摘要
结肠癌是我国高发的恶性肿瘤,占胃肠道恶性肿瘤的第三位,其发病率逐年增高,且有年轻化趋势。结肠癌诊断主要依赖肠镜及腹部CT等手段,故大多数患者明确诊断时已处于癌症晚期失去了手术治疗的机会,因此寻找特异的早期诊断标记及分子治疗靶点十分重要。鱼精蛋白(protamines,PRM)被认为与男性不育有关,最新研究表明,PRM1可能是一个新的结肠癌相关的肿瘤/睾丸(Cancer/Testis,CT)抗原基因。本研究的目的在于通过检测结肠癌组织中PRM1-mRNA和蛋白水平的变化,阐述PRM1在临床诊断结肠癌中的意义及其作为基因治疗靶点的可能性。具体研究如下:
1实验路线
1.1收集结肠癌患者外周血,分离血清;收集结肠癌组织、正常结肠组织。
1.2实时定量PCR比较结肠癌组织与正常结肠组织PRM1mRNA表达水平差异。
1.3PRM1mRNA检测用于结肠癌诊断敏感性分析。
1.4免疫组织化学染色观察PRM1在结肠癌组织中的表达。
1.4.1结肠癌组织PRM1表达水平与正常结肠组织PRM1表达水平差异比较。
1.4.2高、中、低分化结肠腺癌组织PRM1表达水平差异比较。
1.5western blot检测比较结肠腺癌与正常结肠组织PRM1表达水平差异。
1.6ELISA试验检测结肠癌患者血清PRM1水平。
1.6.1结肠癌患者血清PRM1水平与健康人差异比较。
1.6.2不同分期结肠癌患者血清PRM1水平差异比较。
1.6.3高、中、低分化结肠腺癌患者血清PRM1水平差异比较。
1.6.4PRM1血清检测用于结肠癌诊断的阳性率。
1.7ELISA试验检测结肠癌患者血清CEA水平。
1.8血清PRM1、CEA检测用于结肠癌诊断敏感性比较。
2结果
2.1结肠癌PRM1mRNA表达水平高于正常结肠组织
53例结肠癌组织平均PRM1-mRNA含量显著高于正常结肠组织,P=0.01116。将定量PCR反应产物进行琼脂糖凝胶电泳,通过辉度扫描比较结肠癌与正常结肠组织PRM1mRNA表达差异,结果与定量PCR结果一致。
2.2组织PRM1mRNA检测用于结肠癌诊断敏感性分析
以结肠癌组织中PRM1-mRNA含量大于等于正常结肠组织PRM1-mRNA含量的2倍以上记为PRM1-mRNA表达阳性。统计结果显示,53例结肠癌病例PRM1mRNA检测总体阳性率为96.2%(51/53)。
2.3免疫组织化学染色观察PRM1在结肠癌组织中的表达
实验采取免疫组织化学染色的方法检测结肠癌组织表达PRM1表达及确定组织分布特点。结果显示,结肠癌细胞胞浆内及癌细胞围成的腺腔内可见PRM1阳性染色颗粒,正常结肠组织细胞内无明显PRM1阳性染色。低分化腺癌细胞内PRM1染色强度明显高于高分化腺癌细胞。
2.4western blot检测PRM1在结肠癌组织中的表达
实验采取western blot的方法检测结肠癌组织与正常结肠组织表达PRM1水平差异。结果显示,结肠癌组织PRM1水平远远高于正常结肠组织,与免疫组化实验一同证明,结肠癌组织整体PRM1表达水平高于健康结肠组织。
2.5ELISA试验检测结肠癌患者血清PRM1水平
2.5.1结肠癌患者血清PRM1水平与健康人差异比较
ELISA试验检测结肠癌患者与健康成年人血清PRM1水平,结果显示,结肠癌患者血清PRM1水平显著高于健康成年人,P<0.05。
2.5.2PRM1血清检测用于结肠癌诊断的阳性率
根据健康成年人PRM1血清水平的95%可信区间判断结肠癌患者血清水平阳性率,结果显示,结肠癌患者血清PRM1检测阳性率为80%(24/30)。
2.5.3不同分期结肠癌患者血清PRM1水平差异比较
比较T_1N_XM_X、T_2N_XM_X、T_3N_XM_X结肠癌患者血清PRM1水平,结果显示,各组患者血清PRM1水平无明显差异,提示结肠癌患者血清PRM1水平升高程度与肿瘤T分期无关。
2.5.4高、中、低分化结肠腺癌患者血清PRM1水平差异比较
按结肠腺癌细胞病理分化程度比较患者血清PRM1水平差异,结果显示,低分化结肠癌患者血清PRM1水平与中分化结肠癌患者相比,差异无显著性,P>0.05。
2.6ELISA试验检测结肠癌患者血清CEA水平
ELISA试验检测结肠癌患者与健康成年人血清CEA水平,结果显示,结肠癌患者血清CEA平均水平显著高于健康成年人平均水平,P<0.05。
2.7血清PRM1、CEA检测用于结肠癌诊断敏感性比较
临床上常用CEA作为结肠癌等肿瘤的血清学诊断标记,故将PRM1与CEA用于结肠癌诊断的敏感性进行比较,结果显示,血清PRM1用于结肠癌诊断的阳性率为80%(24/30),血清CEA用于结肠癌诊断的阳性率为30%(9/30)。经过统计学检验,p<0.05。证实PRM1用于结肠癌诊断的敏感性高于CEA。
3结论
3.1PRM1mRNA在结肠癌中含量显著高于正常结肠组织。
3.2结肠腺癌细胞及癌细胞围成的腺腔内可见PRM1蛋白阳性染色,且PRM1表达水平与结肠腺癌细胞分化度有关。提示PRM1可能通过自分泌/旁分泌的方式作用于肿瘤细胞。
3.3Western blot实验进一步证明,结肠腺癌组织比正常结肠组织PRM1表达水平显著高于正常肠组织。
3.4结肠癌患者外周血中PRM1水平高于健康成年人,提示PRM1可能通过内分泌的方式作用于远处组织及细胞,具体作用及作用方式有待于进一步研究。
3.5血清PRM1水平用于结肠癌诊断的阳性率高于CEA。
