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目的探讨环氧合酶-2(COX-2)的表达与肝癌的关系,并构建表达人COX-2反义RNA的腺病毒载体,研究其对人肝癌细胞生长的抑制作用。方法采用免疫组织化学法探讨34例肝癌组织COX-2 的表达与肝癌病理特征的关系。采用基因重组法把人COX-2的cDNA片段反向克隆于穿梭质粒pHCMVSP1A,获得pAd-AShcox-2,通过脂质体与pJM17共转染293细胞,经同源重组产生编码COX-2反义RNA的重组腺病毒--Ad-AShcox-2。经聚合酶链反应法鉴定为阳性克隆者大量扩增、纯化,转染人肝癌细胞株SMMC-7402和SMMC-7721,采用免疫细胞化学、细胞集落形成率及流式细胞术检测其对肝癌细胞生长、凋亡及细胞周期分布的影响。结果34例肝癌组织中有28例COX-2高度表达,阳性率达82.4%; COX-2的表达水平与肝癌的病理分级有关,与甲胎蛋白、细胞类型、有无肝内转移无关。成功构建、扩增、纯化得到编码COX-2反义RNA的重组腺病毒Ad-AShcox-2,滴度达1.06×1012PFU/ml;Ad-AShcox- 2转染两种肝癌细胞株后,发现高度表达COX-2的SMMC-7402 COX-2表达水平明显降低,细胞凋亡率明显增加,出现G1期阻滞,与Ad-LacZ组及空白对照组比较差异有统计学意义(P<0.05);而不表达COX- 2的SMMC-7721变化不明显。细胞集落形成实验显示SMMC-7402细胞集落形成率较低(2.7%±0.94%); 而SMMC-7721
Objective To investigate the relationship between cyclooxygenase-2 (COX-2) expression and hepatocellular carcinoma (HCC) and to construct adenoviral vector expressing human COX-2 antisense RNA to study its inhibitory effect on the growth of human hepatoma cells. Methods The expression of COX-2 in 34 HCC tissues and the pathological characteristics of HCC were studied by immunohistochemistry. The cDNA fragment of human COX-2 was reverse cloned into the shuttle plasmid pHCMVSP1A by gene recombination to obtain pAd-AShcox-2. The 293 cells were cotransfected with pJM17 by liposome and homologous recombination resulted in the expression of COX-2 antisense RNA recombinant adenovirus - Ad-AShcox-2. The positive clones identified by polymerase chain reaction (PCR) were amplified, purified and transfected into human hepatocellular carcinoma cell lines SMMC-7402 and SMMC-7721. Immunocytochemistry, colony formation rate and flow cytometry were used to detect the effect on hepatoma cells Growth, apoptosis and cell cycle distribution. Results The expression of COX-2 was highly expressed in 28 of 34 HCC tissues. The positive rate of COX-2 was 82.4%. The expression of COX-2 was related to the pathological grade of HCC and had no relationship with the expression of alpha-fetoprotein, cell type and intrahepatic metastasis. The recombinant adenovirus Ad-AShcox-2 encoding COX-2 antisense RNA was successfully constructed, amplified and purified with a titer of 1.06 × 1012 PFU / ml. After transfecting Ad-AShcox-2 cells with two hepatoma cell lines, The expression of COX-2 in COX-2-induced SMMC-7402 cells was significantly lower than that in Ad-LacZ and blank control groups (P <0.05). However, the changes of SMMC-7721 that did not express COX-2 were not obvious. The colony formation assay showed that the colony formation rate of SMMC-7402 cells was low (2.7% ± 0.94%), while the SMMC-7721