血管紧张素Ⅱ2型受体基因转染对肾间质纤维化的防治意义

Significance of transfected angiotensin Ⅱ type 2 receptor gene on the treatment and prevention of renal interstitial fibrosis

目的 探讨血管紧张素Ⅱ2型受体(AT2R)基因体外转染人肾间质成纤维细胞(hRIF)后对肾间质纤维化进程的影响。方法 构建AT2R基因腺病毒载体(AdCMV-AT2R),体外转染hRIF,用流式细胞仪、BrdU掺入法检测hRIF增殖。用改良Boyden趋化小室、激光共聚焦显微镜查F-actin检测hRIF迁移。用原位末端标记(TUNEL)、bcl-2和bax mRNA表达检测hRIF凋亡。结果 hRIF转染表达AT2R。S期与G_2、M期细胞比例受血管紧张素(Aug)Ⅱ作用后无明显变化(8.1%对13.9%,P>0.05)。BrdU平均掺入量降低54.2%(P<0.05)。迁移细胞数抑制比例达62.2%(P<0.01)。F-actin表达明显受抑制。TUNEL染色法显示大量凋亡细胞、bcl-2表达无明显变化、bax表达增加76.3%(P<0.05)。结论 hRIF体外转染表达AT2R基因后,明显抑制了细胞的增殖与迁移,促进了细胞凋亡,对肾间质纤维化的防治有积极的作用。

Objective To study the effect on the process of renal interstitial fibrosis after transfection of angiotensin Ⅱ type 2 receptor(AT2R) gene to human renal intersitial fibroblasts(hRIF) in vitro. Methods A recombinant adenoviral vector, AdCMV-AT2R containing rat AT2R gene, was constructed by homologous recombination, and then used to transfer AT2R gene to hRIF in vitro. Proliferation of hRIF was detected by flow cytometry and incorporation of bromodeoxyuridine(BrdU). The modified Boyden chamber method was used to test the migration of hRIF. The re-organization of F-actin in hRIF was analyzed by confocal microscopy. Apoptosis in cultured hRIF was quantified by terminal deoxynucleotide transferase-mediated d UTP nick end-labeling(TUNEL) in situ. Results The expression of AT2R mRNA increased obviously in transferred hRIF. As affected by Ang Ⅱ, the ratio of S, G2 and M periods did not change significantly (8. 1% vs 13. 9% , P > 0. 05) . Brd U incorporation reduced by 54. 2% ( P < 0. 05) . The number of hRIF migration decreased by 62. 2% ( P < 0. 01) . The expression of F-actin down-regulated significantly. Numbers of apoptosis cells were detected by TUNEL in transferred groups. The expression of bcl-2 did not change signifiantly, and the expression of bax increased by 76.3% (P < 0. 05) . Conclusions AdCMV-AT2R is able to generate a high level of AT2R expression in cultured hRIF, which significantly inhibits the proliferation and migration, and enhances apoptosis of hRIF. This effect may be of great value in the treatment and prevention of renal interstitial fibrosis.