幽门螺杆菌中性粒细胞激活蛋白对615系胃癌小鼠肿瘤生长的影响

Effect of Hp-NAP on tumor growth of 615 line gastric cancer mice

[目的]通过建立小鼠胃癌模型,探讨幽门螺杆菌中性粒细胞激活蛋白(Hp-NAP)对免疫细胞Th17/Treg的调节作用,以及是否通过影响其平衡来抑制胃癌的发展。[方法](1)18只6周龄SPF级正常雄性615系小鼠,随机分为健康对照组、胃癌阴性处理组(简称胃癌组)、胃癌干预组(简称干预组)。胃癌组及干预组均于小鼠右下肢根部皮下注射7×10^(6)/μl mfc细胞200μl,干预组分别于第0、3、6、9、12天给予瘤旁注射0.4μg/μlHp-NAP 150μl,胃癌组于同一时间给予瘤旁注射150μl磷酸盐缓冲液,第14天处死小鼠并剥离肿瘤。(2)比较肿瘤的大小及检测瘤体内血管内皮生长因子(VEGF)mRNA的表达情况。(3)流式细胞技术检测各组小鼠脾脏组织中Th17、Treg细胞的数量。(4)RT-PCR法检测瘤体组织中IL-17、ROR-γt、foxP3 mRNA表达情况。[结果](1)肿瘤蜡块标本组织切片苏木精-伊红染色可见大量核大深染并有核分裂像的癌细胞;干预组瘤体平均体积(0.291 cm^(3))较胃癌组(0.409 cm^(3))小,而且胃癌组有1例出现腹腔转移和血性腹水形成;干预组瘤体组织中VEGFmRNA表达及血管密度明显低于胃癌组(P<0.01)。(2)胃癌组脾脏组织中Th17、Treg细胞数明显高于健康对照组(P<0.05),干预组Th17细胞数量也明显高于胃癌组(P<0.05);而干预组与胃癌组脾脏组织中Treg细胞数无明显差异(P>0.05)。(3)干预组瘤体组织中IL-17、ROR-γt mRNA表达明显高于胃癌组(P<0.05,P<0.01),而foxP3 mRNA表达无明显差异(P>0.05)。(4)干预组瘤体组织中IL-17蛋白水平较胃癌组明显增高(P<0.05),而TGF-β表达无显著差异。[结论]Hp-NAP可能通过增加外周Th17细胞表达及肿瘤微环境中IL-17的浸润直接或间接抑制了胃癌的进展。

[Objective]To establish a mouse model of gastric cancer to explore the role of Hp-NAP on immune cells of Th17/Treg and whether to inhibit the development of gastric cancer by affecting its balance.[Methods]A total of 18 six week old male SPF 615 mice were randomly divided into healthy control group,gastric cancer negative treatment group(gastric cancer group),gastric cancer intervention group(the intervention group).Both gastric cancer group and intervention group were injected subcutaneously of 7×10^(6)MFC cells 200μl in the right lower limb roots.Mice of intervention group were respectively treated with peritumoral injection of 40μg/100μl/dose of Hp-NAP 150μl on days 0,3,6,9,12;the mice of gastric cancer group were given with peritumoral injection of 150μl sterile PBS at the same time.All of mice were killed at day 14 and tumors were excised and analyzed.First,to assess the growth of tumor,we measured the tumor size and detected the expression of vascular endothelium growth factor(VEGF)mRNA level.Then the number of Th17 and Treg cells in spleen were calculated by Flow cytometry technology.Changes in expression of IL-17,RORγt,foxP3 mRNA levels in tumours were documented by qRT-PCR.[Results]We found that HE staining of tumor specimens showed a number of cancer cells with hyperchromatic nuclei and mitotic figures.The average volume of tumors in group(0.291 cm^(3))with Hp-NAP-treated was smaller than gastric cancer group(0.409 cm^(3)),and there was a mouse with peritoneal metastasis and hemorrhagic ascites in latter group.Expression of VEGF mRNA and the MVD in tumors was significantly lower in intervention group than gastric cancer group(P=0.004).In addition,number of Th17 and Treg cells in gastric carcinoma group was significantly higher than the normal group(P=0.013,P=0.022).Th17 cells of intervention group was obviously higher than that of gastric cancer group(P=0.026).However Treg cells in the spleen had no significant difference between intervention group and gastric cancer group(P>0.05).The expression of IL-17,RORγt mRNA in intervention group was obviously higher than gastric carcinoma group(P=0.033,P=0.002),while expression of foxP3 mRNA had no obvious difference(P=0.059).The level of IL-17 protein in tumor tissue of intervention group was significantly higher than that of gastric cancer group(P=0.021),but the expression of TGF-βhad no significant difference.[Conclusion]Our findings indicate that Hp-NAP increase peripheral Th17 cells and the infiltrating of IL-17 in tumor microenvironment to inhibit the development of gastric cancer directly or indirectly.