复方中药制剂诱导成纤维细胞凋亡的实验研究

目的 观察丹苓冲剂及丹参对成纤维细胞凋亡的诱导作用,为临床上防治增生性玻璃体视网膜病变(PVR)提供新药物.方法 Annexin V-FITC和碘化丙锭(PI)双染法,在流式细胞仪下测定不同质量浓度的丹苓冲剂与丹参作用不同时间后,成纤维细胞的早期凋亡率.用末端脱氧核糖核酸酶介导的dUTP末端标记法(TUNEL法)观察丹苓冲剂与丹参作用48 h后的细胞凋亡情况.结果 50 mg/mL、100 mg/mL丹苓冲剂和3.47、27.75、111 mg/mL丹参作用6 h组与24 h组及1.56 mg/mL丹苓冲剂作用24 h组细胞早期凋亡率与正常对照相比差异均有统计学意义(P＜0.01).丹苓冲剂与丹参作用48 h后,细胞核出现阳性着色.结论 中药丹苓冲剂及其单体成分丹参可以诱导成纤维细胞凋亡,有望成为新的防治PVR的药物.

Danling and Salviae Miltiorrhizae-induced apoptosis in fibroblast

Objective In order to provide new therapy for the prevention of proliferative vitreoretinopathy(PVR),the effect of Danling and Salviae Miltiorrhizae on inducing fibroblast apoptosis was detected in vitro.MethodsThe conjunctival tissue from rabbit was cultured and passaged in DMEM containing 10% fetal bovine serum.Cultured rabbit conjunctival fibroblast was treated with Danling and Salviae Miltiorrhizae.The experimental concentrations of Danling and Salviae Miltiorrhizae were 1.56 mg/mL,50 mg/mL,100 mg/mL and 3.47 mg/mL,27.75 mg/mL,111 mg/mL respectively.Passage 4 of fibroblast was used in this study.After different time points of incubation with the reagents indicated above,the apoptosis rate of fibroblast was detected by flow cytometry.And apoptosis of cultured conjunctival fibroblast was determined by TUNEL method.ResultsCultured fibroblast showed the spindle-like in shape and absent reaction for CK,S-100 and GFAP and positive staining for Vimentin.The number of fibroblast was significantly increased with the incubation time.50 and 100 mg/mL of Danling or 3.47,27.75 and 111 mg/mL Salviae Miltiorrhizae induced apoptosis of fibroblast when compared with the control group(P<0.01)at 6 and 24 hours of incubation.The apoptosis of fibroblast was severer in 1.56 mg/mL Danling group than the control group at 24 hours of incubation(P<0.01).After 48 hours of incubation with different concentrations of Danling and Salviae Miltiorrhizae,part of nucleus of fibroblast showed brown color which was the positive staining after stained by TUNEL assay.Chromatin condensation of chromatin and apoptosis body were detected by light microscope.ConclusionDanling and Salviae Miltiorrhizae induce apoptosis of cultured fibroblast in a time-and concentration-dependent manner.These results imply that Danling and Salviae Miltiorrhizae have potential role in preventing and treating proliferative vitreoretinopathy.