双调蛋白过表达促进小鼠肠癌CT26细胞的体内生长

Overexpression of amphiregulin promotes the growth of mouse colon carcinoma CT26 cells in vivo

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 epidermal growth factor amphiregulin （AREG） on the growth of mouse colon carcinoma CT26 cells and its related mechanisms. Methods： The protein expression level of AREG in different mouse cancer cells was detected by ELISA.Mouse colon carcinoma CT26 cells,melanoma B16 cells and hepatocellular carcinoma LPC-Akt cells were transfected with the recombinant lentiviral plasmid carrying AREG gene,while the ones transfected with empty plasmid were used as the negative controls.After AREG overexpression,the cell proliferation,colony-forming abilities and cell cycle progression in vitro were detected by MTT,colony-forming assay and FCM,respectively.After the homograft mouse model of CT26 cells was constructed,the growth of homograft tumor was observed,the distribution of immune cells in tumor tissues was detected by FCM,furthermore the expression of chemokine was detected by real-time fluorescent quantitative PCR. Results： The levels of AREG expression were relatively low in mouse colon carcinoma CT26 cells,melanoma B16 cells and hepatoma LPC-Akt cells.AREG overexpression did not markedly affect the proliferation,colony-forming abilities and cell cycle progression of these three types of tumor cells in vitro（all P 〉 0.05）.However,in the homograft mouse model of CT26 cells,AREG overexpression significantly promoted the growth of tumor cells in vivo（P 〈 0.01）,decreased the percentage of CD8＋ T cells （P 〈 0.05）,and reduced the mRNA level of CC chemokine 5 ligand （CCL5）（P 〈 0.05） which was related to CD8＋ T cell recruitment. Conclusion： AREG promotes the growth of mouse colon carcinoma CT26 cells in vivo.AREG may affect the tumor microenvironment by regulating the production of chemokine which is related to CD8＋ T cell recruitment.