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Article(id=1208055576609788248, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1208055572495179979, articleNumber=null, orderNo=null, doi=10.11855/j.issn.0577-7402.2022.06.0545, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1629993600000, receivedDateStr=2021-08-27, revisedDate=null, revisedDateStr=null, acceptedDate=1631635200000, acceptedDateStr=2021-09-15, onlineDate=1765952494050, onlineDateStr=2025-12-17, pubDate=1656345600000, pubDateStr=2022-06-28, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1765952494050, onlineIssueDateStr=2025-12-17, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1765952494050, creator=13701087609, updateTime=1765952494050, updator=13701087609, issue=Issue{id=1208055572495179979, tenantId=1146029695717560320, journalId=1189873630562394117, year='2022', volume='47', issue='6', pageStart='533', pageEnd='638', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1765952493070, creator=13701087609, updateTime=1765952764848, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1208056712481841868, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1208055572495179979, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1208056712481841869, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1208055572495179979, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=545, endPage=554, ext={EN=ArticleExt(id=1208055577129881949, articleId=1208055576609788248, tenantId=1146029695717560320, journalId=1189873630562394117, language=EN, title=Effect and mechanism of myotubularin-related protein 7 on proliferation and migration of mouse vascular smooth muscle cells, columnId=1190310110212751762, journalTitle=Medical Journal of Chinese People’s Liberation Army, columnName=Basic Research, runingTitle=null, highlight=null, articleAbstract=

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 (5×1010 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-S6Ser235/236 and p-4EBP1Thr37/46 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-S6Ser235/236 and p-4EBP1Thr37/46 (P<0.05) were decreased in Ad-Mtmr7+PDGF-BB group. Compared with the Ad-Mtmr7+PDGF-BB group, the protein levels of p-S6Ser235/236 and p-4EBP1Thr37/46 (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.

, correspAuthors=Da-Chun Yang, Xiong-Shan Sun, authorNote=null, correspAuthorsNote=
* Yang Da-Chun, E-mail: ;
Sun Xiong-Shan, E-mail:
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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(5×1010 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-S6Ser235/236和p-4EBP1Thr37/46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-S6Ser235/236和p-4EBP1Thr37/46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-S6Ser235/236和p-4EBP1Thr37/46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。

, correspAuthors=杨大春, 孙雄山, authorNote=null, correspAuthorsNote=
杨大春，E-mail：;
孙雄山，E-mail：
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赵维维，硕士研究生，主要从事心血管疾病的基础与临床研究

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赵维维，硕士研究生，主要从事心血管疾病的基础与临床研究

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赵维维，硕士研究生，主要从事心血管疾病的基础与临床研究

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Am J Transl Res, 2017, 9(3):1307-1316., articleTitle=Folic acid inhibits dedifferentiation of PDGF-BB-induced vascular smooth muscle cells by suppressing mTOR/P70S6K signaling, refAbstract=null), Reference(id=1208055593093403388, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208055576609788248, doi=null, pmid=null, pmcid=null, year=2017, volume=9, issue=11, pageStart=1176, pageEnd=null, url=null, language=null, rfNumber=[30], rfOrder=34, authorNames=Yoon MS, journalName=Nutrients, refType=null, unstructuredReference=Yoon MS. The role of mammalian target of rapamycin (mTOR)in insulin signaling[J]. 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A. qRT-PCR检测Mtmr7 mRNA表达水平(n=6)；B. Western blotting检测Mtmr7蛋白表达水平(n=3)；(1)P<0.05，(2)P<0.001

, figureFileSmall=dtKc2sNxGDnvKdMzM8EeWg==, figureFileBig=ojOnQeQ/sCYM6haFdjmCsA==, tableContent=null), ArticleFig(id=1208055582536340034, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208055576609788248, language=EN, label=Fig.2, caption=Effect of over-expressed Mtmr7 on proliferation and migration of PDGF-BB-induced mouse VSMC, figureFileSmall=gM7MnjDcm8mAML0PjxA9ew==, figureFileBig=cHuUM+JlFUddsir/pLbgRQ==, tableContent=null), ArticleFig(id=1208055582611837513, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208055576609788248, language=CN, label=图2, caption=过表达Mtmr7对PDGF-BB诱导的小鼠VSMC增殖和迁移的影响

