【摘要】：目的:DNA甲基化是一種調節(jié)基因表達的重要機制,在腫瘤發(fā)生、發(fā)展中起重要作用。研究表明,在腫瘤診斷、預后及療效判斷方面,DNA甲基化是一種有潛在應用價值的腫瘤標志物。本研究運用自行建立的實時熒光定量PCR甲基化檢測方法,檢測前列腺癌患者外周血游離DNA中谷胱苷肽-S-轉移酶P1(GSTP1)基因啟動子區(qū)域甲基化表達情況,探討其在前列腺癌早期診斷中的價值。方法:收集入院治療的患者的外周血樣本,每位患者各2管,約2ml/管,置于-80℃超低溫冰箱凍存。其中符合入組標準的樣本分別為:行前列腺癌內分泌治療患者58例作為A組,患者年齡波動于61~82歲,平均年齡72歲;因“排尿困難,發(fā)現PSA升高”入院就診的患者42例作為B組,其中23例診斷為前列腺癌(B1),19例診斷為非前列腺癌(B2),B組年齡波動于55~78歲,平均年齡70歲;非前列腺疾病患者34例作為C組,PSA在正常參考值范圍內,為同期因尿道狹窄、腎結石、腎囊腫等良性疾病就診的男性患者,其年齡波動于58~76歲,平均年齡68歲。運用甲基化特異性PCR法對三組患者GSTP1基因的表達情況進行定性檢測,運用定量PCR檢測GSTP1基因的甲基化相對表達量,統(tǒng)計分析其與前列腺癌患者主要臨床病理特征之間的關系。結果:1.A組患者外周血游離DNA中GSTP1基因的甲基化相對表達量(M/U)為0.330973310±0.1848315242,B2組患者的相對表達量為1.585767684±0.8382501872,C組患者的相對表達量為2.354086941±2.1664719163,Welch檢驗P=0.0000.05,所以A、B2、C三組總體均數不全相等。兩兩比較,A、B2兩組總體均數間存在顯著差異(P=0.000);A、C兩組總體均數間存在顯著差異(P=0.000);B2、C兩組總體均數間不存在顯著差異(P=0.230)�？烧J為A組患者GSTP1基因甲基化比值低于B2、C兩組。2.B1組患者GSTP1基因的甲基化相對表達量(M/U)為0.304547000±0.1905957866,與B2組比較分析,Welch檢驗P=0.0000.05,兩組總體均數間存在顯著差異。3.A組患者與B1組患者GSTP1基因的甲基化相對表達量比較分析,采用獨立樣本t檢驗,P=0.8060.05,兩組總體均數間無顯著差異。4.前列腺癌患者GSTP1基因甲基化程度與相應PSA相關性分析,Kendall’tau_b、Spearman’rho兩種非參數方法檢驗,Kendall相關系數為0.198,Spearman相關系數為0.263,均為P0.05,提示前列腺癌患者外周血游離DNA中GSTP1基因甲基化相對表達量與PSA相關性不顯著(相關系數小于0.5)。5.Gleason評分為5~7分和8~10分的GSTP1基因甲基化程度總體均數間無顯著差異(P=0.930),危險度分級為中低危和中高危的GSTP1基因甲基化程度總體均數間無顯著差異(P=0.241)。結論:1.前列腺癌患者和前列腺良性病變患者外周血游離DNA中GSTP1基因均存在甲基化和非甲基化表達;2.前列腺癌患者外周血游離DNA中GSTP1基因甲基化表達量比值(M/U)較低,而良性病變患者(BPH等)的甲基化比值較高,推測是機體對腫瘤產生的反饋性保護作用;3.內分泌治療對前列腺癌患者外周血游離DNA中GSTP1基因甲基化相對表達量無影響;4.前列腺癌患者外周血游離DNA中GSTP1基因甲基化相對表達量與PSA相關性不顯著(r0.5),可將其作為篩查前列腺癌的獨立生物標記物;5.檢測患者外周血GSTP1基因甲基化相對表達量,雖然對前列腺癌的Gleason評分和危險度分級無預測作用,但對提高前列腺癌早期診斷準確率有一定價值。
[Abstract]:Objective: DNA methylation is an important mechanism for regulating gene expression and plays an important role in the occurrence and development of tumor. The study shows that DNA methylation is a potentially valuable tumor marker in the diagnosis, prognosis and evaluation of the tumor. This study uses a real-time fluorescence quantitative PCR methylation detection method established by ourselves to detect the tumor. The methylation of the promoter region of the glucocysteinase -S- transferase P1 (GSTP1) gene in the peripheral blood free DNA of the prostate cancer patients was measured and the value of it was discussed in the early diagnosis of prostate cancer. Methods: the peripheral blood samples of the patients treated by the hospital were collected, each of the patients was 2 tubes, about 2ml/ tube, and stored at -80 C cryopreservation. The standard samples of the group were as follows: 58 patients with prostate cancer endocrine therapy were treated as A group. The age of the patients was 61~82 years old and the average age was 72 years old; 42 cases were treated as B group because of "dysuria and PSA rise", of which 23 were diagnosed as prostate cancer (B1), 19 were diagnosed as non prostate cancer (B2), and the B group fluctuated in 55~7. 8 years old, the average age of 70 years, 34 cases of non prostatic disease patients as C, PSA in the normal reference range, for the same period of urethral stricture, kidney stones, renal cysts and other benign diseases in male patients, whose age fluctuates at the age of 58~76 and the average age of 68 years. The expression of GSTP1 gene in three groups of patients by methylation specific PCR The relative expression of methylation of GSTP1 gene was detected by quantitative PCR, and the relationship between the main clinicopathological features of the prostate cancer patients was statistically analyzed. Results: the relative expression of GSTP1 gene (M/U) was 0.330973310 + 0.1848315242 in the peripheral blood free DNA of the 1.A group, and the relative expression of the B2 group was 1.5857. 