results The proliferation model of PASMCs could be effectively established by PDGF-BB.The proliferation of PASMCs was significantly increased by PDGF-BB at 15-60μg/L for 24 h and 48 h,and the best effect was induced by 30μg/L for 48 h(P<0.05).Flow cytometry results showed that the percentage of G1 phase was significantly lower in PDGF-BB group than that of control group,while the percentages of S phase and G2 phase were significantly increased(P<0.01).PDGF-BB can induce cells to increase IL-6 mRNA and protein secretion and reduce GATA6 mRNA and protein expression levels.Correlation analysis showed that the proliferation of PASMCs was positively correlated with the expressions of IL-6 mRNA and negatively correlated with the expression of GATA6 mRNA(r=0.538,-0.647 respectively,P<0.05).The expression of IL-6 mRNA was negatively correlated with the expression of GATA6 mRNA(r=-0.705,P<0.01).Conclusion PDGF-BB induces proliferation of PASMCs cultured in vitro.IL-6 and GATA6 are involved in the occurrence and development of PDGF-induced PASMCs proliferation.PASMC proliferation is associated with up-regulation of IL-6 mRNA and downregulation of GATA6 mRNA.