肌微管素相关蛋白7在小鼠血管平滑肌细胞增殖和迁移中的作用及机制

Effect and mechanism of myotubularin-related protein 7 on proliferation and migration of mouse vascular smooth muscle cells

目的探究肌微管素相关蛋白7(Mtmr7)在小鼠血管平滑肌细胞(VSMCs)增殖和迁移中的作用及其机制。方法培养小鼠主动脉平滑肌细胞系(MOVAS),用30 ng/ml血小板源性生长因子BB(PDGF-BB)诱导小鼠VSMCs体外增殖和迁移,采用qRT-PCR和Western blotting分别检测PDGF-BB干预后Mtmr7 mRNA和蛋白表达水平的变化。为探究Mtmr7对小鼠VSMCs增殖和迁移的作用,用Mtmr7基因过表达腺病毒(Ad-Mtmr7)感染VSMCs促使Mtmr7过表达,将MOVAS分为对照组、Ad-Mtmr7组、PDGF-BB组、Ad-Mtmr7+PDGF-BB组4组,采用Ki-67细胞免疫荧光染色、CCK-8法检测VSMCs增殖能力,划痕实验检测VSMCs迁移能力,Western blotting检测哺乳动物雷帕霉素靶蛋白复合物1(mTORC1)下游靶标蛋白水平。用5 mg/L胰岛素(insulin)恢复mTORC1活性,将MOVAS分为PDGF-BB组、Ad-Mtmr7+PDGF-BB组、Ad-Mtmr7+PDGF-BB+insulin组3组,再次检测上述指标,观察Mtmr7对小鼠VSMCs增殖和迁移的影响。构建小鼠颈动脉内皮损伤模型,造模28 d后,Western blotting检测Mtmr7蛋白表达水平变化。随机将小鼠分为4组(n=10):假手术组、假手术+Ad-Mtmr7组、颈动脉内皮损伤组、颈动脉内皮损伤+Ad-Mtmr7组。在小鼠手术后立即用Ad-Mtmr7(5×10^(10)pfu/ml)行颈动脉局部孵育30 min,在造模第7、14、21天行尾静脉注射,以实现体内过表达Mtmr7。造模28 d后,采用HE染色检测各组小鼠颈动脉形态及内膜增生程度。结果与对照组比较,PDGF-BB组Mtmr7 mRNA及蛋白表达水平降低(P<0.001或P<0.05),Ki-67阳性细胞率增高,VSMCs相对数量增多(P<0.01或P<0.001),划痕愈合率增高(P<0.001),p-S6^(Ser235/236)和p-4EBP1^(Thr37/46)蛋白表达水平升高(P<0.05);与PDGF-BB组比较,Ad-Mtmr7+PDGF-BB组Ki-67阳性细胞率降低,VSMCs相对数量减少(P<0.01或P<0.001),划痕愈合率降低(P<0.001),p-S6^(Ser235/236)和p-4EBP1^(Thr37/46)蛋白表达水平降低(P<0.05);与Ad-Mtmr7+PDGF-BB组比较,Ad-Mtmr7+PDGF-BB+insulin组p-S6^(Ser235/236)和p-4EBP1^(Thr37/46)蛋白水平升高(P<0.01),Ki-67阳性细胞率增高,VSMCs相对数量增多(P<0.01或P<0.001),划痕愈合率增高(P<0.01)。与假手术组比较,颈动脉内皮损伤组Mtmr7蛋白表达水平明显降低(P<0.01),颈动脉内膜/中膜面积比值增高(P<0.001);而与颈动脉内皮损伤组比较,过表达Mtmr7后,颈动脉内皮损伤+Ad-Mtmr7组颈动脉内膜/中膜面积比值明显降低(P<0.01)。结论过表达Mtmr7可抑制PDGF-BB诱导的小鼠VSMCs增殖和迁移,从而减轻血管损伤后的内膜增生,其机制与Mtmr7降低mTORC1活性密切相关。

