中华心血管病杂志
中華心血管病雜誌
중화심혈관병잡지
Chinese Journal of Cardiology
2015年
6期
537-541
,共5页
龙仙萍%崔璨%陈攀科%汪松%王冬梅%许官学%姚小剑%石蓓
龍仙萍%崔璨%陳攀科%汪鬆%王鼕梅%許官學%姚小劍%石蓓
룡선평%최찬%진반과%왕송%왕동매%허관학%요소검%석배
降钙素基因相关肽%肌细胞,平滑肌%干细胞
降鈣素基因相關肽%肌細胞,平滑肌%榦細胞
강개소기인상관태%기세포,평활기%간세포
Calcitonin gene-related peptide%Myocytes,smooth muscle%Stem cells
目的 探讨转染降钙素基因相关肽(CGRP)的骨髓间充质干细胞(MSC)对血管平滑肌细胞(VSMC)迁移的影响,及活性修饰蛋白1(RAMP1)在其中的作用和机制.方法 分离、培养大鼠MSC和VSMC,分别应用携带CGRP和RAMP1的慢病毒载体转染MSC和VSMC,然后将MSC与血管紧张素Ⅱ(AngⅡ)诱导的VSMC共培养.实验分为5组:(1) AngⅡ对照组(MSC+ VSMC+ AngⅡ);(2) MSCCGRP+组(MSCCGRP++ VSMC+ AngⅡ);(3)MSCCGRP+ RAMP1 组(MSCCGRP++ VSMCRAMP1-+AngⅡ);(4) MSCCGRP+ RAMP1+组:(MSCCGRP++ VSMCRAMP1++ AngⅡ);(5) RAMP1+ (MSC+VSMCRAMP1++ AngⅡ).应用Transwell实验评价VSMC的迁移能力,Western blot检测蛋白的表达水平.结果 Transwell实验结果显示,MSCCGRP+组VSMC迁移数量[(50.8±2.6)个]少于AngⅡ对照组[(71.4±2.3)个],而多于MSCCGRP+ RAMP1+组[(30.4±3.0)个](P均＜0.05);MSCCGRP+ RAMP1-组VSMC迁移数量[(69.0±5.6)个]多于MSCCGRP+ RAMP1+组(P＜0.05),而与AngⅡ对照组差异无统计学意义(P＞0.05);RAMP1+组VSMC迁移数量[(71.6±3.4)个]与AngⅡ对照组差异也无统计学意义(P＞0.05).Western blot结果显示,MSCCGRP+组磷酸化核因子-κB P65(P-P65)蛋白表达水平低于AngⅡ对照组(0.475±0.022比0.642±0.035,P＜0.05);MSCCGRP+ RAMP1-组P-P65蛋白表达水平高于MSCCGRP+ RAMP1+组(0.670±0.030比0.373 ±0.041,P＜0.05),而与AngⅡ对照组差异无统计学意义(P＞0.05);RAMP1+组P-P65蛋白表达量(0.643 ±0.039)与AngⅡ对照组比较差异也无统计学意义(P＞0.05).结论 RAMP1对CGRP抑制VSMC迁移起促进作用,RAMP1-CGRP通过核因子-κB信号通路抑制VSMC迁移.
目的 探討轉染降鈣素基因相關肽(CGRP)的骨髓間充質榦細胞(MSC)對血管平滑肌細胞(VSMC)遷移的影響,及活性脩飾蛋白1(RAMP1)在其中的作用和機製.方法 分離、培養大鼠MSC和VSMC,分彆應用攜帶CGRP和RAMP1的慢病毒載體轉染MSC和VSMC,然後將MSC與血管緊張素Ⅱ(AngⅡ)誘導的VSMC共培養.實驗分為5組:(1) AngⅡ對照組(MSC+ VSMC+ AngⅡ);(2) MSCCGRP+組(MSCCGRP++ VSMC+ AngⅡ);(3)MSCCGRP+ RAMP1 組(MSCCGRP++ VSMCRAMP1-+AngⅡ);(4) MSCCGRP+ RAMP1+組:(MSCCGRP++ VSMCRAMP1++ AngⅡ);(5) RAMP1+ (MSC+VSMCRAMP1++ AngⅡ).應用Transwell實驗評價VSMC的遷移能力,Western blot檢測蛋白的錶達水平.結果 Transwell實驗結果顯示,MSCCGRP+組VSMC遷移數量[(50.8±2.6)箇]少于AngⅡ對照組[(71.4±2.3)箇],而多于MSCCGRP+ RAMP1+組[(30.4±3.0)箇](P均＜0.05);MSCCGRP+ RAMP1-組VSMC遷移數量[(69.0±5.6)箇]多于MSCCGRP+ RAMP1+組(P＜0.05),而與AngⅡ對照組差異無統計學意義(P＞0.05);RAMP1+組VSMC遷移數量[(71.6±3.4)箇]與AngⅡ對照組差異也無統計學意義(P＞0.05).Western blot結果顯示,MSCCGRP+組燐痠化覈因子-κB P65(P-P65)蛋白錶達水平低于AngⅡ對照組(0.475±0.022比0.642±0.035,P＜0.05);MSCCGRP+ RAMP1-組P-P65蛋白錶達水平高于MSCCGRP+ RAMP1+組(0.670±0.030比0.373 ±0.041,P＜0.05),而與AngⅡ對照組差異無統計學意義(P＞0.05);RAMP1+組P-P65蛋白錶達量(0.643 ±0.039)與AngⅡ對照組比較差異也無統計學意義(P＞0.05).結論 RAMP1對CGRP抑製VSMC遷移起促進作用,RAMP1-CGRP通過覈因子-κB信號通路抑製VSMC遷移.
