【作者】 温玉婷；

【导师】 李敏；

【作者基本信息】 兰州大学 ， 病理学与病理生理学， 2011， 硕士

【摘要】 第一部分目的检测IL-17与STAT3在结直肠癌患者组织和外周血中的表达,探讨其与结直肠癌患者临床病理特征和肿瘤血管生成的关系。方法1.采用免疫组织化学SP法检测94例结直肠癌组织和31例正常肠黏膜组织中IL-17、STAT3蛋白的表达,并用CD34标记微血管计算微血管密度(MVD)。2.收集7例结直肠癌患者和6例健康志愿者外周血标本,RT-PCR法检测外周血单个核细胞(PBMC)中IL-17 mRNA的表达。3. ELISA法检测结直肠癌患者和健康人外周血血浆中IL-17蛋白的含量。结果1.免疫组织化学结果显示IL-17主要表达在结直肠癌组织中的肿瘤细胞和间质淋巴细胞胞浆内。IL-17在肿瘤细胞的阳性表达率和在间质淋巴细胞阳性表达数均明显高于其在正常肠黏膜的表达(P<0.01)；IL-17的表达与结直肠癌分化程度和Duke’s分期有关(P<0.05)。STAT3在结直肠癌组织中的表达率显著高于其在正常肠黏膜中的表达(P<0.01),且与肿瘤细胞的分化程度、淋巴结转移和Duke’s分期有关(P<0.05)。IL-17和STAT3在结直肠癌组织中的表达与MVD均有关(P<0.01)。Spearman等级相关分析表明IL-17和STAT3在结直肠癌组织中的表达呈正相关(r=0.282,P=0.006)。2. RT-PCR结果显示结直肠癌患者PBMC中IL-17 mRNA表达水平明显高于健康对照组(P<0.01)。3. ELISA结果显示结直肠癌患者外周血血浆中IL-17蛋白含量高于健康对照组(P<0.05)。结论1.IL-17在结直肠癌组织间质淋巴细胞中表达增加,并且结直肠癌Duke’s分期越高、肿瘤细胞分化程度越低和MVD值越高,IL-17阳性淋巴细胞数量越多,提示IL-17可能来源于间质淋巴细胞,在结直肠癌的血管生成和生长转移过程中可能发挥促进作用。2.IL-17与STAT3在结直肠癌组织的肿瘤细胞中高表达,并与MVD均有关,提示两者可能在结直肠癌的血管生成和肿瘤的发生发展中起到重要的协同作用。3. IL-17 mRNA和蛋白在结直肠癌患者外周血表达上调,提示IL-17可能在结直肠癌的发生过程中起到了重要的调控作用。第二部分目的探讨人结肠癌细胞株SW480对健康人PBMC产生IL-17的影响,并观察不同浓度的人重组白细胞介素17 (rhIL-17)对SW480细胞增殖、侵袭和迁移能力的影响。方法1.体外共培养SW480细胞和健康人PBMC, ELISA法检测不同时间段培养的PBMC上清液中IL-17蛋白的含量。2.体外培养人结肠癌细胞株SW480,用不同浓度的rhIL-17处理细胞,MTT法检测细胞的生长变化。3. Boyden小室法检测不同浓度的rhIL-17处理的SW480细胞侵袭、迁移能力的改变。结果1.SW480细胞处理组中PBMC的IL-17蛋白的含量明显增加,与自发培养组和刺激培养组相比差异具有统计学意义(P<0.05)。2.SW480细胞经不同浓度的rhIL-17处理后,细胞增殖能力没有明显改变。3.rhIL-17以剂量依赖的方式增强SW480细胞的侵袭和迁移能力,与正常对照组相比差异具有统计学意义(P<0.05)。结论1.SW480细胞可能促进PBMC产生内源性IL-17。2.IL-17可能参与了SW480细胞的侵袭、迁移过程。更多还原

