外泌体参与调节高磷诱导的大鼠血管平滑肌细胞钙化

Exosomes are involved in calcification of vascular smooth muscle cells induced by high phosphorus

目的探究外泌体在高磷诱导的大鼠血管平滑肌细胞(vascular smooth muscle cells,VSMC)钙化中的调节作用。方法体外培养VSMC(A7r5细胞),随机分为正常磷组(0.9 mmol/L)、高磷组(2.6 mmol/L)及高磷外泌体诱导组(即高磷组细胞提取的外泌体加入正常培养的VSMC中)。至培养第7天,收集正常磷组、高磷组细胞培养换液过程中获得的培养液,通过超速离心法得到沉淀物,BCA蛋白定量法测定所得沉淀物蛋白含量,分别对所得沉淀物用透射电镜观察沉淀物形态及大小,Western印迹法检测外泌体标志蛋白肿瘤易感基因101蛋白(tumor susceptibility gene 101 protein,TSG101)、CD9;并对外泌体微RNA(microRNA,miRNA,miR)进行提取,实时定量PCR法检测miR-30b、miR-204、miR-211含量。分别培养7 d后,观察高磷外泌体诱导组VSMC对外泌体的摄取过程,观察3组细胞形态,茜素红染色检测细胞钙盐沉积水平,邻甲苯酚络合铜检测细胞钙离子含量,比色法检测细胞碱性磷酸酶含量,Western印迹法检测成骨特异性转录因子(Runx2)蛋白表达情况。结果(1)VSMC培养液经超速离心法所获沉淀物,经电镜形态学鉴定为外泌体,Western印迹法证实外泌体标志蛋白TSG101、CD9表达阳性。(2)外泌体内富含miRNA,经测定,高磷组外泌体中负向调控Runx2转录的miR-30b、miR-204、miR-211表达较正常磷组明显下调(均P<0.05)。(3)高磷培养VSMC 7 d后,钙盐沉积明显,与正常磷组相比,钙含量、碱性磷酸酶活性明显增高(均P<0.05),Runx2表达也明显增高(P<0.05)。(4)将所得高磷组外泌体加入正常培养的VSMC中,外泌体可被VSMC摄取并成功诱导VSMC发生钙化,VSMC钙化指标及Runx2表达水平均明显增高。结论高磷诱导VSMC发生钙化,促进Runx2表达增高,其机制可能是借由VSMC释放外泌体传递细胞信号而实现。

Objective To explore the regulatory role of exosomes in calcification of rat vascular smooth muscle cells(VSMC)induced by high phosphorus.Methods VSMC(A7r5 cells)were cultured in vitro and randomly divided into three groups:normal phosphorus group(0.9 mmol/L),high phosphorus group(2.6 mmol/L)and high phosphorus exosomes induction group(i.e.the exosomes extracted from high phosphorus group were added to VSMC in normal culture).Until the 7th day of culture,the culture medium of normal phosphorus group and high phosphorus group obtained during the change of cell culture medium was collected,and the precipitate was obtained by ultracentrifugation and suspended by phosphate buffered saline.The protein content of the precipitate was determined by BCA protein quantitative method.The precipitates were identified.The structure and size of exosomes were observed by transmission electron microscope.The exosomes marker proteins tumor susceptibility gene 101 protein(TSG101)and CD9 were detected by Western blotting.The miRNA in exosomes was extracted,and the expression of related miRNA(miR-30b,miR-204,miR-211)were observed by real-time quantitative PCR.After 7 days of cultivation,the exosomes uptake process of VSMC in high phosphorus exosomes induction group was observed.The calcium deposition was detected by Alizarin stain,and the calcium content was detected by O-cresol complex copper.The content of alkaline phosphatase was detected by colorimetry.The protein expression of Runx2 was quantified by Western blotting.Results(1)The precipitate obtained by ultracentrifugation of the cell culture fluid was identified as exosomes by electron microscopy morphology.Western blotting confirmed that the expression of the exosomal marker proteins TSG101 and CD9 were positive.(2)The exosomes were rich in miRNAs.The expression of miR-30b,miR-204,miR-211,which negatively regulated the transcription of Runx2,was significantly down-regulated in the high-phosphorus group compared with the normal group(P<0.05).(3)After culturing rat VSMC with high phosphorus for 7 days,calcium salt deposition was obvious.Compared with the normal phosphorus group,calcium content and alkaline phosphatase activity were significantly increased(both P<0.05),and Runx2 expression was also significantly increased(P<0.05).(4)Added the obtained high-phosphorus exosomes to the normal cultured VSMC,the exosomes could be taken up by VSMC and successfully induced VSMC calcification.The levels of cell calcification indicators and Runx2 expression were significantly increased.Conclusions High phosphorus induces calcification of VSMC and promotes the increase of Runx2 expression.The mechanism may be realized by releasing exosomes from VSMC to transmit cell signals.