人趋化因子受体1促进小鼠血管平滑肌细胞增殖的机制研究  

Role of CMKLR1 on mouse vascular smooth muscle cells proliferation and related mechanism

作　　者：刘华东[1] 熊玮[1] 刘启云[1] 李江华[1] 吴美善[1] 张键[2] 董少红[1] 

机构地区：[1]暨南大学第二临床医学院深圳市人民医院心内科,518020 [2]中国科学院深圳先进技术研究院

出　　处：《中华心血管病杂志》2016年第7期605-609,共5页Chinese Journal of Cardiology

基　　金：深圳市重点资助科技计划（201201022）

摘　　要：目的观察人趋化因子受体1（CMKLRl）基因缺陷性小鼠血管平滑肌细胞（VSMC）株中细胞的增殖情况并探讨其作用机制。方法以小鼠CMKLRl基因mRNA序列作为干扰靶点合成短发夹RNA并构建慢病毒载体，将慢病毒感染VSMC建立CMKLRl基因缺陷性细胞株。细胞株构建成功后，以正常VSMC和慢病毒感染VSMC为对照，分别采用荧光定量PCR和Westernblot检测基因缺陷性细胞中CMKLRl的mRNA和蛋白水平。实验分为4组，在正常、慢病毒感染和基因缺陷性VSMC中加入血小板源性生长因子-BB促进细胞增殖，分别为增殖组、对照组和沉默组，另取正常VSMC加入同等体积磷酸盐缓冲液（正常组）。采用细胞计数和5．溴脱氧尿嘧啶核苷（BrdU）掺入法检测各组细胞增殖情况，采用Westernbla检测磷酸化c-Jun氨基末端激酶（p-JNK）的表达水平。结果基因缺陷性VSMC中CMKLRlmRNA的相对表达水平为0．23±0．04，明显低于正常（1．05±0．05）和慢病毒感染VSMC（0．99±0．04）（P均〈0．01）；基因缺陷性VSMC中CMKLRl蛋白的相对表达水平为0．29±0．04，明显低于正常（1．06±0．04）和慢病毒感染VSMC（0．95±0．02）（P〈0．01）。增殖组的细胞数[（50．33±1．20）×10^3／cm。比（42．02±1．53）X10^3／cm2]和BrdUA4‰。值（1．80±0．05比1．55±0．04）均明显高于正常组（P均〈0．05），CMKLRl沉默组的细胞数目[（23．33±2．03）X10^3／cm2]和BrduA值（1．32±0．02）均低于正常组（P均〈0．05），而对照组与增殖组之间差异无统计学意义（P〉0．05）。与正常组VSMC的p-JNK表达水平比较，增殖组VSMC的p-JNK表达水平较高（1．36±0．02比1．03±0．03，P〈0．05），而沉默组VSMC的p-JNK表达水平较低（0．79±0．05比1．034-0．03，P〈0．05）。结论沉默CMKLRl基因表达可通过下调p-JNK表达抑制小鼠血管平滑肌细胞的增殖。Objective To explore the proliferation property of vascular smooth muscle cells （VSMCs） in the stable CMKLR1 gene knock-down mouse VSMCs line and explore related mechanism. Methods The short hairpin RNA sequence targeting to knockdown the coding regions of mouse CMKLR1 mRNA was synthesized and subsequently employed to construct recombinant lentivirus vector. Mouse VSMCs were cultured and infected with the recombinant lentivirus （knockdown VSMCs ）. mRNA and protein CMKLR1 expression in Knockdown VSMCs was measured by real-time PCR and Western blot and compared with those in normal VSMCs （ vehicle VSMCs） and lentivirus control VSMCs （ control VSMCs）. The proliferation of normal, knockdown and control VSMCs was induced by platelet-derived growth factor-BB （PDGF VSMCs ） and measured by cell number counting and BrdU. The phosphorylated c-Jun N-terminal kinase （p-JNK） protein was investigated by Western blot. Results The relative level of CMKLR1 mRNA in knockdown VSMCs （0. 23 ±0. 04 ） was significantly downregulated compared with whichin vehicle VSMCs （ 1.05 ± 0. 05 ） as well as control VSMCs （0. 99± 0. 04） （ P 〈 0. 01 ）. The relative level of CMKLR1 protein in knockdown VSMCs （0. 29± 0. 04） was also significantly decreased, compared with which in vehiele VSMCs （ 1.06 ±0. 04） as well as control VSMCs （0. 95 ±0. 02） （P 〈0. 01 ）. The VSMCs number （（50.33 ± 1.20） x 10^3/cm2） and BrdU A450 value （1.80 ±0.05） in PDGF VSMCs were significantly into＇eased in vehicle VSMCs （ （42. 02 ± 1.53 ） x 10^3/cm2, 1.55 ± 0. 04） （ both P 〈 0. 05 ）. Compared with those in vehicle VSMCs, the VSMCs number （ （23.33±2. 03） x 10^3/cm2 ） and BrdU A450 value （ 1.32 ± 0. 02） in knockdown VSMCs were significantly decreased. The proliferation property between PDGF VSMCs and control VSMCs was similar（ P 〉 0. 05 ）. Compared with the relative level of p-JNK protein （1.03 ±0. 03 ） in vehicle VSMCs, the p-JNK protein level was signifi

关 键 词：肌 平滑 血管 受体 趋化因子 细胞增殖 信号传导 

分 类 号：R54[医药卫生—心血管疾病]