腺病毒介导AT2R基因转染表达抑制肾间质纤维母细胞增殖

EXPRESSION OF THE RAT ANGIOTENSIN TYPE Ⅱ RECEPTOR GENE MEDIATED BY ADENOVIRAL VECTOR INHIBITS THE PROLIFERATION OF CULTURE RENAL INTERSTITIAL FIBROBLASTS

目的 :探讨血管紧张素Ⅱ (AngⅡ ) 2型受体 (AT2R)表达对人肾间质纤维母细胞 (hRIF)增殖的影响。方法 :构建携带AT2R基因的重组复制缺陷型腺病毒载体 (AdCMV -AT2R) ,转染体外培养hRIF并检测AT2RmRNA表达及细胞表达率 ,用细胞周期分析、分裂指数、MTT比色法和 5 -溴尿苷 (BrdU)掺入法检测hRIF增殖。结果 :构建的AdCMV -AT2R体外转染培养hRIF表达率为 90 .5 7%。AT2R峰值表达时 ,增殖期hRIF(S期和G2 -M期 )比率从 3 1.7%降低到 13 .9% (P <0 .0 5 ) ,分裂指数从 3 7.4%降低到 9.6% (P <0 .0 1) ,MTT吸光度和BrdU掺入量分别降低 60 .1%和 5 4.2 % (P <0 .0 1)。结论 :AT2R转染表达可显著抑制体外培养hRIF的增殖 。

Objective:To study the effect on the proliferation of human renal interstitial fibroblasts (hRIF) after transfected angiotensin Ⅱ (AngⅡ) type 2 receptor (AT2R). Method:The recombinant adenoviral vector,AdCMV-AT2R, containing rat AT2 receptor gene was constracted by homologous recombination, and then it was used to transfer AT2 receptor gene to hRIF in vitro. The expression of AT2R mRNA was detected by RT-PCR. The rate of expression and the change of cell cycle in hRIF were analysed by flow cytometry. These assays including cell devision index, incorporation of bromodeoxyuridine(BrdU) and 3-(4,5-dimethyl- thiazol-2-yl)2,5-diphenytetrazolium bromide (MTT) were used to determine the proliferation of hRIF, respectively. Result:RT-PCR showed that the expression of AT2R mRNA increased obviously in transferred hRIF, and the peak value of expression rate is about 90.57% at 48 hours. When the expression of AT2R is at peak value, the ratio of S, G2 and M periods was reduced from 31.7% to 13.9%(P<0.05) and the index of cell division from 37.4% to 9.6%(P<0.01). The OD values of MTT and BrdU incorporation were reduced by 60.1% and 54.2% respectively(P<0.01). Conclusion:Our study indicates that AdCMV-AT2R can generate high level expression of AT2 receptor in cultured rat HRIF and its expression can significantly inhibit the proliferation of hRIF in vitro. It may be a new selection for the gene therapy of chronic renal failure.