血管平滑肌细胞坏死释放IL-1β及其对IL-6和细胞增殖的影响

Effect of IL-1β Released by Necrotic Cells on IL-6 and Proliferation of Vascular Smooth Muscle Cells

【目的】观察坏死的血管平滑肌细胞对周围正常细胞IL-6表达和增殖能力的影响，同时探讨其可能的作用机制。【方法】采用无血清无糖低氧条件制备血管平滑肌细胞坏死模型，收集坏死细胞培养上清液干预正常细胞。实验设置无血清低糖DMEM培养的对照组（control组）、坏死上清干预组（NCS组）、坏死上清与IL-1拮抗剂联合干预组（NCS＋IL—IRA组）、坏死上清与IL-6拮抗剂联合干预组（NCS＋IL-6RA组）、IL-1β干预组（IL-1β组）以及IL-6干预组（IL-6组）。CCK8和BrdUELISA方法检测细胞增殖能力变化；ELISA方法和RT-PCR方法检测各组细胞培养上清液中IL-6分泌及细胞中IL-6mRNA表达情况；Westernblot方法检测各组细胞p21Ras、ERK、p-ERK蛋白的表达。【结果】与对照组相比，NCS组和IL-1β组处理下血管平滑肌细胞IL-6表达有所升高（P〈0．05），NCS组、IL-1β组和II．一6组细胞增殖能力存在显著性增加（P〈0．05），同时，p21Ras和p-ERK的表达也存在增加趋势，而NCS＋II，1RA组和NCS＋IL-6RA组细胞增殖能力较NCS组及IL-10组、IL-6组细胞增殖能力有所下降（P〈0．05），p21RaS和p-ERK的表达也相应有所降低。【结论】坏死的血管平滑肌细胞可能通过释放IL-1β促进了周围正常细胞IL-6的表达，并通过Ras／ERK／C／EBP信号通路促进细胞增殖。

[Objective] To observe the effect of necrotic vascular smooth muscle cell（VSMC） on the expression of IL-6 and the proliferation of surrounding normal cells, and to explore the possible mechanism. [Methods] VSMC necrosis model was established under hypoxia and serum- and glucose-deprived condition. The necrotic cell supernatant（NCS） was collected to intervene normal cells. There were non serum and low- glucose DMEM control group, NCS group, NCS combined with IL-1 antagonist group, NCS combined with IL 6 antagonist group, IL-113 group and IL-6 group. CCK 8 and BrdU ELISA assay were used to determine the proliferation of VSMC. The protein secretion and mRNA expression of IL-6 were detected by ELISA and RT- PCR, and the protein expression of p21Ras, ERK and p-ERK were evaluated by Western blot. [Results] Com- pared with the control group, the expression of IL-6 in VSMC treated with NCS and IL-1β were increased（ P 〈0.05）, and the proliferation ability of NCS group, IL-1β group and IL-6 group were increased markedly（ P 〈0.05）. Meanwhile, the expression of p21Ras and p-ERK were also elevated. However, the proliferation ability of NCS＋IL-1 RA group and NCS-I-IL-6 RA group were decreased in comparison with NCS group and IL-1β group（ P d0.05）, and the expression of p21Ras and p-ERK were also declined. [Conclusion] Necrotic VSMC may promote the expression of IL-6 through the release of IL-1β, and promote the proliferation of surrounding normal cells through Ras/ERK/C/EBP signal pathway.