精-甘-天冬-丝氨酸4肽对肝星状细胞增殖及凋亡的影响

Effects of Arg-Gly-Asp-Ser tetrapeptide on proliferation and apoptosis of hepatic stellate cells in vitro

目的 :探讨精 -甘 -天冬 -丝氨酸 (RGDS) 4肽对纤维连接蛋白 (FN)刺激的肝星状细胞 (HSCs)增殖、凋亡及caspase - 3表达的影响。方法 :应用体外HSCs培养技术 ,采用 [3 H]-胸腺嘧啶核苷 ([3 H]-TdR)掺入法测定HSCs增殖 ;膜联蛋白 (Annexin -V) /碘化丙啶 (PI)双标记流式细胞术、TUNEL、扫描电镜及透射电镜等方法测定HSCs凋亡 ;采用甲苯胺兰染色方法测定细胞粘附率 ;应用流式细胞方法测定caspase - 3蛋白表达。结果 :① 2 5mg·L-1、5 0mg·L-1、10 0mg·L-1浓度RGDS 4肽剂量、时间依赖性抑制HSCs增殖 ,P <0 0 1。②RGDS 4肽对HSCs凋亡的诱导作用亦呈剂量和时间依赖关系 ,P <0 0 1。扫描电镜、透射电镜观察 ,RGDS 4肽组出现典型的凋亡征象。③RGDS 4肽作用于HSCs2h ,2 5mg·L-1、5 0mg·L-1、10 0mg·L-1组粘附抑制率分别是 8 82 %、2 9 4 1%、4 5 5 9% ,而RGES4肽组的粘附抑制率仅为 4 4 1% ,P <0 0 1。④RGDS 4肽处理组caspase - 3表达明显高于FN、RGES 4肽组。结论 :RGDS 4肽剂量和时间依赖性抑制HSCs增殖并诱导其凋亡。RGDS 4肽抑制增殖及诱导凋亡效应 ,依赖于caspase -3,也与其抗粘附作用有关。

AIM:To investigate the effects of Arg-Gly-Asp-Ser (RGDS) tetrapeptide on proliferation,apoptosis and caspase 3 expression in FN-stimulated HSCs in vitro. METHODS:[ 3H]-thymidine incorporation,Annexin-V/Propidium Iodide double-labeled flow cytometry(FCM),TUNEL,scanning electron microscope and transmission electron microscopy were employed to estimate the influence of RGDS on proliferation and apoptosis of HSCs. The adhesion rates were observed by toluidine blue colorimetric assay. The expression of caspase-3 protein was detected by FCM. RESULTS:①Compared with control and FN groups,RGDS tetrapeptide at concentrations of 25 mg·L -1 ,50 mg·L -1 and 100 mg·L2 1 inhibited the proliferation of HSCs ( P<0.0 1), and the inhibition rates of 100 mg·L -1 at 12 h,24 h and 48 h were 62.73%,74.23%,80.22%,respectively.②RGDS tetrapeptide induced the HSC apoptosis in dose-dependent and time-dependent manners( P<0.01 ). Observed with scanning electron microscope,the cell bodies and cellular processes of HSCs exposed to RGDS tetrapeptide were seen to be diminished. Microvilli on the cell surface decreased,became short even disappeared. Observed with transmission electron microscopy,the chromatins condensed, shrunk and aggregated along inside of nuclear membrane to exist in the form of ball,petal and crescent. Sometimes,apoptotic bodies formed. ③After exposure of HSCs to RGDS tetrapeptide for 2 h,the inhibition rates of adhesion were 8.82%,29.41% and 45.59%,respectively,but that of RGES group was only 4.41%, P <0.01. ④ The expression of caspase 3 was obviously higher in RGDS tetrapeptide group than that in FN group,RGES tetrapeptide. CONCLUSION:These results suggest that RGDS tetrapeptide may inhibit proliferation and induce apoptosis of HSCs in both dose-dependent and time-dependent manners in vitro, which may be related to the abrogation of cell adhesion and caspase 3.