重组人p53腺病毒抑制肝癌HepG_2细胞增殖机制的研究

目的探讨重组人p53腺病毒(rA d-p53)抑制肝癌H epG2细胞增殖的作用机制。方法以高、中、低浓度(分别为3×1012VP、3×1011VP和3×1010VP)的rA d-p53处理肝癌H epG2细胞(分别为观察Ⅰ、Ⅱ、Ⅲ组),用免疫组化法检测周期素D1(cy linD1)、周期素E(cyclinE)蛋白在肝癌H epG2细胞中的表达,用M TT比色法观察rA d-p53对肝癌H epG2细胞生长的抑制作用,用流式细胞仪检测细胞周期分布;另设空白对照组。结果①rA d-p53对H epG2细胞有明显的抑制效应,且具有时间、剂量依赖关系。②观察Ⅰ、Ⅱ、Ⅲ组G1期细胞分别上升至75.8%、70.1%、66.5%,明显高于对照组(P<0.05);观察Ⅰ、Ⅱ组S期细胞分别下降至13.2%、20%,明显低于对照组(P<0.05)。③观察Ⅰ、Ⅱ、Ⅲ组肝癌H epG2细胞cyclinD1蛋白水平分别减少45.6%、35.1%和26.3%,与对照组比较P均<0.05;cyclinE蛋白水平分别减少33.3%、27.1%和18.1%,与对照组比较P均<0.05。结论rA d-p53对肝癌H epG2细胞有明显的抑制效应,其抑制效应具有时间、剂量依赖关系,其机制可能是通过抑制下游cy-clinD 1、cyclinE表达阻止细胞进入S期。

Objective： To investigate the anti-proliferative effect of recombinant Human Ad-p53 Injection（rAd-p53） on carcinoma of the liver cell line HepG2 from the mechanism of arrest. Methods： 3×10^12VP（the high concentration group）, 3×10^11VP （the moderate concentration group） and 3×10^10VP （the low concentration group）rAd-p53 were transferred into carcinoma of the liver cell line HepG2 respectively as observation Ⅰ, Ⅱ and Ⅲ groups. Expression of cyclin D1 and cyclin E proteins in carcinoma of the liver cell line were detected by immunohistochemistry assay and the MTT assay was used to observe the inhibitory effect of rAd-p53 on growth of carcinoma of the liver cell cine HepG2. Cell cycle distribution was assayed by flow cytometry. Results： ① Recombinant Ad-p53 produced obvious time-and-dose-dependent inhibitory effect on HepG2 cells. ② The percentage of G1 phase cells increased to 75.8%,70.1%,66.5% respectively in the Ⅰ , Ⅱ and Ⅲ groups,which had significant difference （P〈0.05）. Percentage of S phase cells decreased to 13.2% 2 0% in the Ⅰ group and Ⅱ group,which had significant difference （P〈0. 05）. ③ Immunohistochemistry assay showed that the level of cyclin D1 protein decreased 45.6%, 35.1% and 26.3% respectively and the level of cyclinE protein decreased 33.3% ,27.1% and 18.8% respectively in the Ⅰ, Ⅱ, Ⅲ groups,which had significant differences compared with the control group （P〈0.05）. Conclusion：Recombinant Ad-p53 can produce obvious time-and-dose-dependent inhibitory effect on HepG2 cells. The mechanism may be inhibition of the expression of cyclin D1 and cyclin E which block cells go into nhase S.