3′-Me-DAB诱导大鼠肝癌发生过程中p53及相关基因mRNA的定量分析

背景与目的:有关肝癌中 p53基因突变及 p53蛋白表达异常已有报道 , 但其在 mRNA水平上的变化尚不清楚 . 为了解在肝癌发生、发展和预后过程中 p53、谷胱甘肽转硫酶 P( glutathione S-transferase P,GST-P) 、甲胎蛋白 ( α -fetoprotein,AFP) 和白蛋白 mRNA水平的变化 , 本研究定量分析癌前病变和癌灶中 GST-P、 AFP和白蛋白 mRNA的量 . 方法:在 3′ -甲基 -4-二甲胺偶氮苯 (3′ -methyl-4-dimethylaminoazobenzene,3′ -Me-DAB)诱发 F344大鼠肝癌过程中 , 用激光捕获显微取样仪 (LCM)准确获得大鼠肝脏中微小癌灶或癌前病变组织后 , 采用 LightCyclerTM V3 System 实时 (real-time)RT-PCR定量分析这些病灶组织中 mRNA水平 . 结果:在实验的第 6、 12和24周,癌前病变组织中p53mRNA量均显著高于正常组织(P≤0.001)。从第6周到第24周,癌前病变组织中p53mRNA逐渐降低(P<0.01)。癌组织中p53mRNA含量高于正常组织,低于同期癌前病变组织(P=0.028和0.0136)。第24周的癌前病变组织或癌组织中细胞核呈p53强染色。各实验期,癌前病变中GST-PmRNA量明显高于正常组织和癌组织(P<0.001)。癌组织中AFPmRNA的表达量显著高于癌前病变组织和正常组织(P<0.001),白蛋白mRNA的表达量显著低于这两种组织(P<0.01)。GST-P和AFP分别在癌前病变组织和癌组织中呈灶状强表达。结论：GST-P和AFPmRNA分别在癌前病灶组织和癌组织中强表达,是这些病变组织的重要标志物。p53mRNA在早期肝癌前病变和癌灶中显示高水平的表达,而较晚期p53蛋白升高。

Quantitative analysis of p53 and related genes mRNA in rat hepatocarcinogenesis induced by 3'-Me-DAB

BACKGROUND & OBJECTIVE: p53 gene mutations and abnormal expression of p53 in hepatocarcinoma have been reported, but alteration in mRNA level is not yet understood. In order to find out the alteration in mRNA levels of p53, glutathione S-transferase P (GST-P), alpha-fetoprotein (AFP), and albumin in genesis, developing, and prognosis of hepatocarcinoma, quantitative analysis of mRNA levels of p53, GST-P, AFP, and albumin in prehepatocarcinoma and hepatocarcinoma foci was performed. METHODS: During hepatocarcinogenesis of F344 rat induced by 3'-methyl-4-dimethylamino-azobenzene (3'-Me-DAB), the levels of these mRNAs were quantitatively analyzed by LightCycler V3 System real-time RT-PCR after capturing accurately micro-foci in prehepatocarcinoma and hepato-carcinoma of rats with laser capture microdissection (LCM). RESULTS: At the 6th, 12th and 24th experiment weeks, the p53 mRNA levels in all of prehepatocarcinoma foci were markedly higher than those in the adjacent normal tissues (all of P < or = 0.001), and gradually decreased from the 6th week to the 24th week (P < 0.01). The content of p53 mRNA in hepatocarcinoma foci was higher than that in normal tissue (P = 0.028 and 0.013), but lower than that in prehepatocarcinoma foci. At 24th weeks, the sections of livers exhibited intensive immunostaining of p53 protein in prehepatocarcinoma and hepatocarcinoma foci. At any time-point of experiment, GST-P mRNA levels in prehepatocarcinoma foci were significantly higher than those in the adjacent normal liver tissue and hepatocarcinoma foci (all of P < 0.001). The concentration of AFP mRNA was the highest (P < 0.001) and that of albumin mRNA was the lowest (P < 0.01) in hepatocarcinoma foci as compared with adjacent normal tissue and prehepatocarcinoma foci. The GST-P protein and AFP protein were expressed strongly in prehepatocarcinoma and hepatocarcinoma foci, respectively. CONCLUSION: GST-P and AFP mRNA overexpressed in prehepatocarcinoma and hepatocarcinoma foci respectively will be profitable markers for diagnosis during hepatocarcinogenesis. The p53 mRNA highly expressed at early stage of prehepatocarcinoma and hepatocarcinoma, but the increasing concentration of p53 protein was found at later stage.