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目的:探讨上皮细胞生长因子双调蛋白(amphiregulin,AREG)对小鼠肠癌CT26细胞生长的影响及其相关机制。方法:采用ELISA法检测不同小鼠肿瘤细胞中AREG蛋白的表达水平。将携带AREG基因的重组慢病毒载体转染至小鼠肠癌CT26细胞、黑素瘤B16细胞和肝癌LPC-Akt细胞,同时以转染空载体细胞作为阴性对照。采用MTT法、克隆形成实验和FCM法分别检测AREG过表达后细胞的体外增殖和克隆形成能力以及细胞周期进程。构建AREG过表达CT26细胞的小鼠移植瘤模型,观察小鼠体内移植瘤的生长情况,并且采用FCM法和实时荧光定量PCR法分别检测移植瘤组织中免疫细胞的分布情况以及趋化因子的表达水平。结果:小鼠肠癌CT26、黑素瘤B16和肝癌LPC-Akt细胞中AREG相对低表达。过表达AREG后,上述3种肿瘤细胞的体外增殖能力、克隆形成能力及细胞周期进程均无明显变化(P值均>0.05)。然而,在CT26细胞的小鼠移植瘤模型中,AREG过表达可明显促进肿瘤细胞的体内生长(P<0.01),下调肿瘤组织中CD8~+T细胞所占比例(P<0.05),并降低与CD8+T细胞招募相关的CC趋化因子配体5(CC chemokine ligand 5,CCL5)的转录水平(P<0.05)。结论:AREG能够促进小鼠肠癌CT26细胞的体内生长,推测其作用机制可能与调控CD8+T细胞招募相关趋化因子,从而影响肿瘤微环境有关。
Objective: To investigate the effect of amphiregulin (AREG) on the growth of mouse colon cancer CT26 cells and its related mechanisms. Methods: The expression of AREG protein in different mouse tumor cells was detected by ELISA. Recombinant lentiviral vector carrying AREG gene was transfected into CT26 cells of mice colon carcinoma, B16 cells of melanoma and LPC-Akt cells of liver cancer. Meanwhile, empty vector was transfected into the cells as a negative control. MTT assay, colony formation assay and FCM method were used to detect the proliferation and colony-forming ability of cells after AREG overexpression and the cell cycle progression. The transplanted tumor model of AREG-overexpressing CT26 cells was constructed and the growth of transplanted tumor in mice was observed. The distribution of immune cells and the expression of chemokines were detected by FCM and real-time fluorescence quantitative PCR Level. Results: AREG was relatively low expressed in mouse colon cancer CT26, melanoma B16 and liver cancer LPC-Akt cells. After over-expression of AREG, the proliferation, clonality and cell cycle progression of the above three tumor cells showed no significant changes (P> 0.05). However, the overexpression of AREG significantly promoted the growth of tumor cells in vivo (P <0.01) and downregulated the percentage of CD8 + T cells (P <0.05) in CT26 cells and decreased Transcription levels of CC chemokine ligand 5 (CCL5) correlated with CD8 + T cell recruitment (P <0.05). CONCLUSION: AREG can promote the growth of mouse colon cancer CT26 cells in vivo. It is speculated that AREG may be involved in the regulation of CD8 + T cell recruitment related chemokines and thus the tumor microenvironment.