凝血酶对人皮肤成纤维细胞增殖作用的研究

目的：观察不同浓度的凝血酶对成纤维细胞的刺激作用,以及阿加曲班对这种刺激的抑制作用。方法：采用贴壁法进行人皮肤成纤维细胞原代培养,并传代、鉴定。用MTT法检测不同浓度凝血酶刺激后的成纤维细胞活力。用氯胺T法检测不同浓度凝血酶刺激后的细胞上清中羟脯氨酸含量。用免疫组化的方法检测凝血酶刺激后的成纤维细胞中α-平滑肌肌动蛋白（α-SMA）的表达情况。在培养液中加入阿加曲班后重复上述检测。结果：①培养的细胞为典型的成纤维细胞形态,波形蛋白单克隆抗体免疫组化染色呈阳性;②加入不同浓度凝血酶的各组吸光值均高于不加凝血酶组（A组）（P〈0.01）,加入阿加曲班和凝血酶的各组吸光值与A组无显著性差异（P〉0.05）;③加入不同浓度凝血酶的各组细胞上清中羟脯氨酸含量均高于不加凝血酶组（A组）（P〈0.01）,加入阿加曲班和凝血酶的各组上清中羟脯氨酸含量与A组相比无统计学差异（P〉0.05）;④凝血酶刺激后的成纤维细胞中有α-SMA的表达,加入阿加曲班后此种表达被抑制。结论：凝血酶能诱导成纤维细胞增殖、胶原分泌量增加,能刺激成纤维细胞表达α-SMA,使成纤维细胞转变为肌成纤维细胞。阿加曲班能抑制凝血酶的这种作用。

Study of thrombin on proliferation of fibroblasts Isolated from mini pig's skin

Objective Observe the stimulatory functions of thrombin different concentration on fibroblasts,and the inhibitory actions of argatroban on the stimulatory functions.Methods The minipig skin fibroblasts were cultured in monolayer.The vigor of fibroblasts which were stimulated by different concentration of thrombin was detected by MTT.The contents of hydroxyproline in supernatant were detected by chloramines T method.The expressions of α-SMA in fibroblasts which were stimulated by thrombin,were detected by immunohistochemistry staining.After argatroban was infused into the culture solution,the detections mentioned above were repeated.Results ① The cells appeared to be the typical fibroblast,the immunohistochemistry staining of α-SMA in the cells were positive.②Compared with the group without thrombin,the OD value of the groups contained different concentration of thrombin were increased（P﹤0.01）,the OD value of the groups contained different concentration of thrombin and argatroban had no changes（P﹥0.05）.③Compared with the group without thrombin,the hydroxyproline content in supernatant of the groups contained different concentration of thrombin were increased（P0.01）,the hydroxyproline content in supernatant of the groups contained different concentration of thrombin and argatroban had no changes（P0.05）.④α-SMA expressed in the fibroblast stimulated by thrombin.The expression of α-SMA was inhibited by argatroban.Conclusions Thrombin can induce fibroblast＇s proliferation,make fibroblast product more collagen,increase the expression of α-SMA in fibroblast,transform fibroblast to myofibroblast.Argatroban can inhibit these effects of thrombin.