中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2015年
20期
1619-1624
,共6页
石蓓%陈攀科%龙仙萍%赵然尊%邓文文
石蓓%陳攀科%龍仙萍%趙然尊%鄧文文
석배%진반과%룡선평%조연존%산문문
降钙素基因相关肽%间充质干细胞%血管平滑肌细胞%增殖%血管狭窄
降鈣素基因相關肽%間充質榦細胞%血管平滑肌細胞%增殖%血管狹窄
강개소기인상관태%간충질간세포%혈관평활기세포%증식%혈관협착
Calcitonin gene-related peptide%Mesenchymal stem cells%Vascular smooth muscle cells%Proliferation%Restenosis
目的 探讨降钙素基因相关肽(CGRP)修饰的间充质干细胞(MSCs)对血管平滑肌细胞(VSMCs)增殖和血管狭窄的影响.方法 培养应用MSCs与VSMCs,Lv-CGRP-EGFP转染MSCs,采用逆转录-聚合酶链反应(RT-PCR)和酶联免疫吸附实验(ELISA)法检测MSCs中CGRP的表达.MSCs与VSMCs共培养后,实验分为MSCsCGRP+/++ VSMCs组、MSCsCGRP-/-+ VSMCs和MSCsPBS+VSMCs组,应用3-(4,5-二甲基-2-噻唑)-2,5-二苯基溴化四唑(MTT)法检测VSMCs增殖能力.构建大鼠颈动脉球囊损伤模型,携带慢病毒的MSCs移植动物模型中,细胞移植后7d,Western印迹法检测血管中CGRP表达,免疫荧光染色检测损伤血管CD31的表达;细胞移植后28 d,苏木精-伊红染色法(H-E染色)检测损伤血管新生内膜/中膜面积;免疫组化法检测增殖细胞核抗原(PCNA)的表达.结果 病毒转染后48 h,与PBS组比较,CGRP修饰的MSCs中CGRP的mRNA(0.135±0.049比25.306±0.110)和蛋白水平(1.953±0.039比19.483±0.421)均增高(均P＜0.05).细胞共培养后24、48、72、96 h,MTT染色显示,与MSCsPBS+ VSMCs组比较,MSCsCGRP+/++ VSMCs组中VSMCs的增殖活性明显减弱[24 h(0.54±0.06比0.44±0.06),48 h(0.69 ±0.11比0.54±0.07),72 h(0.87±0.08比0.70 ±0.11),96 h(1.42±0.28比1.10±0.24)均P＜0.05].体内研究显示:细胞移植后7d,与对照组比较,MSCs-CGRP移植组中CGRP表达水平明显增加(23.953±3.039比50.451±5.311)(均P＜0.05),免疫荧光染色检测显示MSCs-CGRP组内膜有CD31连续性表达,而对照组没有.细胞移植后28 d,HE染色显示,与对照组(0.99±0.06)比较,MSCs-CGRP组(0.48±0.03)血管增生内膜面积明显减小(均P＜0.05),免疫组化结果显示,MSCs-CGRP组中血管内膜的PCNA表达量较对照组明显降低(78.4％±10.2％比25.1％±6.0％,P＜0.05).结论 转染CGRP的MSCs能分泌CGRP蛋白和表达CGRP mRNA,CGRP修饰MSCs更能抑制VSMCs增殖,减轻血管损伤后新生内膜面积.
目的 探討降鈣素基因相關肽(CGRP)脩飾的間充質榦細胞(MSCs)對血管平滑肌細胞(VSMCs)增殖和血管狹窄的影響.方法 培養應用MSCs與VSMCs,Lv-CGRP-EGFP轉染MSCs,採用逆轉錄-聚閤酶鏈反應(RT-PCR)和酶聯免疫吸附實驗(ELISA)法檢測MSCs中CGRP的錶達.MSCs與VSMCs共培養後,實驗分為MSCsCGRP+/++ VSMCs組、MSCsCGRP-/-+ VSMCs和MSCsPBS+VSMCs組,應用3-(4,5-二甲基-2-噻唑)-2,5-二苯基溴化四唑(MTT)法檢測VSMCs增殖能力.構建大鼠頸動脈毬囊損傷模型,攜帶慢病毒的MSCs移植動物模型中,細胞移植後7d,Western印跡法檢測血管中CGRP錶達,免疫熒光染色檢測損傷血管CD31的錶達;細胞移植後28 d,囌木精-伊紅染色法(H-E染色)檢測損傷血管新生內膜/中膜麵積;免疫組化法檢測增殖細胞覈抗原(PCNA)的錶達.結果 病毒轉染後48 h,與PBS組比較,CGRP脩飾的MSCs中CGRP的mRNA(0.135±0.049比25.306±0.110)和蛋白水平(1.953±0.039比19.483±0.421)均增高(均P＜0.05).細胞共培養後24、48、72、96 h,MTT染色顯示,與MSCsPBS+ VSMCs組比較,MSCsCGRP+/++ VSMCs組中VSMCs的增殖活性明顯減弱[24 h(0.54±0.06比0.44±0.06),48 h(0.69 ±0.11比0.54±0.07),72 h(0.87±0.08比0.70 ±0.11),96 h(1.42±0.28比1.10±0.24)均P＜0.05].體內研究顯示:細胞移植後7d,與對照組比較,MSCs-CGRP移植組中CGRP錶達水平明顯增加(23.953±3.039比50.451±5.311)(均P＜0.05),免疫熒光染色檢測顯示MSCs-CGRP組內膜有CD31連續性錶達,而對照組沒有.細胞移植後28 d,HE染色顯示,與對照組(0.99±0.06)比較,MSCs-CGRP組(0.48±0.03)血管增生內膜麵積明顯減小(均P＜0.05),免疫組化結果顯示,MSCs-CGRP組中血管內膜的PCNA錶達量較對照組明顯降低(78.4％±10.2％比25.1％±6.0％,P＜0.05).結論 轉染CGRP的MSCs能分泌CGRP蛋白和錶達CGRP mRNA,CGRP脩飾MSCs更能抑製VSMCs增殖,減輕血管損傷後新生內膜麵積.
