【作者】 刘徽；

【导师】 林治华；

【作者基本信息】 重庆理工大学 ， 微生物与生化药学， 2011， 硕士

【摘要】 目的:研究IL-17在结直肠癌中的表达以及其和肿瘤微血管密度的关系,初步探讨IL-17促进肿瘤血管生成的作用机理。方法:1.收集55例结直肠癌患者的病理切片,采用免疫组织化学的方法,观察IL-17在结直肠癌组织中的表达情况。2.收集55例结直肠癌患者的病理切片,分析IL-17的分布与结直肠癌微血管密度(MVD)的相关性。首先采用免疫组织化学的方法,观察血管内皮标志CD31在结直肠癌组织中的分布,并进行微血管密度计数。然后运用独立样本T检验,统计分析IL-17与结直肠癌组织MVD的相关性。3.分析结直肠癌中IL-17与VEGF-A的关系。首先,收集55例结直肠癌患者的病理切片,采用免疫组织化学的方法,在结直肠癌组织中观察VEGF-A的表达情况。然后用卡方检验分析IL-17与VEGF-A的相关性。同时,流式分析结肠癌细胞株IL-17受体(IL-17R)的表达,选择高表达IL-17R的细胞株。以50ng/mL的IL-17A刺激细胞后,用荧光定量PCR检测刺激前后的VEGF-A基因的表达差异。4.分析结直肠癌细胞株在IL-17刺激前后后趋化因子CXCL6/CCL2的表达情况。以50ng/mL的IL-17刺激IL-17R高表达的细胞株,荧光定量PCR检测刺激前后趋化因子CXCL6/CCL2的基因表达差异。结果:1.在结直肠癌组织中,我们发现,IL-17的阳性染色主要分布于肿瘤间质和癌巢周围,且伴有炎性细胞的浸润。IL-17的阳性表达为28例,阴性表达为27例。在大肠管状腺瘤和正常的大肠粘膜组织中,IL-17为阴性表达。2.以血管内皮标记物CD31染肿瘤血管内皮,参考Weidner等人方法,统计肿瘤MVD,然后研究其与IL-17表达的相关性。发现IL-17高表达时,MVD值为90.00±32.66;IL-17低表达时,MVD值为66.89±32.83。运用独立样本T检验统计得出结论:肿瘤MVD与IL-17的表达呈正相关(P=0.012)。3.免疫组化对结直肠癌组织中的VEGF-A进行染色,运用卡方检验分析IL-17的表达与VEGF的相关性,结论为IL-17在结直肠癌组织中的表达与VEGF的表达相关(P<0.05)。流式筛选IL-17R高表达的结直肠癌细胞株,选择细胞株LS174T和LOVO(其IL-17受体的表达分别为98.05%和97.63%),以IL-17刺激这两株细胞3h后,其VEGF基因的表达为未刺激的3.8倍和4.87倍。说明IL-17可以上调结直肠癌细胞的VEGF。所以得出结论,IL-17通过上调结直肠癌细胞的VEGF表达进而促进肿瘤血管生成。4.以IL-17刺激高表达IL-17R的结直肠癌细胞株LOVO, LS174T和HCT116 3h后发现CXCL6的基因表达上升为0h的5.27倍,2.61倍和1.3倍,CCL2的基因表达上升为0h的1.17倍,3.4倍和10.37倍。因此,我们得出结论为IL-17通过上调趋化因子CXCL6和CCL2促进肿瘤血管的生成。结论:IL-17在结直肠癌组织中的表达与肿瘤的恶化程度相关。进一步究其原因为IL-17通过上调结直肠癌细胞的VEGF和趋化因子CXCL6和CCL2促进肿瘤血管的生成,进而促进了肿瘤的发生发展和侵袭转移。更多还原

【Abstract】 PurposeThe aim of this study is to determine the IL-17 expression in colorectal cancer (CRC) and to examine the correlation between IL-17 expression and MVD. Thus we initially demonstrate the mechanism of IL-17 promoting tumor angiogenesis in CRC.Methods1. We collected 55 cases of pathological sections obtained from human CRC tissue and determined the IL-17 expression by immunohistochemisty.2. We collected 55 cases of pathological sections obtained from human CRC tissue and examined the correlate between IL-17 expression and MVD in CRC tissue. First, we determined the distribution of CD31, which was the endothelial marker and measured the microvessel density (MVD) in tumor. Then we analyzed the correlation between the level of IL-17 expression and MVD by Student’s T test.3. Analyzed the correlation between the level of IL-17 expression and VEGF-A expression. First, we determined the expression of VEGF-A in CRC pathological sections. Then we analyzed the correlation between the level of IL-17 expression and VEGF-A expression by Pearson’sχ2 test. Second, the IL-17R overexpressed cell lines were selected from CRC cell lines by flowcytometry tests. Eventually, we determined the expression of VEGF gene between the normal cell lines and IL-17 stimulated cell lines using real-time PCR.4. Analyzed the expression of chemokines CXCL6/CCL2 between the normal cell lines and IL-17 stimulated cell lines. We cultured the IL-17R overexpressed cell lines and determine the expression of these two chemokines between the normal cell lines and IL-17 stimulated cell lines by using real-time PCR.Results1. We found in CRC tissue that IL-17 positive staining was located in peritumor (in tumor stroma) and intratumor, followed with infiltrating inflammatory cells. The positive level of IL-17 expression is 28 cases. The IL-17 didn’t expressed in colon adenoma and normal mucosal tissue.2. Tumor vascular endothelium was stained with CD31 and measured MVD according to Weidner method. We analyzed the correlation between IL-17 expression and MVD. When IL-17 was high expression in tissue, the MVD was 90.00±32.66;when IL-17 was low in tissue, MVD was 66.89±32.83. Thus, statistical analysis by Student’s T test, we got the conclusion was that MVD was correlated with IL-17 expression (P=0.012).3. We used immunohistochemisty stain of VEGF-A and statistical analyzed. So the conclusion was IL-17 expression correlated with VEGF, statistical analyzed by Pearson’sχ2 test (P<0.05). We seleted IL-17R highexpressed CRC cell lines examined by flowcytometry test. The IL-17R expression of LS174T and LOVO were 98.05% and 97.63%. Then we used these two cell lines stimulated with IL-17(50ng/mL). After stimulation 3 hours, the gene expressions of VEGF were 3.8 and 4.87 times than the same non-stimulated ones, respectively. Therefore, IL-17 could enhanced proangiogenic activity in CRC tissue and cell lines mediated by upregulating VEGF.4. We cultured LOVO, LS174T and HCT116 and stimulated with IL-17(50ng/mL). Three hours later, we found that the gene expressions of CXCL6 were 5.27, 2.61 and 1.3 times than the same non-stimulated ones and the gene expressions of CCL2 were 1.17, 3.4 and 10.37 times. So IL-17 unregulated CXCL6 and CCL2 and further enhanced proangiogenic activity in CRC cell lines.ConclusionIL-17 expression in CRC was correlated with tumor malignancy. So we did a further study and get the conclusion, which was IL-17 promoted proangiogenic activity in CRC by upregaulating the expression of VEGF and chemokines (CXCL6 and CCL2).更多还原