A. Ki-67细胞免疫荧光染色检测小鼠VSMC增殖能力(×400)(n=3)；B. CCK-8法检测小鼠VSMC增殖能力(n=12)；C. 划痕实验检测小鼠VSMC迁移能力(×40)(n=3)；(1)P<0.01，(2)P<0.001

, figureFileSmall=gM7MnjDcm8mAML0PjxA9ew==, figureFileBig=cHuUM+JlFUddsir/pLbgRQ==, tableContent=null), ArticleFig(id=1208055582695723602, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208055576609788248, language=EN, label=Fig.3, caption=Effect of over-expressed Mtmr7 on mTORC1 activity in PDGF-BB-induced mouse VSMC (Western blotting, n=3), figureFileSmall=qU75Bfp5VMVlxvPDQQlf/Q==, figureFileBig=a8rKxsHudCIbeS3tsNrP6A==, tableContent=null), ArticleFig(id=1208055582779609687, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208055576609788248, language=CN, label=图3, caption=过表达Mtmr7对PDGF-BB诱导的小鼠VSMC中mTORC1活性的影响 (Western blotting，n=3)

(1)P<0.05，(2)P<0.01

, figureFileSmall=qU75Bfp5VMVlxvPDQQlf/Q==, figureFileBig=a8rKxsHudCIbeS3tsNrP6A==, tableContent=null), ArticleFig(id=1208055582888661595, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208055576609788248, language=EN, label=Fig.4, caption=Effect of insulin on proliferation and migration of PDGF-BB-induced mouse VSMCs after restoring mTORC1 activity, figureFileSmall=eIRvYj8qYN0zTVM+BZjbew==, figureFileBig=ta0qW1EuQMEluowYYkC+Uw==, tableContent=null), ArticleFig(id=1208055582959964769, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208055576609788248, language=CN, label=图4, caption=胰岛素恢复mTORC1活性对PDGF-BB诱导的小鼠VSMCs增殖和迁移的影响

A. Western blotting检测各组VSMCs中S6、p-S6Ser235/236和4EBP1、p-4EBP1Thr37/46蛋白表达水平(n=3)；B. Ki-67细胞免疫荧光染色检测小鼠VSMCs增殖能力(×400)(n=3)；C. CCK-8法检测小鼠VSMCs增殖能力(n=5)；D. 划痕实验检测小鼠VSMCs迁移能力(×40)(n=3)；(1)P<0.05，(2)P<0.01，(3)P<0.001

, figureFileSmall=eIRvYj8qYN0zTVM+BZjbew==, figureFileBig=ta0qW1EuQMEluowYYkC+Uw==, tableContent=null), ArticleFig(id=1208055583048045160, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208055576609788248, language=EN, label=Fig.5, caption=Expression of Mtmr7 after vascular endothelial injury in mice (Western blotting, n=3), figureFileSmall=T34TtiV4E+KepUT2D1S/Dw==, figureFileBig=0wmeoyyZULyN92bp5D0U2g==, tableContent=null), ArticleFig(id=1208055583136125550, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208055576609788248, language=CN, label=图5, caption=小鼠血管内皮损伤后Mtmr7的表达变化(Western blotting，n=3)

(1)P<0.01

, figureFileSmall=T34TtiV4E+KepUT2D1S/Dw==, figureFileBig=0wmeoyyZULyN92bp5D0U2g==, tableContent=null), ArticleFig(id=1208055583291314801, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208055576609788248, language=EN, label=Fig.6, caption=Effect of overexpression of Mtmr7 on intimal hyperplasia after vascular endothelial injury in mice(HE ×200) (n=10), figureFileSmall=YJBGLrCLWmYDbbUmkoWK8A==, figureFileBig=l0Wsi7eBoCfY0zl2WHSf9w==, tableContent=null), ArticleFig(id=1208055583400366710, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208055576609788248, language=CN, label=图6, caption=体内过表达Mtmr7对小鼠血管内皮损伤后内膜增生的影响(HE ×200)(n=10)

