重组TGF_α-PE40对血管平滑肌细胞增殖的抑制作用(英文)

Inhibitory effect of recombinant TGF_α-PE40 on vascular smooth muscle cell proliferation

目的:探讨基因重组转换生长因子α-绿脓杆菌外毒素融合蛋白(TP40)对血管平滑肌细胞(SMC)增殖的抑制作用。方法:用RNA印迹法和免疫组化检测原代培养增殖期SMC及静止期SMC表皮生长因子受体(EGFR)的表达,用结晶紫染色法和[~3H]亮氨酸掺入法检测TP40对增殖期SMC和静止期SMC增殖及蛋白质合成的抑制作用,用过量EGF(TP40浓度的100倍)竞争拮抗TP40的细胞毒作用。结果:增殖期SMC EGFR的mRNA及受体蛋白质的表达显著高于静止期SMC。TP40浓度为10及100μg·L^(-1)时,对增殖期SMC增殖及蛋白质合成的抑制作用较静止期SMC强(P<0.01),对[~3H]亮氨酸掺入抑制的IC_(50)及其95％可信限分别为8.01(5.05-12.69)和121.95(90.98-163.47)μg·L^(-1)。过量EGF能完全拮抗TP40的细胞毒作用。结论:增殖期SMC能高水平地表达EGFR,TP40对增殖期SMC的增殖具有导向抑制作用,作用部位为细胞的EGFR。

AIM: To study inhibitory effect of recombinant transforming growth factor α- Pseudomonas exotoxin fusion protein (TP40) on proliferation of the cultured vascular smooth muscle cells (SMC). METHODS: Expression of epidermal growth factor receptor (EGFR) mRNA and EGFR in cultured proliferating and quiescent SMC was analyzed with Northern blot and immunohistochemistry. Inhibitory effects of TP40 on SMC proliferation and protein synthesis were analyzed with crystal violet staining and [3H]leucine incorporation. Competition assays were performed by the addition of 100-fold excess of EGF. RESULTS: Expression of EGFR mRNA and EGFR in rapidly proliferating SMC increased than that in quiescent SMC. When the concentration of TP40 was 10 or 100 μg·L-1, inhibitory effects of TP40 on rapidly proliferating SMC proliferation and protein synthesis were much higherthan that on quiescent SMC ( P < 0.01), and the IC50 of [3H]leucine incorporation against rapidly proliferating and quiescent SMC were 8.01 (5.05-12.69) and 121.95 (90.98 - 163.47) μg·L-1. Excess EGF completely blocked inhibitory effects of TP40. CONCLUSION: The rapidly proliferating SMC express EGFR at a high level. TP40 selectively inhibited the proliferation of rapidly proliferating SMC. The cytotoxic effects of TP40 were specifically mediated by EGFR.