shRNA 沉默趋化素对小鼠主动脉平滑肌细胞增殖能力的影响及可能的机制

Effect of shRNA silencing of Chemerin on proliferation of mouse aortic smooth muscle cells and possible mechanisms

摘要:

目的 探讨shRNA 沉默趋化素（Chemerin）对小鼠主动脉平滑肌细胞增殖能力的影响及可能的 机制。方法 构建Chemerin 基因RNA 干扰慢病毒载体，培养小鼠主动脉平滑肌细胞（ASMC），分成4 组： Sham 组、PDGF 组、对照序列组和Chemerin 沉默组。Chemerin 沉默组和对照序列组分别转染携带Chemerin 干扰基因和对照基因序列的慢病毒，分别向PDGF 组、对照序列组和Chemerin 沉默组细胞中加入PDGFBB， Sham 组则加入等量的PBS。利用实时荧光定量聚合酶链反应（qRT-PCR）检测各组细胞中Chemerin 基因表达，细胞计数实验和BrdU 掺入法测定各组细胞增殖能力，Western blot 法检测各组细胞中Chemerin、 ERK1/2、p-ERK1/2、JNK 和p-JNK 蛋白表达。结果 Chemerin 沉默组细胞中Chemerin 基因和蛋白相对表 达量分别为（0.35±0.09）和（0.32±0.09），与其他3 组比较，差异有统计学意义（P <0.05），均低于其他3 组， 且对照序列组和PDGF 组均高于Sham 组；Chemerin 沉默组细胞数和吸光度A 值分别为（34.27±3.08）×103 个/cm2 和（1.26±0.07），与其他3 组比较，差异有统计学意义（P <0.05），均低于其他3 组，且对照序列组和 PDGF 组均高于Sham 组；Chemerin 沉默组细胞中ERK1/2 和JNK 蛋白相对表达量高于对照序列组和PDGF 组， 低于Sham 组，Chemerin 沉默组细胞中p-ERK1/2 和p-JNK 蛋白相对表达量低于其他3 组，而对照序列组和 PDGF 组高于Sham 组，均差异有统计学意义（P <0.05）。结论 特异性沉默Chemerin 基因可阻止ASMC 的 增殖作用，其机制可能与抑制ERK1/2 和JNK 信号通路有关。

Abstract:

Objective To investigate the effect of shRNA silencing of Chemerin on the proliferation of aortic smooth muscle cells (ASMCs) in mice and the possible mechanisms. Methods Chemerin gene RNA interference lentiviral vector was constructed. The mouse ASMCs were cultured and divided into four groups: sham group, PDGF group, control sequence group and Chemerin-silenced group. ASMCs in the Chemerin-silenced group and the control sequence group were transfected with lentiviruses carrying the Chemerin interference gene and the control gene sequence, respectively. PDGF-BB was added to the PDGF group, the control sequence group and the Chemerin silenced group, respectively. The sham group was added with the same amount of PBS. The expressions of Chemerin gene were detected by qRT-PCR. The cell proliferative ability was measured using cell counting test and BrdU incorporation method. The expressions of Chemerin, ERK1/2, p-ERK1 2, JNK and p-JNK proteins were analyzed by Western blot. Results The relative expression levels of Chemerin mRNA and protein in the Chemerin-silenced group were (0.35 ± 0.09) and (0.32 ± 0.09), respectively, which were lower than those of the remaining 3 groups (P < 0.05), and they were statistically higher in the control sequence group and the PDGF group than in the sham group (P < 0.05). The number of cells and A value in the the Chemerin-silenced group were (34.27 ± 3.08) ×103/cm2 and (1.26 ± 0.07), which were lower than those in the remaining 3 groups (P < 0.05), and they were significantly higher in the control sequence group and the PDGF group than in the sham group (P < 0.05). The relative expression levels of ERK1/2 and JNK proteins in the Chemerin-silenced group were higher than those in the control sequence group and the PDGF group, but lower than those in the sham group; the relative expression levels of p-ERK1/2 and p-JNK proteins in the Chemerin-silenced group were lower than those in the remaining 3 groups, while they were statistically higher in the control sequence group and the PDGF group than in the sham group (P < 0.05). Conclusions Specific silencing of Chemerin gene could prevent the proliferation of ASMCs, the mechanism might be related to inhibition of ERK1/2 and JNK signaling pathway.