(1)P<0.01，(2)P<0.001

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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
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赵维维 1 , 杨大春 1, 2, * , 孙雄山 2, *
解放军医学杂志 | 基础研究 2022,47(6): 545-554
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解放军医学杂志 | 基础研究 2022, 47(6): 545-554
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
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赵维维1, 杨大春1, 2, * , 孙雄山2, *
作者信息
  • 1西南交通大学医学院，成都 610031
  • 2西部战区总医院心血管内科，成都 610083

    • 赵维维，硕士研究生，主要从事心血管疾病的基础与临床研究

通讯作者:

杨大春，E-mail：;
孙雄山，E-mail：
Effect and mechanism of myotubularin-related protein 7 on proliferation and migration of mouse vascular smooth muscle cells
Wei-Wei Zhao1, Da-Chun Yang1, 2, * , Xiong-Shan Sun2, *
Affiliations
  • 1College of Medicine, Southwest Jiaotong University, Chengdu 610031, China
  • 2Cardiovascular Department, General Hospital of PLA Western Theater Command, Chengdu 610083, China
出版时间: 2022-06-28 doi: 10.11855/j.issn.0577-7402.2022.06.0545
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摘要
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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(5×1010 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-S6Ser235/236和p-4EBP1Thr37/46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-S6Ser235/236和p-4EBP1Thr37/46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-S6Ser235/236和p-4EBP1Thr37/46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。

血管平滑肌细胞  /  肌微管素相关蛋白7  /  细胞增殖  /  细胞迁移  /  再狭窄

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 (5×1010 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-S6Ser235/236 and p-4EBP1Thr37/46 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-S6Ser235/236 and p-4EBP1Thr37/46 (P<0.05) were decreased in Ad-Mtmr7+PDGF-BB group. Compared with the Ad-Mtmr7+PDGF-BB group, the protein levels of p-S6Ser235/236 and p-4EBP1Thr37/46 (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.

