Long Xianping, Cui Can, Chen Panke, Wang Song, Wang Dongmei, Xu Guanxue, Yao Xiaojian, Shi Bei
Deparment of Cardiology, First Affiliated Hospital, Zunyi Medical College, Zunyi 563003, China.
Deparment of Cardiology, First Affiliated Hospital, Zunyi Medical College, Zunyi 563003, China; Email:
Zhonghua Xin Xue Guan Bing Za Zhi. 2015 Jun;43(6):537-41.
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.
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) MSC(CGRP+) group (MSC(CGRP+) + VSMC + Ang II); (3) MSC(CGRP+) RAMP1(-) group (MSC(CGRP+) + VSMC(RAMP1-) + Ang II); (4) MSC(CGRP+) RAMP1(+) group (MSC(CGRP+) + VSMC(RAMP1+) + Ang II); (5) RAMP1(+) group (MSC + VSMC(RAMP1+) + 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.
VSMC migration number was significantly lower in MSC(CGRP+) group compared with Ang II group (50.8 ± 2.6 vs. 71.4 ± 2.3, P < 0.05), but higher than in MSC(CGRP+) RAMP1(+) group (50.8 ± 2.6 vs. 30.4 ± 3.0, P < 0.05). When RAMP1 expression reduced in VSMC, compared with MSC(CGRP+) RAMP1(+) group, VSMC migration increased in the MSC(CGRP+) RAMP1(-) group compared to MSC(CGRP+)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 MSC(CGRP+) 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 MSC(CGRP+)RAMP1(-) group was higher than that in MSC(CGRP+) RAMP1(+) group (0.670 ± 0.030 vs. 0.373 ± 0.041, P < 0.05), and there was no difference between MSC(CGRP+)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).
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.
探讨降钙素基因相关肽（CGRP）修饰的骨髓间充质干细胞（MSC）对血管平滑肌细胞（VSMC）迁移的影响及其相关机制。
从大鼠体内分离并培养MSC和VSMC，用高表达慢病毒载体将CGRP转染至MSC，用受体活性修饰蛋白1（RAMP1）的高表达慢病毒载体和RAMP1的沉默表达慢病毒载体转染VSMC。然后将MSC与VSMC共培养。实验组如下：（1）Ang II组（MSC + VSMC + Ang II）；（2）MSC（CGRP+）组（MSC（CGRP+）+ VSMC + Ang II）；（3）MSC（CGRP+）RAMP1（-）组（MSC（CGRP+）+ VSMC（RAMP1-）+ Ang II）；（4）MSC（CGRP+）RAMP1（+）组（MSC（CGRP+）+ VSMC（RAMP1+）+ Ang II）；（5）RAMP1（+）组（MSC + VSMC（RAMP1+）+ Ang II）。采用Transwell实验检测平滑肌细胞的迁移，采用蛋白质免疫印迹法检测各组细胞的蛋白表达。
与Ang II组相比，MSC（CGRP+）组的VSMC迁移数量显著降低（50.8±2.6对71.4±2.3，P<0.05），但高于MSC（CGRP+）RAMP1（+）组（50.8±2.6对30.4±3.0，P<0.05）。当VSMC中RAMP1表达降低时，与MSC（CGRP+）RAMP1（+）组相比，MSC（CGRP+）RAMP1（-）组的VSMC迁移增加（69.0±5.6对30.4±3.0，P<0.05），且与Ang II组相似（69.0±5.6对71.4±2.3,P>0.05），与RAMP1（+）组相似（71.6±3.4）。根据蛋白质免疫印迹结果，MSC（CGRP+）组的P-P65蛋白表达低于Ang II组（0.475±0.022对0.642±0.035，P<0.05）。MSC（CGRP+）RAMP1（-）组的P-P65蛋白表达高于MSC（CGRP+）RAMP1（+）组（0.670±0.030对0.373±0.041，P<0.05），且MSC（CGRP+）RAMP1（-）组与Ang II组之间无差异（P>0.05）。RAMP1（+）组（0.643±0.039）与Ang II组之间的P-P65蛋白表达相似（P>0.05）。
CGRP通过RAMP1抑制VSMC迁移。NF-κB和RAMP1在CGRP对VSMC迁移的抑制作用中起关键作用。因此，RAMP1-CGRP信号通路通过NF-κB信号通路抑制VSMC迁移。