Objective To investigate the role and mechanism of myotubularin-related protein 7(Mtmr7)in proliferation and migration of mouse vascular smooth muscle cells(VSMCs).Methods The mouse aortic smooth muscle cell line(MOVAS)was cultured.30 ng/ml of platelet-derived growth factor-BB(PDGF-BB)was used to induce proliferation and migration of VSMCs in vitro.The changes of mRNA and protein expression levels after PDGF-BB intervention in Mtmr7 was assessed by qRT-PCR and Western blotting.To explore the role of Mtmr7 in proliferation and migration of mouse VSMCs,the adenovirus carrying Mtmr7(Ad-Mtmr7)was used to infect VSMCs for overexpression of Mtmr7.MOVAS was divided into control group,Ad-Mtmr7 group,PDGF-BB group and Ad-Mtmr7+PDGF-BB group.The proliferation capacity of VSMCs was analyzed by Ki-67 immunofluorescence staining and cell counting kit-8(CCK-8)assay.The migration capacity was assessed by scratch assay.The downstream target protein levels of mammalian rapamycin target protein complex 1(mTORC1)were determined by Western blotting.Insulin(5 mg/L)was used to restore the activity of mTORC1.MOVAS were divided into PDGF-BB group,Ad-Mtmr7+PDGF-BB group and Ad-Mtmr7+PDGF-BB+insulin group.The proliferation,migration and protein levels were measured by methods as the same mentioned above.A model of carotid endothelial injury was established.At 28 days after operation,the protein level of Mtmr7 was determined by Western blotting.The mice were randomly divided into 4 groups(10 each):sham group,sham+Ad-Mtmr7 group,carotid endothelial injury group and carotid injury+Ad-Mtmr7 group.To overexpress Mtmr7,Ad-Mtmr7(5×10^(10)pfu/ml)was injected into the carotid artery immediately after operation and then partly incubated for 30 min.At 7th,14th and 21st day after operation,the mice were injected the adenovirus via tail vein.Twenty-eight days after modeling,the morphology of carotid artery and the degree of intimal hyperplasia were analyzed by HE staining.Results Compared with control group,the mRNA and protein levels of Mtmr7 were obviously reduced(P<0.001 and P<0.05),the rate of Ki-67 positive cells and the relative number of VSMCs increased(P<0.01 and P<0.001),the rate of wound healing and the protein expression levels of p-S6^(Ser235/236)and p-4EBP1^(Thr37/46)increased(P<0.001 and P<0.05)in PDGF-BB group.Compared with the PDGF-BB group,the rate of Ki-67 positive cells,the relative number of VSMCs(P<0.01 or P<0.001),the rate of wound healing(P<0.001)and the protein levels of p-S6^(Ser235/236)and p-4EBP1^(Thr37/46)(P<0.05)were decreased in Ad-Mtmr7+PDGF-BB group.Compared with the Ad-Mtmr7+PDGF-BB group,the protein levels of p-S6^(Ser235/236)and p-4EBP1^(Thr37/46)(P<0.01),the rate of Ki-67 positive cells,the relative number of VSMCs(P<0.01 or P<0.001)and the rate of wound healing(P<0.01)were increased in Ad-Mtmr7+PDGF-BB+insulin group.Compared with the sham group,the protein expression level of Mtmr7 decreased significantly(P<0.01)and the ratio of intima/media area increased(P<0.001)in carotid endothelial injury group.Compared with the carotid endothelial injury group,after overexpression of Mtmr7,the ratio of intima/media area decreased significantly(P<0.01)in carotid endothelial injury+Ad-Mtmr7 group.Conclusion Overexpression of Mtmr7 may inhibit the proliferation and migration of VSMCs induced by PDGF-BB in mice,alleviating intimal hyperplasia after vascular injury,which is closely related to the mTORC1 activity reduced by Mtmr7.