vascular smooth muscle cells  /  myotubularin-related protein 7  /  cell proliferation  /  cell migration  /  restenosis
赵维维, 杨大春, 孙雄山. 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. 解放军医学杂志, 2022 , 47 (6) : 545 -554 . DOI: 10.11855/j.issn.0577-7402.2022.06.0545
Wei-Wei Zhao, Da-Chun Yang, Xiong-Shan Sun. Effect and mechanism of myotubularin-related protein 7 on proliferation and migration of mouse vascular smooth muscle cells[J]. Medical Journal of Chinese People’s Liberation Army, 2022 , 47 (6) : 545 -554 . DOI: 10.11855/j.issn.0577-7402.2022.06.0545
正文
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血管成形术已成为治疗冠心病最重要的治疗方法[1]，但血管再狭窄严重制约了血管重建手术的远期预后[2]。作为血管壁主要构成成分的血管平滑肌细胞(vascular smooth muscle cells，VSMCs)，其过度增殖和迁移是血管损伤后内膜增生、再狭窄的主要病理机制[3-4]。虽然抗增殖药物在防治血管内膜增生中有一定效果，但仍有5%~10%的患者会发生再狭窄[5]。因此，进一步揭示血管内膜增生、再狭窄发生的潜在机制并寻找新的干预靶点是当今临床和科研工作的重点。肌微管蛋白为高度保守的蛋白家族，是一大类广泛存在于人类的有脂质脱磷酸作用的蛋白，在细胞增殖、迁移、血管病理生理调控中发挥重要作用[6]。肌微管素相关蛋白7(myotubularin-related protein 7，Mtmr7)是肌微管蛋白家族的关键成员之一，在脑、肝、肾和睾丸等组织中均有表达[7]。既往研究发现，Mtmr7可参与肿瘤发生[8]、肌形成[9]、生殖细胞发育[10]等过程，被证实具有抑制多种细胞增殖的作用。然而，Mtmr7在血管及VSMCs中的表达及功能目前尚不明确。本研究使用血小板源性生长因子BB(platelet-derived growth factor BB，PDGF-BB)诱导VSMCs增殖和迁移[11]，并建立小鼠颈动脉内皮损伤模型[12]，探讨Mtmr7对VSMCs增殖和迁移的作用及机制，以期为防治血管再狭窄提供新思路。
1 材料与方法
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1.1 实验动物及主要材料、试剂
46只8~10周龄雄性野生型SPF级C57BL/6J小鼠，体重20~23 g，购自中国成都达硕生物有限公司。实验前小鼠适应性喂养2周，饲养室内环境保持恒温恒湿(温度22~25 ℃，湿度60%~65%)，给予规律光暗照射各12 h，自由摄食、饮水。实验过程符合国家及单位有关实验动物的管理及使用规定。小鼠主动脉平滑肌细胞系(MOVAS)购自中国深圳豪地华拓公司。DMEM高糖培养液、0.25%胰蛋白酶、PBS、双抗购自美国HyClone公司。胎牛血清(FBS)购自美国Gibco公司。PDGF-BB购自美国R&D公司。DMSO购自美国Sigma公司。腺病毒由中国上海吉凯基因科技有限公司构建。RNA提取试剂、RNA反转录试剂盒、荧光定量PCR试剂盒购自日本TaKaRa公司。qRT-PCR引物由北京擎科生物科技有限公司合成。GAPDH单克隆抗体购自中国武汉三鹰生物技术有限公司。Mtmr7多克隆抗体购自美国Invitrogen公司。Ki-67抗体购自中国北京博奥森生物有限公司。核糖体蛋白(S6)抗体、磷酸化(p)-S6Ser235/236抗体、4E结合蛋白1(4EBP1)抗体、p-4EBP1Thr37/46抗体购自美国CST公司。蛋白提取试剂盒、BCA蛋白定量试剂盒、山羊抗兔二抗、山羊抗鼠二抗、苏木精-伊红染色试剂盒、细胞增殖及毒性检测(CCK-8)试剂盒、重组人胰岛素购自北京索莱宝生物有限公司。ECL发光液购自美国Millipore公司。
1.2 方法
1.2.1 过表达腺病毒的构建