Colon cancer is one high incidence of malignancy in China, accounting for thethird in total malignant tumors of the gastrointestinal. Patients with colon cancerincreased year by year, and are getting younger and younger. Diagnosis of coloncancer mainly depends on the means of colonoscopy and abdominal CT, so mostpatients lost opportunity of surgical treatment when they get clearly diagnosis, solook for specific early diagnostic markers and molecular therapeutic targets is veryimportant. Protamine (PRM) is considered to be related with male infertility, thelatest research shows that, PRM1may be a new colon cancer/testis (Cancer/Testis,CT) antigen gene. The purpose of this study is to elaborated the significance ofPRM1in the clinical diagnosis of colon cancer and the possibility of PRM1as genetherapy targets of colon cancer through the detection of PRM1-mRNA and proteinlevels change in colon cancer tissue. Specific studies are as follows:
Experimental Routes:
1.1Collecte peripheral blood of patients with colon cancer, the serum wasseparated; collected colon cancer tissue and normal colon tissues.
1.2Compare the differences of PRM1mRNA expression level of colon cancertissue and normal colon tissue via Real-time quantitative PCR.
1.3Analysis sensitivity of PRM1mRNA detection for the diagnosis of coloncancer.
1.4Immunohistochemical staining observe PRM1expression in colon cancertissue
1.4.1Compare PRM1expression level in colon cancer tissue and normalcolon tissue PRM1expression levels.
1.4.2Compare difference of PRM1expression in high differentiated adenocarcinoma, medium differentiated colon adenocarcinoma andpoorly differentiated colon adenocarcinoma.
1.5Compare the differences of PRM1expression level between colonadenocarcinoma and normal colon tissue by western blot.
1.6Detect serum PRM1levels of patients with colon adenocarcinoma.
1.6.1Compare the differences serum levels between patients with colonadenocarcinoma and health controls.
1.6.2Compare the differences serum levels between patients who withadenocarcinoma in different stages.
1.6.3Compare the differences serum levels between well, moderate andpoorly differentiated colon adenocarcinoma.
1.6.4The positive rate of serum PRM1for colon cancer diagnosis.
1.7ELISA assay detected serum CEA level in colon cancer.