목적 탐토강개소기인상관태(CGRP)수식적간충질간세포(MSCs)대혈관평활기세포(VSMCs)증식화혈관협착적영향.방법 배양응용MSCs여VSMCs,Lv-CGRP-EGFP전염MSCs,채용역전록-취합매련반응(RT-PCR)화매련면역흡부실험(ELISA)법검측MSCs중CGRP적표체.MSCs여VSMCs공배양후,실험분위MSCsCGRP+/++ VSMCs조、MSCsCGRP-/-+ VSMCs화MSCsPBS+VSMCs조,응용3-(4,5-이갑기-2-새서)-2,5-이분기추화사서(MTT)법검측VSMCs증식능력.구건대서경동맥구낭손상모형,휴대만병독적MSCs이식동물모형중,세포이식후7d,Western인적법검측혈관중CGRP표체,면역형광염색검측손상혈관CD31적표체;세포이식후28 d,소목정-이홍염색법(H-E염색)검측손상혈관신생내막/중막면적;면역조화법검측증식세포핵항원(PCNA)적표체.결과 병독전염후48 h,여PBS조비교,CGRP수식적MSCs중CGRP적mRNA(0.135±0.049비25.306±0.110)화단백수평(1.953±0.039비19.483±0.421)균증고(균P＜0.05).세포공배양후24、48、72、96 h,MTT염색현시,여MSCsPBS+ VSMCs조비교,MSCsCGRP+/++ VSMCs조중VSMCs적증식활성명현감약[24 h(0.54±0.06비0.44±0.06),48 h(0.69 ±0.11비0.54±0.07),72 h(0.87±0.08비0.70 ±0.11),96 h(1.42±0.28비1.10±0.24)균P＜0.05].체내연구현시:세포이식후7d,여대조조비교,MSCs-CGRP이식조중CGRP표체수평명현증가(23.953±3.039비50.451±5.311)(균P＜0.05),면역형광염색검측현시MSCs-CGRP조내막유CD31련속성표체,이대조조몰유.세포이식후28 d,HE염색현시,여대조조(0.99±0.06)비교,MSCs-CGRP조(0.48±0.03)혈관증생내막면적명현감소(균P＜0.05),면역조화결과현시,MSCs-CGRP조중혈관내막적PCNA표체량교대조조명현강저(78.4％±10.2％비25.1％±6.0％,P＜0.05).결론 전염CGRP적MSCs능분비CGRP단백화표체CGRP mRNA,CGRP수식MSCs경능억제VSMCs증식,감경혈관손상후신생내막면적.
Objective To explore the role of calcitonin gene-related peptide (CGRP) in the proliferation of vascular smooth muscle cells (VSMCs) and restenosis.Methods The high-expression CGRP lentivirus [Lenti-green fluorescent protein (GFP)-CGRP] was constructed.And mesenchymal stem cells (MSCs) were transfected with Lenti-GFP-CGRP,Lenti-GFP and phosphate buffer saline (PBS).Reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to determine the CGRP gene and protein expression level of smooth muscle cells in each group respectively.Then MSCs were co-cultured with VSMCs.Experimental groups were MSCsCGRP+/+ + VSMCs,MSCsCGHP-/-+ VSMCs and MSCsPBS + VSMCs groups.The method of methyl thiazolyl tetrazolium (MTT) was employed to detect the proliferation of smooth muscle cells.Sacculus damaged atherosclerotic carotid was prepared according to the previous study.MSCs were transfected with Lv-CGRP-EGFP and Lv-CGRP and then transplanted into rat model.At Day 7 post-transplantation,injured carotid artery was harvested to detect the C expression of GRP by Western blot and the expression of CD31 by immunofluorescence.At Day 28 post-transplantation,injured carotid artery was harvested to assess organization morphology by hematoxylin and eosin stain and detect the expression of proliferating cell nuclear antigen (PCNA) by immunohistochemical stain.Results As compared with control and Lenti-GFP groups,the expressions of CGRP protein and CGRP mRNA increased at 72 h after transfecting with Lenti-GFP-CGRP (P ＜0.05).After 72-hour co-culturing with VSMCs,the proliferation ability of VSMCs was the lowest in MSCsCGRP+/+ + VSMCs group versus other three groups (P ＜ 0.05).At Day 7 post-transplantation,as compared with control and Lenti-GFP groups,the expression of CGRP proteins increased significantly in MSCs-CGRP group (P ＜ 0.05).A continuous expression of CD31 was found in damaged carotid intima in MSCs-CGRP group,but not in control group.At Day 28 post-transplantation,the area of intimal hyperplasia was smaller in MSCs-CGRP group than that in control and Lenti-GFP groups.Also the expression of PCNA decreased more in MSCs-CGRP group than control and Lenti-GFP groups (P ＜ 0.05).Conclusions CGRP protein and CGRP mRNA are expressed after CGRP transfection.And CGRP can suppress the proliferation of VSMCs and reduce intimal hyperplasia so as to facilitate a recovery of damaged endothelium.