由上海吉凯基因科技有限公司构建重组Mtmr7基因过表达腺病毒(Ad-Mtmr7)和空载体腺病毒，Ad-Mtmr7所携带基因序列编号为NM-001040699。根据该公司腺病毒使用操作手册，制备5×104个/ml细胞悬液，将细胞悬液铺于孔板中，当细胞融合度达60%左右，将病毒稀释至1×108 pfu/ml，以最佳感染复数(MOI)=100时的病毒量，使用携带Mtmr7基因过表达腺病毒或空载腺病毒感染VSMCs，12 h后更换新鲜完全培养基，继续培养24 h后进行后续实验。
1.2.2 细胞培养及分组
MOVAS采用含10%FBS、1%双抗的DMEM高糖培养液培养，置于细胞培养箱中，培养条件为5% CO2、37 ℃。每2~3 d更换一次培养液，待细胞生长至约80%融合时，用0.25%胰酶消化后进行1:3或1:4传代。选择处于对数生长期的细胞进行实验。构建小鼠VSMCs增殖和迁移模型，用30 ng/ml PDGF-BB诱导小鼠VSMCs体外增殖和迁移[11]，所有实验均以DMSO为溶剂。另取MOVAS细胞分为对照组、PDGF-BB组、Ad-Mtmr7组和Ad-Mtmr7+PDGF-BB组。用5 mg/L胰岛素(insulin)恢复哺乳动物雷帕霉素靶蛋白复合物1(mammalian target of rapamycin complex 1，mTORC1)的活性[13]，将MOVAS细胞分为PDGF-BB组、Ad-Mtmr7+PDGF-BB组和Ad-Mtmr7+PDGF-BB+insulin组，观察小鼠VSMCs增殖和迁移情况。
1.2.3 Western blotting检测Mtmr7及mTORC1下游靶标蛋白水平
提取各组VSMCs(详见1.2.2)蛋白检测Mtmr7及mTORC1下游靶标蛋白水平，提取血管组织蛋白检测Mtmr7蛋白水平，按照蛋白提取试剂盒说明书操作。BCA法测定蛋白浓度，标准化各组蛋白样品上样量。蛋白样品进行SDS-PAGE电泳分离，然后转至PVDF膜上，5%脱脂奶粉或胎牛血清中室温封闭1 h[14]。加入相应的Mtmr7抗体、S6抗体、p-S6Ser235/236抗体、4EBP1抗体、p-4EBP1Thr37/46抗体和GAPDH抗体，抗体稀释比均为1:1000，置于4 ℃摇床上孵育过夜。次日，室温复温30 min后TBST洗膜，再用二抗(1:5000)室温摇床上孵育1 h。TBST洗膜，滴加ECL发光液后曝光。采用ImageJ软件分析条带灰度值。
1.2.4 qRT-PCR检测Mtmr7 mRNA水平
根据RNA提取试剂盒说明书步骤提取PDGF-BB、DMSO处理后的VSMCs总RNA。标准化RNA浓度，将RNA反转录为cDNA后进行qRT-PCR扩增目的基因和内参基因，以GAPDH为内参，用2–ΔΔCt法计算Mtmr7的相对表达量。引物序列如下：Mtmr7，上游5'-GTGGAAGAATCGAGCTGACTAC-3'，下游5'-CCCCTTTTCAAAGCGGTTATACA-3'；GAPDH，上游5'-AGGTCGGTGTGAACGGATTTG-3'，下游5'-TGTAGACCATGTAGTTGAGGTCA-3'。
1.2.5 Ki-67细胞免疫荧光染色检测VSMCs增殖能力
在24孔板中放入爬片，加入细胞悬液，按1.2.2分组处理后倒掉培养液，PBS洗3次；4%多聚甲醛溶液固定30 min，PBS洗3次；0.3%的TritonX-100室温通透15 min，PBS洗3次；吸水纸吸干，山羊血清室温封闭30 min；吸水纸吸掉封闭液，每张玻片滴加足量的Ki-67抗体(1:100)并放入4 ℃湿盒中过夜。次日，复温30 min后PBS洗3次；随后在暗室中滴加荧光二抗(1:100)，湿盒37 ℃孵育1 h；滴加DAPI复染核5 min，PBS洗3次；吸水纸吸干液体后用含抗荧光淬灭剂的封片液进行封片，在荧光显微镜下观察并采集图像[15]。
1.2.6 CCK-8法检测VSMCs增殖能力
用完全培养液制备2×105个/ml的细胞悬液，每孔100 μl接种于96孔板中，每组设置5~10个复孔。按1.2.2分组干预后，待细胞增殖至80%左右取出96孔板，每孔加入CCK-8溶液10 μl，遮光锡纸包裹后继续孵育1.5 h，采用酶标仪检测450 nm处的吸光度(OD)值，并绘制细胞生长曲线，计算并比较各组VSMCs相对数量。
1.2.7 划痕实验检测VSMCs迁移能力
将VSMCs接种于6孔板中(1×105个/孔)，按1.2.2分组进行腺病毒处理后，培养细胞生长至细胞单层融合，用无菌200 μl吸头垂直于细胞培养板底划线。用PBS洗去多余的悬浮细胞，在显微镜下拍摄0 h照片。用无血清培养液培养，根据分组加入PDGF-BB或DMSO或胰岛素处理，置于细胞培养箱中继续培养至24 h，在显微镜下拍摄24 h照片。ImageJ软件分析结果。