1.8Compare the sensitivity of serum PRM1, CEA for diagnosis colonadenocarcinoma.
2. Result
2.1The PRM1mRNA levels in the colon cancer is higher than that in normalcolon tissues
The average PRM1-mRNA content of53cases of colon cancer tissue wassignificantly higher than that in normal colon tissue, P=0.01116. The same resultsconsistent with the results of the quantitative PCR was got through the luminancescanning when quantitative PCR reaction product was subjected to agarose gelelectrophoresis and compared PRM1mRNA expression difference between coloncarcinoma and normal colon tissue.
2.2Sensitivity analysis of PRM1mRNA detection for colon cancer diagnosis
PRM1-mRNA expression was defined positive when PRM1-mRNAcontent in colon cancer tissue was higher or equal to2times than that in normalcolon tissue. Statistics results showed that the PRM1mRNA positive rate was96.2% (51/53)
2.3Immunohistochemical staining assay detected PRM1expressions incolon cancer tissue
PRM1expression and tissue distribution characteristics were detected viaimmunohistochemistry. The results showed that PRM1positive staining particleswere found in the cytoplasm of colon cancer cells and glandular cavity surroundedby cancer cells. PRM1staining intensity in poorly differentiated adenocarcinomacells was significantly higher than the well-differentiated adenocarcinoma cells.
2.4Detected PRM1expression in colon cancer tissue via western blot
The experiments used western blot to detect PRM1expression levels of coloncancer tissue and normal colon tissue. The results showed that the PRM1level incolon cancer tissue were far higher than that in the normal colon tissue. Western blotexperiment together with the immunohistochemistry experiment proved the PRM1expression level was higher in colon cancer tissue than that in normal colon tissue.
2.5ELISA test detected serum PRM1level of patients with colon cancer
2.5.1Compare serum PRM1level with colon cancer with that in healthypeople.
Detected serum PRM1levels in patient with colone cancer and healthy adultsby ELISA assay, the results showed that the serum PRM1level of patients withcolon cancer were significantly higher than that in healthy adults, P <0.05.
2.5.2The positive rate of serum PRM1used for colon cancer diagnosis
Using the confidence interval for the group of healthy subjects as the cut-offvalue, the sensitivity of PRM1as markers for colon adenocarcinoma were calculatedto investigate its diagnostic efficacies.Ap-value≤0.05was considered to bestatistically significant.The results showed that the serum positive rate of patientswith colon cancer was80%(24/30)
2.5.3Compared PRM1levels of patients with different stages colon cancer
Compare serum PRM1level of patients with T_1N_XM_X、T_2N_XM_X、T_3N_XM_X colon cancer, the results showed that there was no significant differences in serumPRM1level of patients who in different colon stages.
2.5.4Compared PRM1levels of patients with different differentiation
Compared serum PRM1level according to Pathological differentiation of colonadenocarcinoma cells. The results showed that there was no significant differencesin serum PRM1level of patients with different differentiation, P>0.05.
2.6Detected serum CEA level of patients with colon cancer via ELISA test
Using ELISA test to detect serum CEA level of patients with colon cancer andhealthy adult, the results showed that the average serum CEA level of patients withcolon cancer was significantly higher than that of healthy adults (P<0.05)
2.7serum PRM1, CEA detection for colon cancer diagnostic sensitivity
Serum CEA was commonly as a serological diagnostic marker for colon cancerand other tumors, so we compared the diagnostic sensitivity of serum PRM1withCEA as a colon cancer diagnostic marker, the results showed that the diagnosispositive rate of PRM1was80%(24/30) and positive rate of CEA was30%(9/30).
3. Conclusions
3.1PRM1mRNA levels in colon cancer were significantly higher than that innormal colon tissue.
3.2PRM1positive staining particles were found in the cytoplasm of coloncancer cells and glandular cavity surrounded by cancer cells. PRM1performs bothautocrine and paracrine function.
3.3PRM1level in colon cancer tissue was far higher than that in the normalcolon tissue with western blot.
3.4The serum PRM1level of patients with colon cancer is significantly higherthan that of healthy adults. PRM1performs endocrine function.
3.5Serum PRM1level is more sensitive than serum CEA.
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