67684 + 0.8382501872, the relative expression of patients in group C was 2.354086941 + 2.1664719163, Welch test P=0.0000.05, so A, B2, C three groups were not all equal. 22 compared, A, B2 two groups there were significant differences (P=0.000), A, C two groups there were significant differences (P=0.000); two groups of the total number of two groups did not exist obvious There was a difference (P=0.230). The methylation ratio of GSTP1 gene in group A patients was lower than that of B2, and the relative expression of GSTP1 gene methylation (M/U) in group C two was 0.304547000 + 0.1905957866, compared with the B2 group and Welch test P=0.0000.05. There were significant differences in the methylation of the two groups. Relative expression analysis, using independent sample t test, P=0.8060.05, there was no significant difference between the total number of the two groups. The degree of methylation of GSTP1 gene in.4. prostate cancer patients was related to the corresponding PSA correlation analysis, Kendall 'tau_b, Spearman' Rho two non parametric methods, the Kendall correlation coefficient was 0.198, the Spearman correlation coefficient was 0.263, all P. 0.05, the correlation between the relative expression of GSTP1 gene methylation and PSA in the peripheral blood free DNA of the prostate cancer patients was not significant (the correlation coefficient was less than 0.5). There was no significant difference between the GSTP1 gene methylation degree of the 5~7 and 8~10 scores (P=0.930), and the risk grade was the middle and low risk and the middle risk of the GSTP1 gene methylation. There was no significant difference between the total number of degrees (P=0.241). Conclusion: 1. the GSTP1 genes in peripheral blood free DNA of the prostate cancer patients and benign prostatic lesions all have methylation and non methylation expression; 2. the GSTP1 gene methylation expression ratio (M/U) in the peripheral blood free DNA of the prostate cancer patients is lower than that of the benign disease patients (BPH and so on). The higher the ratio of the substrate, it is presumed to be the feedback protective effect of the body on the tumor; 3. the endocrine therapy has no effect on the relative expression of GSTP1 gene methylation in the peripheral blood free DNA of the patients with prostate cancer; 4. the relative expression of the methylation in the free DNA of the peripheral blood of the prostate cancer patients is not significantly associated with the PSA (r0.5). In order to screen the independent biomarkers of prostate cancer, 5. the relative expression of GSTP1 gene methylation in peripheral blood of the patients has no predictive effect on the Gleason score and risk classification of prostate cancer, but it is of certain value in improving the accuracy of early diagnosis of prostate cancer.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R737.25

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