1.2.8 小鼠颈动脉内皮损伤模型的建立及分组
雄性野生型C57BL/6J小鼠按照参考文献[12]的方法建立小鼠颈动脉内皮损伤模型：小鼠称重后用0.3%戊巴比妥钠(30 mg/kg)腹腔注射麻醉，固定于手术台上；小鼠颈部皮肤脱毛，消毒、铺单，行颈部正中切口1.0~1.5 cm；分离出左侧颈总、颈内、颈外动脉，结扎颈外动脉远心端，夹闭颈总动脉近心端和颈内动脉，从颈外动脉近心端插入粗糙导丝(0.38 mm)至颈总动脉，来回旋转导丝以损伤血管内皮，拔出导丝，结扎穿刺段血管并松开血管夹，缝合包扎。行假手术的小鼠不用导丝损伤颈动脉内皮，其余操作与行颈动脉内皮损伤手术的小鼠相同。分别取假手术及颈动脉内皮损伤小鼠3只用于Western blotting检测Mtmr7蛋白水平。为观察Mtmr7在血管内皮损伤后内膜增生中的作用，将其余40只小鼠随机分为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。假手术+Ad-Mtmr7组与颈动脉内皮损伤+Ad-Mtmr7组在小鼠手术后立即用Ad-Mtmr7(5×1010 pfu/ml)行颈动脉局部孵育30 min，在造膜第7、14、21天行尾静脉注射，以实现Mtmr7过表达；假手术组与颈动脉内皮损伤组则用同等剂量空载腺病毒处理。造膜28 d后，用0.3%戊巴比妥钠(100 mg/kg)腹腔注射处死小鼠，取出颈总动脉，以备HE染色使用。
1.2.9 HE染色观察小鼠颈动脉形态及内膜增生情况
取出假手术组、假手术+Ad-Mtmr7组、颈动脉内皮损伤组、颈动脉内皮损伤+Ad-Mtmr7组小鼠颈总动脉，用4%多聚甲醛固定、脱水、石蜡包埋、切片。二甲苯脱蜡，梯度乙醇脱水，苏木精染色，盐酸乙醇分化，氨水返蓝，伊红染色，自来水冲洗，脱水，透明，晾干，封片，于显微镜下观察。
1.3 统计学处理
采用SPSS 20.0软件进行统计分析。计量资料以$\bar{x}±s$表示，符合正态分布且方差齐，两组间比较采用独立样本t检验，多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。采用双侧检验，P<0.05为差异有统计学意义。
2 结果
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2.1 PDGF-BB对小鼠VSMCs中Mtmr7表达的影响
qRT-PCR结果显示，与对照组比较，PDGF-BB组Mtmr7 mRNA水平明显降低(P<0.001，图1A)；Western blotting检测结果显示，与对照组比较，Mtmr7蛋白表达水平也明显降低(P<0.05，图1B)。
2.2 过表达Mtmr7对PDGF-BB诱导的小鼠VSMCs增殖和迁移的影响
Ki-67细胞免疫荧光染色结果显示，PDGF-BB组Ki-67阳性细胞率明显高于对照组(P<0.01)，而Ad-Mtmr7组Ki-67阳性细胞率与对照组比较差异无统计学意义(P>0.05)；与PDGF-BB组比较，Ad-Mtmr7+PDGF-BB组Ki-67阳性细胞率明显降低(P<0.01，图2A)。CCK-8法检测结果与Ki-67细胞免疫荧光染色结果相似，与对照组比较，PDGF-BB组VSMCs相对数量明显增多(P<0.001)，Ad-Mtmr7组VSMCs相对数量变化不大(P>0.05)；与PDGF-BB组比较，Ad-Mtmr7+PDGF-BB组VSMCs相对数量明显减少(P<0.001，图2B)。划痕实验结果显示，与对照组比较，PDGF-BB组划痕愈合率增高(P<0.001)，Ad-Mtmr7组划痕愈合速度无明显差异(P>0.05)；与PDGF-BB组比较，Ad-Mtmr7+PDGF-BB组划痕愈合率明显降低(P<0.001，图2C)。
2.3 过表达Mtmr7对PDGF-BB诱导的小鼠VSMCs中mTORC1活性的影响
Western blotting结果显示，与对照组比较，PDGF-BB组p-S6Ser235/236和p-4EBP1Thr37/46蛋白表达水平明显升高(P<0.05)，Ad-Mtmr7组p-S6Ser235/236和p-4EBP1Thr37/46蛋白表达水平无明显变化(P>0.05)；与PDGF-BB组比较，Ad-Mtmr7+PDGF-BB组p-S6Ser235/236和p-4EBP1Thr37/46蛋白表达水平明显降低(P<0.05，图3)。
2.4 胰岛素恢复mTORC1活性对PDGF-BB诱导的小鼠VSMCs增殖和迁移的影响
