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目的:观察重组人源性色素上皮衍生因子(Pigment epithelium-derived factor,PEDF)对缺氧/复氧(H/R)后平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖和凋亡的影响。方法:以组织贴块法培养VSMCs后,随机分为3组,即对照组、H/R模型组及模型+PEDF组。模型+PEDF组又按PEDF的浓度(50、100、200及400 ng/ml)分为4组。用MTT比色法检测VSMCs增殖的变化;用Hoechst/PI双染色法和流式细胞仪法(FCM)检测VSMCs的凋亡;用Western blot法测定凋亡相关蛋白Bcl-2、Bax的表达。结果:①MTT比色法检测显示,缺氧、复氧可促进VSMCs增殖;PEDF能够抑制VSMCs增殖,以200 ng/ml的PEDF作用更显著。Hoechst/PI双染色法和FCM检测显示,经100、200 ng/ml的PEDF作用后,VSMCs的凋亡率较对照组和H/R组明显增高(P<0.01)。与对照组和H/R组比较,Western blot检测显示,100、200 ng/ml的PEDF能够下调Bcl-2蛋白表达并上调Bax蛋白表达(P<0.05,P<0.01)。结论:H/R后,VSMCs增殖增多,PEDF可抑制H/R后VSMCs增殖,其作用可能与Bcl-2、Bax通路促进VSMCs凋亡有关。
Objective: To observe the effects of recombinant human derived pigment epithelium-derived factor (PEDF) on the proliferation and apoptosis of vascular smooth muscle cells (VSMCs) after hypoxia / reoxygenation (H / R). Methods: VSMCs were cultured by tissue patch method and randomly divided into 3 groups: control group, H / R model group and model + PEDF group. The model + PEDF group was divided into 4 groups according to the concentration of PEDF (50, 100, 200 and 400 ng / ml). The proliferation of VSMCs was detected by MTT assay. The apoptosis of VSMCs was detected by Hoechst / PI double staining and flow cytometry (FCM). The expressions of Bcl-2 and Bax were detected by Western blot. Results: ① MTT colorimetric assay showed that hypoxia and reoxygenation could promote the proliferation of VSMCs; PEDF could inhibit the proliferation of VSMCs with PEDF at 200 ng / ml. Hoechst / PI double staining and FCM showed that the apoptotic rate of VSMCs was significantly higher than that of the control group and H / R group (P <0.01) after 100,200 ng / ml PEDF treatment. Compared with control group and H / R group, Western blot showed that 100,200 ng / ml PEDF could down-regulate the expression of Bcl-2 protein and up-regulate Bax protein expression (P <0.05, P <0.01). Conclusion: After H / R, the proliferation of VSMCs is increased. PEDF can inhibit the proliferation of VSMCs after H / R, which may be related to the apoptosis of VSMCs induced by Bcl-2 and Bax.