siRNA抑制HMGB1表达对AngⅡ诱导的CFs增殖及胶原分泌的影响

The inhibitory effects of HMGB1 siRNA on the proliferation and collagen secretion of cardiac fibroblasts induced by angiotensin Ⅱ

目的：探讨siRNA干扰高迁移率族蛋白B1（HMGB1）表达能否有效抑制 AngⅡ诱导的心成纤维细胞（CFs）的增殖及胶原分泌，从而为心肌纤维化的发生提供新的认识，为心肌纤维化的治疗提供新的思路。方法酶消化法分离小鼠心脏成纤维细胞，差速贴壁法去除心肌细胞。波形蛋白和 DDR2免疫细胞化学染色鉴定成纤维细胞，将CFs分为：A组（正常对照组）：培养时不加干预药物； B组（AngⅡ组）：加 AngⅡ10－6 mol／L；C 组（AngⅡ＋HMGB1siRNA 组）：HMGB1siRNA 转染CFs后加 AngⅡ10－6 mol／L；D 组（AngⅡ＋阴性对照 siRNA 组）：阴性对照 siRNA 转染 CFs 后加 AngⅡ10－6mol／L。各组细胞培养48 h后，采用CCK8比色法检测 CFs 细胞增殖， ELISA法检测上清液胶原含量，RT －PCR 法测定细胞 HMGB1mRNA 表达。结果使用酶消化法和差速贴壁法获得的高纯度细胞符合成纤维细胞的形态特征并且波形蛋白与 DDR2免疫细胞化学染色阳性；与 A 组比较，B组CFs增殖，上清液胶原含量均明显增加（P ＜0．05）；HMGB1mRNA 表达明显上调（P ＜0．05），与 B 组比较，C组CFs增殖，上清液胶原含量显著降低（P ＜0．05），HMGB1 mRNA表达显著下调（P ＜0．05）。 B组与D组差异无统计学意义（P ＞0．05）。结论HMGB1参与了 AngⅡ诱导的CFs增殖及胶原表达过程，siRNA 干扰 HMGB1表达可有效抑制AngⅡ诱导的CFs的增殖及胶原分泌。

Objective To investigate the inhibitory effects of high mobility group protein 1 （HMGB1） siRNA on the proliferation of cardiac fibroblasts （ CFs） and its collagen secretion after angiotensin Ⅱ （AngⅡ） induction.Methods CFs were isolated from the heart of mice through enzymatic digestion, while cardiac myocytes were removed by the pre-plate technique.The obtained cells were identified by immunochemical staining for vimentin and DDR2.Then, the cells were divided into four groups according to their various treatments： Group A （without treatment）, Group B （10^-6 mol/L AngⅡ）, Group C （10^-6 mol/L AngⅡ ＋HMGB1 siRNA）, and Group D （10^ 06 mol/L AngⅡ ＋negative control siRNA）. Each group was cultivated for 48 h.The proliferation of CFs was detected by CCK 8.The quantity of collagen in the su-pernatant was measure by ELISA.The expression of HMGB1 mRNA was determined by RT -PCR.Results The cells obtained after enzymatic digestion and preplate enrichment were determined as CFs due to their morphological characteris -tics and positive immunochemical staining for vimentin and DDR2.Compared with Group A, CFs in Group B grew faster while secreting remarkably increased amounts of collagen , in addition obvious up -regulated levels of HMGB1 mRNA （all P 〈0.05）.Compared with Group B, CFs in Group C grew slower, wile secreting substantially reduced quantities of collagen and HMGB1 mRNA （all P 〈0.05）.No significant differences were found between Groups B and D （P 〉0.05）.Conclusion HMGB1 is involved in the growth and collagen production of CFs induced by AngⅡ, which can be efficiently suppressed by HMGB1 siRNA.