人肝癌HepG2和Hep3B细胞的异质性与生物学行为关系的初探

Preliminary research on the relationship between heterogeneity and biological behavior of human hepatocellular carcinoma HepG2 and Hep3B cells

目的:比较人肝癌HepG2和Hep3B细胞异质性,探讨其与耐药、侵袭、迁移生物学特性的关系。方法:以p53野生型HepG2和p53缺失型Hep3B细胞为研究对象,采用四甲基偶氮唑盐比色法(MTT法)分析细胞对顺铂(DDP)、紫杉醇(PTX)、依托泊苷(VP-16)、索拉非尼(So)的敏感性,细胞计数试剂盒(CCK-8)法和平板克隆形成实验测定细胞的生长曲线及克隆形成能力,划痕、Transwell实验检测细胞的迁移和侵袭能力,建立人肝癌裸鼠皮下移植瘤模型,记录小鼠生长情况及出瘤时间,苏木精—伊红法(HE法)观察瘤组织及各脏器的病理学改变,蛋白免疫印迹法(western blotting法)检测细胞和组织中p53、骨髓细胞白血病-1(Mcl-1)、Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤(Bcl-2)、穹窿主体蛋白(MVP)、多药耐药蛋白1(MDR1)、基质金属蛋白酶-2(MMP2)、E钙粘蛋白(E-cadherin)的表达差异。结果:MTT结果显示,相对HepG2细胞,Hep3B细胞对化疗药物的敏感性较低(P<0.05,P<0.01,P<0.001);Hep3B细胞具有更高的克隆、侵袭和迁移能力(P<0.05,P<0.01,P<0.001);在裸鼠皮下移植瘤模型中,Hep3B组随着时间的延长体重出现明显下降,出瘤时间也明显长于HepG2(P<0.001),且在种植了Hep3B的裸鼠中,出现了癌细胞肝转移现象;与HepG2细胞相比,Hep3B细胞Bax、p53蛋白表达下调,Bcl-2、Mcl-1、MVP、MDR1、MMP2、E-cadherin蛋白表达上调(P<0.05,P<0.001),移植瘤组织的p53及MMP2、E-cadherin的表达与细胞的表达趋势一致(P<0.01,P<0.001)。结论:Hep3B细胞具有更明显的耐药、抗凋亡和侵袭、迁移能力,本研究可为临床上两种不同生物学特性的肝细胞癌治疗方案的确定提供实验依据。

Objective:To compare the heterogeneity of human hepatocellular carcinoma HepG2 and Hep3B cells,and to explore the relationship between the heterogeneity and the biological characteristics of drug resistance,invasion and migration.Methods:HepG2(p53 wild-type)and Hep3B(p53 deficient)cells were used as research subjects.The sensitivity of the two cell lines to cisplatin(DDP),paclitaxel(PTX),etoposide(VP-16)and sorafenib(So)was analyzed by tetramethylazolium salt colorimetric method(MTT).Cell counting kit-8(CCK-8)assay and plate clone formation assay were used to detect the cellular growth curve and clone formation ability.The migration and invasion abilitites of cells were detected by scratch and Transwell assay.Subcutaneous transplantation tumor models of human hepatocellular carcinoma in nude mice were established,and the growth and tumor emergence time of mice were recorded.The transplanted tumor tissues and each viscera with HE to observe the pathological changes.Western blotting was conducted to detect the expression differences of p53,myelocytic leukemia-1(Mcl-1),Bcl-2 associated X protein(Bax),B-cell lymphoma(Bcl-2),major vault protein(MVP),multidrug-resistant protein 1(MDR1),matrix metalloproteinase-2(MMP2),and E-cadherin in cells and tissues.Results:MTT results showed that Hep3B cells were less sensitive to chemotherapy drugs than HepG2 cells(P<0.05,P<0.01,P<0.001)and Hep3B cells revealed stronger abilities in proliferation,migration and invasion(P<0.05,P<0.01,P<0.001).In the model of subcutaneous tumor transplantation in nude mice,the body weight decreased significantly with the extension of time,the time of tumor emergence was also longer than that of HepG2(P<0.001),and liver metastasis of cancer cells was observed in nude mice implanted with Hep3B.Compared with HepG2 cells,the protein expressions of Bax and p53 in Hep3B cells decreased,the protein expressions of Bcl-2,Mcl-1,MVP,MDR1 and Ecadherin significantly increased(P<0.05,P<0.001)and the expressions of p53,MMP2 and E-cadherin in transplanted tumor tissue were consistent with the expression trend of cells(P<0.01,P<0.001).Conclusion:Hep3B cells show more obvious drug resistance,anti-apoptosis,invasion and migration abilities.This study can provide experimental basis for the determination of two clinical treatments of hepatocellular carcinoma with different biological characteristics.