甲基化水平和免疫细胞表面Tim-3表达水平与前列腺癌的关系研究

发布时间：2018-04-19 23:10

本文选题：前列腺癌 + 临床病理特征　；参考：《新疆医科大学》2016年博士论文

【摘要】：目的:研究前列腺癌患者组织中基因的甲基化水平、免疫细胞表面Tim-3表达水平与前列腺癌临床病理特征的关系,探讨甲基化和Tim-3在前列腺癌发生、侵袭和转移中的作用。方法:1)选取PITX2、WNT5a、SPARC、EPB41L3、TPM4等基因作为目的基因,然后通过甲基化特异性PCR方法分别检测35例良性前列腺增生组织中甲基化水平,接着再对157例临床DNA样本进行筛选,分析甲基化水平与前列腺癌临床病理特征的关系;2)利用流式细胞分析法检测35例良性前列腺增生症患者和157例前列腺癌患者血清中免疫细胞Tim-3表达水平,分析血清中Tim-3表达水平与前列腺癌临床病理特征的关系;采用免疫组织化学方法检测组织中Tim-3在35例良性前列腺增生症和157例前列腺癌标本中的表达水平,统计分析组织中Tim-3表达水平与前列腺癌临床病理特征的关系;3)采用western blot法检测转移潜能前列腺癌LNCa P细胞中Tim-3的表达水平,分析其表达水平与前列腺癌侵袭转移能力;采用Si RNA干扰前列腺癌LNCa P细胞中Tim-3的表达,MTT检测细胞增殖,Transwell小室实验检测前列腺癌LNCa P细胞侵袭能力变化。结果:1)前列腺癌患者组织中PITX2,WNT5a,SPARC,EPB41L3,和TPM4甲基化水平明显升高,提示这些基因甲基化水平与前列腺癌发生发展有一定的关系。根据不同PSA水平结果显示PSA10ng/ml、10~20ng/ml、20ng/ml组PITX2甲基化率分别为25.10%、28.64%及35.82%,不同PSA组中PITX2及WNT5a基因甲基化水平比较,差异有统计学意义(P0.05);Glenson评分≤7分组和≥8分组,PITX2基因甲基化率分别为26.31%、33.16%,两组间比较差异有统计学意义(P0.05);根据不同TNM分期分组为≤T2期、Tx N1期及M1期组,PITX2基因甲基化率为23.13%、26.64%、38.37%,组间比较差异有统计学意义(P0.05)。多因素分析发现PITX2甲基化水平与前列腺癌患者的关系最密切。2)前列腺癌患者外周血中CD4+T、CD8+T细胞表面Tim-3表达水平分别为(4.77±3.56)%,(5.90±4.91)%,良性前列腺增生的表达量为(0.96±0.54)%,(0.73±0.62)%,差异有统计学意义(P0.05);前列腺增生和前列腺癌组中Tim-3免疫组化表达水平评分比较差异有统计学意义(P0.05);前列腺癌免疫组化染色Tim-3表达水平据不同PSA水平结果显示,PSA10ng/ml、10~20ng/ml、20ng/ml组Tim-3免疫组化评分分别为3.92±0.75分、7.63±0.81分及10.37±0.35分,差异有统计学意义(P0.05);根据不同Glenson评分,分为≤7分组和≥8分组,Tim-3免疫组化评分为11.02±0.96分,5.62±0.69分,差异有统计学意义(P0.05);根据不同TNM分期分组为≤T2期、Tx N1期及M1期组,Tim-3免疫组化评分为4.27±0.38,7.34±0.71,10.31±0.82,差异有统计学意义(P0.05)3)Tim-3对人前列腺癌细胞增殖、侵袭能力结果提示,对照组和过表达组和干扰组三组细胞:过表达组的Tim-3的表达水平显著高于对照组,而干扰组Tim-3表达显著低于对照组(P0.05)。结论:1)前列腺癌患者组织PITX2基因甲基化水平高于良性前列腺增生;PITX2甲基化水平与PSA水平、Gleason评分及TNM分期有关,提示PITX2甲基化水平与前列腺癌的存在密切关系;2)前列腺癌患者血清及组织中Tim-3表达水平均高于良性前列腺增生;Tim-3表达水平与PSA水平、Gleason评分及TNM分期有关,提示Tim-3表达水平与前列腺癌存在密切的关系;3)Tim-3表达下调能抑制前列腺癌细胞的生长,Tim-3是前列腺癌的重要调节因子;Tim-3表达水平与前列腺癌侵袭、转移能力有关,可作为前列腺癌侵袭、转移的分子标记物;下调Tim-3能抑制前列腺癌细胞的体外侵袭和迁移能力。
[Abstract]:Objective: To study the methylation level of gene in the tissues of prostate cancer patients, the relationship between the expression level of Tim-3 and the clinicopathological features of prostate cancer, and to explore the role of methylation and Tim-3 in the occurrence, invasion and metastasis of prostate cancer. Methods: 1) select PITX2, WNT5a, SPARC, EPB41L3, TPM4 and other genes as the target genes, and then pass through the gene as the target gene. Methylation specific PCR method was used to detect the methylation level in 35 cases of benign prostatic hyperplasia, and then 157 clinical DNA samples were screened to analyze the relationship between the methylation level and the clinicopathological features of the prostate cancer. 2) 35 cases of benign prostatic hyperplasia and 157 cases of prostate cancer were detected by flow cytometry. The expression level of immune cell Tim-3 in serum, the relationship between the expression of Tim-3 in serum and the clinicopathological features of prostate cancer, and the expression level of Tim-3 in 35 specimens of benign prostatic hyperplasia and 157 cases of prostate cancer by immunohistochemical method, and the level of Tim-3 expression in the analysis tissue and prostate cancer The relationship between the clinicopathological features; 3) the expression level of Tim-3 in the LNCa P cells of metastatic potential prostate cancer was detected by Western blot method, and the expression level of the prostate cancer and the invasion and metastasis of prostate cancer were analyzed. Si RNA interfered with the expression of Tim-3 in the LNCa P cells of the prostate cancer, and the MTT detected the proliferation of the cell, and the Transwell chamber test detected the prostate cancer. Results: 1) 1) the levels of PITX2, WNT5a, SPARC, EPB41L3, and TPM4 methylation in the tissues of the patients with prostate cancer were significantly elevated, suggesting that the levels of methylation of these genes were related to the development of prostate cancer. According to the results of PSA, the PITX2 methylation rate of PSA10ng /ml, 10~20ng/ml and 20ng/ml groups was 25.10%, respectively. 