【Abstract】 Part OneObjectivesTo explore the relationship between the expression of IL-17 and STAT3 in blood and tissue of CRC, and to investigate their possible relationships with the clinic pathological features and the angiogenesis.Methods1. Immunohistochemistry (SP method) was used to detect the expression of IL-17 and STAT3 proteins in 94 specimens of colorectal carcinoma tissues and 31 cases normal mucosa membrane. Microvessel density (MVD) was counted based on CD34 marked endothelial cells of nicrovessels.2. RT-PCR was used to detect the expression level of IL-17 mRNA in peripheral blood mononuclear cell (PBMC) of 7 colorectal carcinoma patients and 6 health people.3. ELISA was used to detect the expression level of IL-17 protein in plasma of 7 colorectal carcinoma patients and 6 health people.Results1. Immunohistochemical data showed that IL-17 protein significantly expressed in cytoplasm of tumor cells and stromal lymphocytes of CRC tissues. The positive expressions of IL-17 in tumor cells and stroma lymphocytes were significantly higher than in normal mucous membrane (P<0.01). The expression of IL-17 was related to cancer cell differentiation and Duke’s stage (P<0.05). Compared with corresponding normal tissues, the levels of STAT3 were significantly increased in tumors of CRC patients (P<0.01). The expression of STAT3 in CRC was related to cancer cell differentiation, metastasis of lymphnode and Duke’s stage (P<0.05). MVD was closely correlated to the expression of IL-17 and STAT3 (P<0.01). and there was a stable positive relationship among the two proteins (r=0.282,P=0.006). 2. The expression level of IL-17 mRNA in patients" PBMC was higher than control group (P<0.01).3. The expression level of IL-17 protein in patients’ plasma was higher than control group (P<0.05).Conclusion1. The expression of IL-17 is up-regulated in stromal lymphocytes of CRC tissues, and the prevalence of intratumoral IL-17-producing cells correlate with the poorer differentiation and the higher Duke’s stage. Moreover, the level of IL-17-producing cells are positively correlated the intratumoral MVD in tumor. It is suggested that IL-17 is derived from stromal lymphocytes in tumor. IL-17 may play an important role in the angiogenesis and metastasis of CRC.2. IL-17 and STAT3 expressing are significantly higher in CRC tissues, and MVD is closely correlated to these factors. It is suggested that the two factors may play an important synergism role in the angiogenesis and carcinogenesis of CRC.2. The expression level of IL-17 mRNA and protein are up-regulated in peripheral blood of CRC patients. It is implied that IL-17 is probably play an important regulation role involved in the pathogenesis of CRC.Part TwoObjectivesTo investigate the influence of human colon carcinoma cell line SW480 on IL-17 secreted by PBMC of healthy people in vitro and to examine the effect of recombinant human IL-17 (rhIL-17) on the proliferation, invasion and motility of SW480.Methods1. ELISA was used to detect the expression level of IL-17 protein in supernatants of PBMC cocultured with SW480 in vitro.2. Human colon carcinoma cell line SW480 cultured were treated respectively with different concentrations of rhIL-17. The effects of rhIL-17 on the proliferation property of SW480 were detected by MTT colorimetric assay.2. Boyden chamber were used to detect the effects of rhIL-17 with different concentrations on the invasion and motility of SW480.Results 1. IL-17 levels in supernatants from SW480 cocultures were higher than that of in medium alone and OKT3-preactivated PBMC (P<0.01).2. Human colon carcinoma cell line SW480 cultured in vitro were treated respectively with different concentrations of rhIL-17. The effects of rhlL-17 on the proliferation property of SW480 were detected by MTT colorimetric assay.3. Boyden chamber were used to detect the effects of rhIL-17 with different concentrations on the invasion and motility of SW480.Conclusion1. Tumor cells may facilitate the generation of IL-17 by PBMC.2. IL-17 may regulate the invasion and motility of the SW480 cells.更多还原