【摘要】 目的研究血管紧张素(AngⅡ)1型受体(AT1)拮抗剂洛沙坦(losartan)对人胰腺星状细胞增殖和迁移的影响及其抗胰腺纤维化的作用。方法组织贴壁法从人胰腺癌组织中原代分离纯化人胰腺星状细胞(hPSC),体外培养10d后细胞活化。应用放射免疫分析法测定细胞培养上清液和细胞匀浆中AngⅡ的含量,免疫细胞化学和原位杂交方法检测PSC上的AT1表达。应用AngⅡ(10-8mol/L)和洛沙坦梯度浓度设计多种组合处理PSC细胞。BrdU掺入法和TUNEL法分别检测细胞增殖和凋亡。Wound-healing分析法观察细胞的迁移能力。Real-timePCR、Westernblot和免疫荧光方法检测PSC中型胶原和p38的表达。结果人PSC存在AT1的表达,而细胞培养上清和细胞匀浆中未能检测到AngⅡ。洛沙坦能够时效和量效性地诱导细胞凋亡(10-5mol/L时最明显),能减轻Ang所导致的细胞迁移和型胶原的分泌,抑制PSCp38的表达。结论AngⅡ主要依靠旁分泌而并非自分泌途径,通过AT1受体对PSC发挥作用,而洛沙坦通过抑制AngⅡ同AT1结合而发挥抗纤维化效应。更多还原

【Abstract】 Objective To investigate the effects of AT1 (Type 1 angiotensin Ⅱ receptor) antagonist (Losartan) on apoptosis, proliferation and migration of human pancreatic stellate cells (hPSCs), and study its anti-fibrotic effects. Methods hPSCs were isolated using an outgrowth method from pancreatic samples of patients with pancreatic carcinoma. The cells became myofibroblast phenotype after 10 days culture. Using radioimmunoassay technique the concentration of AngⅡ was detected in culture medium and cell homogenate. Immunocytochemistry and in situ hybridization methods were utilized to test AngⅡ expression in hPSCs. Effects of Losartan on hPSCs proliferation and apoptosis were investigated using BrdU incorporation, TUNEL and cell migration was observed by phasecontrast microscopy and Wound-healing assay when cells were treated with Losartan. Immunofluorescence and Western blot were applied to quantify the expression of type Ⅰ collagen in hPSCs. The expression of p38 in hPSCs was detected by real-time PCR and Western blot. Results There existed AT1 expression in hPSCs, while no AngⅡ was detected in culture medium and cell homogenate. Losartan induced cell apoptosis in a dose-dependent manner and time-dependent manner (apparently at 10 -5 mol/L). No pro-proliferative effect was observed under the same condition. Losar-tan of 10 -5 mol/L also alleviated the motion capability and typeⅠ collagen content in hPSCs compared with AngⅡ treatment and non-treatment control groups. Also, the expression of p38 in hPSCs was inhibited.Conclusions These findings suggest that paracrine, not autocrine, of AngⅡ effects on apoptosis, proliferation and migration hPSCs, which was mediated by AT1 expressed on cells, while Losartan may exert anti-fibrotic effects by inhibiting hPSCs motion, and probably partly by inducing apoptosis.更多还原