Western blotting检测结果显示，与PDGF-BB组比较，Ad-Mtmr7+PDGF-BB组p-S6Ser235/236和p-4EBP1Thr37/46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-S6Ser235/236和p-4EBP1Thr37/46蛋白表达水平升高(P<0.01，图4A)。Ki-67细胞免疫荧光染色结果显示，与PDGF-BB组比较，Ad-Mtmr7+PDGF-BB组Ki-67阳性细胞率降低(P<0.001)；与Ad-Mtmr7+PDGF-BB组比较，Ad-Mtmr7+PDGF-BB+insulin组Ki-67阳性细胞率增高(P<0.01，图4B)。CCK-8法检测结果显示，与PDGF-BB组比较，Ad-Mtmr7+PDGF-BB组VSMCs相对数量减少(P<0.001)；与Ad-Mtmr7+PDGF-BB组比较，Ad-Mtmr7+PDGF-BB+insulin组VSMCs相对数量增多(P<0.001，图4C)。划痕实验结果显示，与PDGF-BB组比较，Ad-Mtmr7+PDGF-BB组划痕愈合率降低(P<0.01)；与Ad-Mtmr7+PDGF-BB组相比，Ad-Mtmr7+PDGF-BB+insulin组划痕愈合率增高(P<0.01，图4D)。
2.5 小鼠血管内皮损伤后Mtmr7蛋白的表达变化
Western blotting检测结果显示，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，图5)。
2.6 体内过表达Mtmr7对小鼠血管内皮损伤后内膜增生的影响
HE染色结果显示，与假手术组比较，颈动脉内皮损伤组颈动脉内膜/中膜面积比值增大(P<0.001)，假手术+Ad-Mtmr7组颈动脉内膜/中膜面积比值无明显变化(P>0.05)；而与颈动脉内皮损伤组相比，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，图6)。
3 讨论
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血管再狭窄是由于新内膜增生引起的血管腔直径减小，传统上将其定义为支架内或支架边缘5 mm以内冠状动脉节段直径狭窄>50%[16]。血管再狭窄是局部血管损伤后的一种修复反应，其形成机制较复杂，涉及血管弹性回缩、血小板沉积、血栓形成、炎症反应、细胞外基质沉积、血管重塑、VSMCs过度增殖和迁移等多种因素[17-18]。相关研究表明，VSMCs增殖是再狭窄的一个重要病理改变，介入治疗过程中可发生血管损伤，血管中膜的VSMCs由正常的收缩型细胞转化为合成型细胞，合成并分泌血小板源性生长因子(PDGF)、肿瘤坏死因子-α(TNF-α)、胰岛素样生长因子1(IGF-1)、血管紧张素 Ⅱ(Ang Ⅱ)、白细胞介素-1(IL-1)、IL-6等多种生长因子和细胞因子，从而产生、分泌大量胶原和细胞外基质，最终引起血管再狭窄[19-20]。在上述过程中多种细胞因子分泌增加，促进了介入术后的炎性细胞浸润，以及血管内皮和VSMCs的增殖和迁移[21]。此外，再狭窄形成的相关分子机制涉及Ras/有丝分裂原活化蛋白激酶(MAPK)、Src激酶、磷脂酰肌醇-3-激酶/蛋白激酶B(PI3K/Akt)、磷脂酶C-γ和两面神激酶/信号转导子和转录激活子(JAK/STAT)等多个分子及细胞信号通路[22]。针对以上分子及细胞信号通路，研究人员进行了多种尝试，主要是利用抗增生药物的药物洗脱支架，但仍不能完全有效降低再狭窄的发生率，因此，仍需继续深入研究调控再狭窄形成的潜在机制。
Mtmr7是肌微管蛋白家族的关键成员之一。肌微管蛋白家族由15种蛋白质组成，包括肌管蛋白1(Mtm1)和肌微管素相关蛋白1至14(Mtmr1-14)，各有其独特的功能[23]，其中9个被证实具有磷酸酶催化活性；多个家族成员与神经肌肉疾病、代谢综合征、肥胖症、癌症的发生相关，可调节细胞的增殖和分化、自噬、分裂等过程[6]。Mtmr7可参与肿瘤发生、肌形成、生殖细胞发育等过程，并被证实具有抑制多种细胞增殖的作用，如Mtmr7蛋白在人结直肠癌细胞中低表达，可抑制人结直肠癌细胞的增殖[8]，沉默Mtmr7可增强C2C12成肌细胞的增殖和早期分化[9]，Mtmr7缺乏可促进体外精原干细胞的增殖[10]。因此，推测Mtmr7也具有抑制VSMCs增殖的作用。
既往研究提示，PDGF-BB可显著诱导VSMCs的增殖和迁移[24]。本研究利用PDGF-BB诱导小鼠VSMCs增殖和迁移，发现PDGF-BB可降低小鼠VSMCs中Mtmr7的表达水平，提示Mtmr7可能在过度增殖、迁移的VSMCs中发挥了一定的调控作用。为进一步明确Mtmr7在小鼠VSMCs增殖和迁移中的作用，本研究采用腺病毒感染VSMCs实现Mtmr7过表达，结果发现Ki-67阳性细胞率、VSMCs相对数量、划痕愈合率均降低，提示过表达Mtmr7可明显抑制PDGF-BB诱导的小鼠VSMCs增殖和迁移。但Mtmr7调控VSMCs增殖和迁移的机制目前尚未见报道。
