IL-33亚型肝癌稳定转染细胞系的构建及体外生物学功能检测

The construction and biological function assessment of HCC cell lines expressing IL-33 isoform

目的构建小鼠全长IL-33、分泌型IL-33、入核型IL-33肝癌细胞的稳转细胞系并检测其对肝癌细胞增殖、凋亡、细胞周期的影响。方法通过PCR的方法克隆小鼠全长IL-33、分泌型IL-33、入核型IL-33基因并将其连接到慢病毒载体pRRL-Venus中。利用293T细胞包装慢病毒并分别感染小鼠肝癌细胞系hepa1-6,经流式分选单个YFP阳性细胞,培养得到稳定表达全长、分泌型、入核型IL-33的hepa1-6稳转细胞系。通过一系列体外实验研究小鼠全长,分泌型,入核型IL-33对hepa1-6细胞生物学功能的作用。包括CCK8检测细胞增殖,Annexin V和7AAD的流式染色检测细胞凋亡,PI流式染色检测细胞周期。结果成功克隆了小鼠全长IL-33、分泌型IL-33、入核型IL-33基因并成功构建了hepa1-6稳转细胞系;全长IL-33促进肝癌细胞增殖,分泌型和入核型IL-33抑制肝癌细胞增殖;全长IL-33能够抑制早期和晚期细胞凋亡,分泌型IL-33对细胞凋亡无明显影响,入核型IL-33对早期的细胞凋亡没有作用,但是能够抑制晚期的细胞凋亡。分泌型IL-33稳转hepa1-6细胞的细胞周期G1期比例增加,而小鼠全长IL-33和入核型IL-33对hepa1-6稳转细胞的细胞周期均无明显作用。结论成功构建了小鼠全长IL-33、分泌型IL-33、入核型IL-33 hepa1-6稳转细胞系;小鼠全长IL-33促进肝癌细胞生长,抑制肝癌细胞的凋亡;分泌型和入核型IL-33均能抑制肝癌细胞的增殖;分泌型IL-33稳转细胞增加G1期细胞比例,入核型IL-33抑制晚期细胞凋亡。这些结果为进一步研究全长IL-33、分泌型IL-33、入核型IL-33在肝细胞肝癌发展中的作用及其机制奠定了基础。

This study aimed to construct HCC cell lines stable expressing full-length-IL-33（fIL-33）,secreted-IL-33（sIL-33）, or nuclear-IL-33（nIL-33）,and to assess the effect of fIL-33, sIL-33, nIL-33 on tumor cell growth, apoptosis and cell cycle. Firstly, fIL-33, sIL-33, nIL-33 were cloned into lentivirus vector pRRL-Venus. Then lentivirus was generated by transfecting venus-fIL-33, venus-sIL-33, venus-nIL-33 or venus into the 293 T packaging cell lines. Hepa1-6 cells were infected with 1 ml lentiviral supernatant for 24 hours. Single cells were sorted into 96-well plates by flow cytometry 72 h later. A series of in vitro experiments were carried out to determine the biological function of fIL-33, sIL-33 and nIL-33, including cell proliferation by CCK8, apoptosis by Annexin V and 7 AAD, cell cycle by PI flow cytometry. Data showed that the fIL-33, sIL-33, and nIL-33 steady transfected cell lines were successfully established. fIL-33 promoted hapa1-6 cells growth while sIL-33 and nIL-33 inhibited hepa1-6 cells proliferation;fIL-33 inhibited both early and late apoptosis of tumor cells while nIL-33 only inhibited late apoptosis, and sIL-33 did not affect apoptosis of hepa1-6 cells. sIL-33 elevated the percentage of G1 period tumor cells significantly. We did not observe obvious effect of fIL-33 and nIL-33 on cell cycle. These results founded a solid foundation for further investigation of fIL-33, sIL-33 and nIL-33 in the development of hepatocellular carcinoma.