28.64% and 35.82%, the difference of PITX2 and WNT5a gene methylation levels in different PSA groups was statistically significant (P0.05); Glenson score was less than 7 groups and 8 groups, and the methylation rate of PITX2 gene was 26.31%, 33.16%, respectively, and two groups were statistically significant (P0.05); according to the different TNM stages, the group was less than T2, Tx N1 phase and M1 period group, The rate of gene methylation was 23.13%, 26.64%, 38.37%. The difference between the groups was statistically significant (P0.05). The multifactor analysis found that the level of PITX2 methylation was most closely related to the prostate cancer patients. The expression level of Tim-3 in the peripheral blood of the prostate cancer patients was (4.77 + 3.56)%, (5.90 + 4.91)%, and benign prostatic hyperplasia, respectively, in the peripheral blood of the prostate cancer patients. The amount of expression was (0.96 + 0.54)%, (0.73 + 0.62)%, and the difference was statistically significant (P0.05). The difference of Tim-3 immunohistochemical expression level in prostatic hyperplasia and prostate cancer group was statistically significant (P0.05), and the expression level of Tim-3 in prostate cancer immunohistochemical staining showed that PSA10ng/ml, 10~20ng/ml, 20ng/ml group Tim-3, according to the results of different PSA levels. The score of immunohistochemistry was 3.92 + 0.75, 7.63 + 0.81 and 10.37 + 0.35. The difference was statistically significant (P0.05). According to the different Glenson scores, it was divided into less than 7 groups and more than 8 groups. The Tim-3 immunohistochemical score was 11.02 + 0.96, 5.62 + 0.69, and the difference was statistically significant (P0.05). According to the different TNM stages, the group was less than T2, Tx N1 period and M1 Stage group, Tim-3 immunohistochemical score was 4.27 + 0.38,7.34 + 0.71,10.31 + 0.82, the difference was statistically significant (P0.05) 3) Tim-3 on human prostate cancer cell proliferation, invasive ability results suggested that the control group and overexpression group and interference group three groups of cells: the expression level of Tim-3 in the over expression group was significantly higher than the control group, while the Tim-3 expression in the interference group was significantly lower. In the control group (P0.05). Conclusion: 1) the methylation level of PITX2 gene in the tissues of the prostate cancer patients is higher than that of the benign prostatic hyperplasia; the level of PITX2 methylation is related to the level of PSA, the Gleason score and the TNM staging, suggesting that the level of PITX2 methylation is closely related to the existence of prostate cancer; and 2) the level of Tim-3 expression in the serum and tissue of the prostate cancer patients is higher than that of the prostate cancer patients. Benign prostatic hyperplasia; Tim-3 expression level is related to PSA level, Gleason score and TNM staging, suggesting that the expression level of Tim-3 is closely related to prostate cancer; 3) down regulation of Tim-3 expression can inhibit the growth of prostate cancer cells, Tim-3 is an important regulator of prostate cancer; the level of Tim-3 expression is related to the invasion and metastasis of prostate cancer. It can be used as a molecular marker for invasion and metastasis of prostate cancer, and down-regulation of Tim-3 can inhibit the invasion and migration ability of prostate cancer cells in vitro.

【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.25

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