mTORC1是一种蛋白激酶复合物，由mTOR与Raptor、DEPTOR、PRA40、mLST8构成，对雷帕霉素的作用十分敏感[25]，在控制细胞生长、增殖、迁移、新陈代谢等生物学过程中具有重要作用[26]。S6和4EBP1是mTORC1复合体的两个主要下游效应靶标，其磷酸化水平可反映mTORC1的活性[26]。既往研究发现，mTORC1的激活可使VSMCs增殖活性增强[27]，而mTORC1失活则可抑制新内膜增生和再狭窄的发生[28]。mTORC1在PDGF-BB诱导的VSMCs增殖、迁移中发挥重要作用，PDGF-BB可通过增强mTORC1活性来促进VSMCs的增殖和迁移[11-12,29]。此外，有研究发现，Mtmr7对结肠癌细胞的增殖抑制效应与mTORC1的失活密切相关[8]。因此，为了进一步了解Mtmr7调控VSMCs增殖和迁移的机制，本研究首先检测了Mtmr7对mTORC1活性的影响，发现过表达Mtmr7可降低S6和4EBP1的磷酸化水平，提示mTORC1的活性受到抑制。有文献报道，胰岛素可通过PI3K/AKT途径激活mTORC1[30]。本研究也发现，应用胰岛素后p-S6Ser235/236和p-4EBP1Thr37/46蛋白表达水平明显升高，提示胰岛素恢复了mTORC1的活性，而mTORC1活性恢复后，过表达Mtmr7对小鼠VSMCs增殖和迁移的抑制作用被逆转，这与以往研究中胰岛素通过激活mTORC1促进细胞增殖和迁移的结论[31]一致。据此推论，过表达Mtmr7可通过降低mTORC1活性来抑制小鼠VSMCs的增殖和迁移。
在体外研究结果的基础上，为了进一步验证Mtmr7在血管内皮损伤后内膜增生中的作用，本研究采用机械性损伤血管内皮法建立了小鼠颈动脉内皮损伤模型。实验结果发现，在颈动脉内皮损伤后Mtmr7蛋白表达水平降低，提示Mtmr7可能在内膜增生中发挥有益的调控作用。此外，本研究采用过表达Mtmr7腺病毒颈动脉局部孵育和尾静脉注射的方法实现体内过表达Mtmr7，结果显示，血管局部过表达Mtmr7可减轻血管损伤后的管腔狭窄和内膜增生，提示体内过表达Mtmr7可抑制小鼠血管损伤后的内膜增生。
综上所述，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，这可能是防止再狭窄发生发展的一种潜在机制，有望成为防治血管再狭窄的新靶点。但由于本研究主要集中于体外实验，体内实验研究较为局限，尤其是Mtmr7减轻血管损伤后管腔狭窄和内膜增生的具体机制是否与体外研究结果一致仍需进一步探讨。
基金
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  • 国家自然科学基金(82100419)
  • 国家自然科学基金(81770299)
文献
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2022年第47卷第6期
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doi: 10.11855/j.issn.0577-7402.2022.06.0545
  • 接收时间：2021-08-27
  • 首发时间：2025-12-17
  • 出版时间：2022-06-28
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  • 收稿日期：2021-08-27
  • 录用日期：2021-09-15
基金
National Natural Science Foundation of China(82100419)
国家自然科学基金(82100419)
National Natural Science Foundation of China(81770299)
国家自然科学基金(81770299)
作者信息
    1西南交通大学医学院，成都 610031
    2西部战区总医院心血管内科，成都 610083

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Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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