목적 탐토전염강개소기인상관태(CGRP)적골수간충질간세포(MSC)대혈관평활기세포(VSMC)천이적영향,급활성수식단백1(RAMP1)재기중적작용화궤제.방법 분리、배양대서MSC화VSMC,분별응용휴대CGRP화RAMP1적만병독재체전염MSC화VSMC,연후장MSC여혈관긴장소Ⅱ(AngⅡ)유도적VSMC공배양.실험분위5조:(1) AngⅡ대조조(MSC+ VSMC+ AngⅡ);(2) MSCCGRP+조(MSCCGRP++ VSMC+ AngⅡ);(3)MSCCGRP+ RAMP1 조(MSCCGRP++ VSMCRAMP1-+AngⅡ);(4) MSCCGRP+ RAMP1+조:(MSCCGRP++ VSMCRAMP1++ AngⅡ);(5) RAMP1+ (MSC+VSMCRAMP1++ AngⅡ).응용Transwell실험평개VSMC적천이능력,Western blot검측단백적표체수평.결과 Transwell실험결과현시,MSCCGRP+조VSMC천이수량[(50.8±2.6)개]소우AngⅡ대조조[(71.4±2.3)개],이다우MSCCGRP+ RAMP1+조[(30.4±3.0)개](P균＜0.05);MSCCGRP+ RAMP1-조VSMC천이수량[(69.0±5.6)개]다우MSCCGRP+ RAMP1+조(P＜0.05),이여AngⅡ대조조차이무통계학의의(P＞0.05);RAMP1+조VSMC천이수량[(71.6±3.4)개]여AngⅡ대조조차이야무통계학의의(P＞0.05).Western blot결과현시,MSCCGRP+조린산화핵인자-κB P65(P-P65)단백표체수평저우AngⅡ대조조(0.475±0.022비0.642±0.035,P＜0.05);MSCCGRP+ RAMP1-조P-P65단백표체수평고우MSCCGRP+ RAMP1+조(0.670±0.030비0.373 ±0.041,P＜0.05),이여AngⅡ대조조차이무통계학의의(P＞0.05);RAMP1+조P-P65단백표체량(0.643 ±0.039)여AngⅡ대조조비교차이야무통계학의의(P＞0.05).결론 RAMP1대CGRP억제VSMC천이기촉진작용,RAMP1-CGRP통과핵인자-κB신호통로억제VSMC천이.
Objective To investigate the impact of calcitonin gene-related peptide (CGRP) modified bone marrow mesenchymal stem cell (MSC) on the migration of vascular smooth muscle cell (VSMC) and related mechanisms.Methods The MSC and VSMC were isolated from rats and cultured,CGRP was transfected to MSC with the high expression lentivirus vector,VSMC was transfected with high expression lentivirus vector of receptor activity modifying protein 1 (RAMP1) and the silence expression lentivirus vector of RAMP1.Then MSC was co-cultured with VSMC.Experimental groups were as follows:(1) Ang Ⅱ group (MSC + VSMC + Ang Ⅱ);(2) MSCCGRP+ group (MSCCGRP+ + VSMC + Ang Ⅱ);(3)MSCCGRP+ RAMP1-group (MSCCGRP+ + VSMCRAMP1-+ Ang Ⅱ);(4) MSCCGRP+ RAMP1 + group (MSCCGRP+ + VSMCRAMP1 + + Ang Ⅱ);(5) RAMP1 + group (MSC + VSMCRAMP1 + + Ang Ⅱ).Transwell assay was applied to detect the migration of smooth muscle cells,Western blot was applied to detect the protein expression of cells in various groups.Results VSMC migration number was significantly lower in MSCCGRP+ group compared with AngⅡ group (50.8 ±2.6 vs.71.4 ±2.3,P ＜0.05),but higher than in MSCCGRP+ RAMP1 + group(50.8 ± 2.6 vs.30.4 ± 3.0,P ＜ 0.05).When RAMP1 expression reduced in VSMC,compared with MSCCGRP+ RAMP1 + group,VSMC migration increased in the MSCCGRP+ RAMP1-group compared to MSCCGRP + RAMP1 + (69.0 ± 5.6 vs.30.4 ± 3.0,P ＜ 0.05) and was similar to Ang Ⅱ group (69.0±5.6 vs.71.4±2.3,P＞0.05) and RAMP1+group (71.6 ±3.4).According to the result of Western blot,P-P65 protein expression in MSCCGRP+ group was lower than that in Ang Ⅱ group(0.475 ± 0.022 vs.0.642 ±0.035,P ＜0.05).P-P65 protein expression in MSCCGRP+ RAMP1-group was higher than that in MSCCGRP+ RAMP1 + group (0.670 ± 0.030 vs.0.373 ± 0.041,P ＜ 0.05),and there was no difference between MSCCGRP + RAMP1-group and Ang Ⅱ group (P ＞ 0.05).P-P65 protein expression was similar between RAMP1 + group (0.643 ± 0.039) and Ang Ⅱ group (P ＞ 0.05).Conclusions CGRP inhibits VSMC migration through RAMP1.NF-κB and RAMP1 play crucial role in the inhibiting effects of CGRP on VSMC migration.Thus,RAMP1-CGRP signaling inhibits VSMC migration through NF-κB signal pathways.
