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目的:比较Wnt/β-catenin信号分子在HepG2和L02细胞中的表达,探讨Wnt/β-catenin信号转导通路在肝癌发生中的作用机制,为肝癌的防治提供新的思路。方法:选取Wnt/β-catenin信号转导通路中上下游关键因子Wnt1、Wnt4、β-catenin、cyclin D1以及c-myc等,应用RT-PCR的方法观察他们在正常肝脏L02细胞和肝癌HepG2细胞中的转录水平。用免疫细胞化学染色方法和Western blot检测研究Wnt/β-catenin信号转导通路中最关键的成员β-catenin在上述2个细胞株中的定位和定量表达。结果:在正常的L02肝细胞中,用RT-PCR的方法未检测到Wnt1、Wnt4、cyclin D1以及c-myc的mRNA转录,只有β-catenin的基因被转录表达。同时用免疫细胞化学染色观察到β-catenin在L02细胞膜处存在表达。而在HepG2肿瘤细胞中,不仅检测到β-catenin的基因转录,同时也检测到Wnt1、cyclin D1以及c-myc的mRNA转录,只有Wnt4未转录。用免疫细胞化学的方法观察β-catenin在肿瘤细胞中的表达明显增强,表现为胞膜着色减弱而胞质甚至是胞核的阳性染色。采用Western blot检测也验证了β-catenin蛋白在肿瘤细胞中的表达水平明显高于正常细胞。结论:Wnt/β-catenin信号转导通路在人的肝癌细胞HepG2中存在异常活性,Wnt1可能是导致信号通路激活的始动因素之一。
OBJECTIVE: To compare the expression of Wnt / β-catenin signaling in HepG2 and L02 cells and to explore the mechanism of Wnt / β-catenin signaling pathway in the pathogenesis of hepatocellular carcinoma (HCC) and to provide new ideas for the prevention and treatment of HCC. Methods: Wnt / β-catenin signal transduction pathway Wnt1, Wnt4, β-catenin, cyclin D1 and c-myc were detected by RT-PCR in normal liver L02 cells and HepG2 hepatoma cells In the transcription level. Immunocytochemical staining and Western blot were used to detect the localization and quantitative expression of β-catenin, the most critical member of the Wnt / β-catenin signaling pathway, in these two cell lines. RESULTS: In normal L02 hepatocytes, mRNA transcripts of Wnt1, Wnt4, cyclin D1 and c-myc were not detected by RT-PCR. Only β-catenin gene was transcribed. Meanwhile, the expression of β-catenin in L02 cell membrane was observed by immunocytochemical staining. In HepG2 tumor cells, not only β-catenin gene transcription but also Wnt1, cyclin D1 and c-myc mRNA transcripts were detected. Only Wnt4 was not transcribed. The expression of β-catenin in tumor cells was observed by immunocytochemistry. The staining of the cytoplasm and even the nucleus showed weakening of the cell membrane. Western blot also verified that the expression level of β-catenin protein in tumor cells was significantly higher than that in normal cells. CONCLUSION: Wnt / β-catenin signaling pathway is abnormally active in HepG2 human hepatocellular carcinoma cells. Wnt1 may be one of the initiating factors that lead to the activation of signaling pathway.