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CGRP修饰的间充质干细胞及其CGRP受体对血管平滑肌细胞增殖及表型转化的影响

Effects of CGRP and CGRP receptor modified mesenchymal stem cells on proliferation and phenotypic transformation of vascular smooth muscle cells

摘要:

目的 观察降钙素基因相关肽(CGRP)修饰的骨髓间充质干细胞(MSCs)与血管平滑肌细胞(VSMCs)共培养后,MSCs分泌的CGRP对VSMCs增殖及表型转化的影响及其具体的作用机制.方法 将携带CGRP的慢病毒载体转染MSCs(MSCsCGRP+/+),单纯慢病毒及PBS转染MSCs作为对照,应用ELISA法检测CGRP蛋白表达;携带RAMP1及shRAMP1的慢病毒载体转染VSMCs,实验分为Lenti-GFP-hRAMP1转染组(RAMP1+/+组)、Lenti-GFP-shRAMP1转染组(RAMP1-/-组)、Lenti-GFP转染组(GFP4/+组)和PBS转染对照组,应用Western印迹技术检测各组VSMCs中RAMP1蛋白表达.然后将MSCsCGRP+/+与过表达RAMP1及沉默RAMP1的VSMCs共培养,实验分为MSCs+VSMCs组、MSCsCGRP+/++ VSMCs组、MSCsCGRP+/++ VSMCsRAMP1+/+组、MSCsCGRP+/++ VSMCs RAMP1-/-4组;应用流式细胞术检测细胞增殖周期,MTT法检测细胞增殖率,Western印迹技术检测VSMCs收缩蛋白(α-SM-actin)和合成型骨桥蛋白(OPN)的表达.结果 过表达CGRP的MSCs其培养基中CGRP的表达量较对照组明显增加(19.530±0.498比3.133 ±0.160 and 3.120 ±0.001,P<0.05);过表达RAMP1及shRAMP1的慢病毒载体转染VSMCs后,RAMP1+/+组的RAMP1表达最多,而RAMP1在RAMP1-/-组表达最少.MSCs与VSMCs共培养后72 h,与MSCsCGRP+/++ VSMCs组比较,MSCsCGRP+/+ VSMCsRAMP1+/+组VSMCs增殖率明显增强(0.270±0.263与0.413±0.070,P<0.05),停留在静止期的细胞数相对增加[(93.51±0.38)%与(84.48 ±0.31)%,P<0.05],α-SM-actin表达显著增加(102 946±3847与51 759 ±635,P<0.05),OPN表达明显降低(26 026±2595与44 201 ±2811,P<0.05);然而沉默VSMCs中的RAMP1后,MSCsCGRP+/+与VSMCs共培养后72 h,CGRP抑制VSMCs的增殖能力较过表达RAMP1的VSMCs明显减弱(0.601±0.04与0.270±0.263,P<0.05),静止期的细胞数量减少[(78.57±0.68)%与(93.51±0.38)%,P<0.05],OPN蛋白表达增加(53 933 ±1192与26 026±2595,P<0.05),α-SM-actin表达减少(34 400±2179与102 946±3847,P<0.05).结论 CGRP转染MSCs后,细胞能分泌目的CGRP蛋白.CGRP修饰MSCs能明显抑制VSMCs的表型转化及细胞增殖,这可能与CGRP受体表达上调增强CGRP效应性有关.

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abstracts:

Objective To explore the effects and mechanism of secretory calcitonin gene-related peptide (CGRP) and CGRP receptor modified mesenchymal stem cells on proliferation and phenotypic transformation of vascular smooth muscle cell.Methods Firstly (Lenti-GFP-CGRP,referred to CGRP +/+)MSCs were transfected with high expression lentivirus vector of CGRP (MSCsCGRP+/+).Protein secretion in the above-mentioned MSCsCGRP+/+ supernatant was detected with enzyme-linked immunosorbent assay (ELISA).And then MSCsCGRP+/+ was co-cultured with VSMCsRAMP1 +/+ and VSMCsRAMP1-/-respectively.Experimental groups were as follows:MSCs + VSMCs MSCsCGRP+/+ + VSMCs,MSCsCGRP+/+ + VSMCsRAMP1 +/+ and MSCsCGRP+/+ + VSMCs RAMP1-/-Flow cytometry was applied to detect the cycle variation of smooth muscle cells,thiazolyl blue tetrazolium bromide method for detecting the proliferation of smooth muscle cells and Western blot for examining the expression changes of spectrin α-SM-actin and synthetic protein OPN in each group respectively.Results After the transfection of CGRP+/+,MSCsCGRP+/+ secreted and expressed CGRP protein,the secretory volume of CGRP protein in MSCsCGRP+/+ increased significantly compared with the control group(19.530 ± 0.498 vs 3.133 ± 0.160 and 3.120 ± 0.001,P <0.05).After a 72 h co-culturing with VSMCs,the proliferation of VSMCs in MSCsCGRP+/+ + VSMCsRAMP1 +/+ group declined significantly (0.270 ± 0.263 vs 0.413 ± 0.070,P < 0.05) and the number of cells staying in G0 phase significantly increased(93.51% ±0.38% vs 84.48% ±0.31%,P <0.05),the expression of contractile phenotype protein α-SM-actin increased and intermediate phenotype OPN declined significantly as compared with MSCsCGRP+/+ + VSMCs group { (α-SM-actin 102 946 ± 3847 vs 51 759 ±635,P < 0.05),OPN (26 026 ± 2595 vs 44 201 ± 2811,P < 0.05) } ; but compared with MSCsCGRP +/+ +VSMCsRAMP1+/+ group,the proliferation of VSMCs in MSCsCGRP+/+ + VSMCsRAMP1-/-group significantly increased(0.601 ± 0.04 vs 0.270 ± 0.263,P < 0.05) while the number of cells staying in GO phase significantly declined(78.57% ± 0.68% vs 93.51% ± 0.38%,P < 0.05).The expression of contractile phenotype protein α-SM-actin declined while intermediate phenotype OPN increased significantly in MSCsCGRP+/+ + VSMCsRAMP1-/-group { α-SM-actin (34 400 ± 2179 vs 102 946 ± 3847,P < 0.05),OPN (53 933 ± 1192 vs 26 026 ± 2595,P < 0.05) }.Conclusions CGRP gene-modified MSCs may secrete target protein stably.And CGRP-modified MSCs inhibits VSMCs phenotypic transformation and cell proliferation.It is probably associated with enhanced CGRP effect due to an up-regulation of CGRP receptor.

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