检测粪便脱落细胞中miR-135b表达筛查结直肠进展期腺瘤及TNMⅠ期Ⅱ期癌的意义研究
发布时间：2018-07-16 16:35
【摘要】：研究背景:结直肠癌(CRC)是全球范围内最常见的恶性肿瘤之一,目前我国结直肠癌患者发现多属晚期,五年生存率低,而结直肠进展期腺瘤(CRA)是结直肠癌最重要的癌前病变。大量数据显示,TNM I期结直肠癌可通过外科手术治愈,II期率的五年生存率为78%,III期和IV期的五年生存率不足40%,探索TNM I、II期CRC和CRA的最佳筛查方法具有较高的临床意义。最近研究发现,粪便脱落细胞mi R-135b在结直肠癌、结直肠腺瘤的表达量高于正常对照,差异具有统计学意义,但诊断意义较低,可能与当前采用的粪便脱落细胞富集方法导致的细胞富集率低有关。我们首次采用了h NOK/h Sef单克隆抗体-叶酸磁珠法,富集清肠液中的肠道上皮脱落细胞,并评估该方法的富集效率。同时以TNM I、II期CRC、进展期腺瘤和正常对照为研究对象,评估粪便脱落细胞mi R-135b诊断结直肠进展期腺瘤和TNM I、II期癌的诊断意义及其与临床病理特点的相关性。目的:1.验证mi R-135b在结直肠癌癌组织与癌旁正常粘膜组织的表达量差异,及其与TNM分期的关系。2.评估hNOK/hSef单克隆抗体-叶酸纳米磁珠法富集粪便脱落细胞的效率。3.评估粪便脱落细胞mi R-135b诊断TNM I、II期CRC和CRA的诊断意义及其与临床病理特点的相关性。方法:1.抽取北京军区总医院消化内窥镜室2014年9月至2015年12月期间行结肠镜检查并经病理学确诊的CRC患者18例,不伴有其他恶性肿瘤,未进行手术、放化疗等任何治疗。在行结肠镜检查时留取癌组织及癌旁正常黏膜组织,离体后立刻放入盛有RNALater RNA stablization reagent的冻存管中,置于㧟80℃冰箱中保存。提取结直肠癌癌组织、癌旁正常组织的总RNA,电泳检测提取到的总RNA完整性,实时定量PCR(real-time PCR,q RT-PCR)测定其中mi R-135b的相对表达量。验证mi R-135b在结直肠癌患者癌组织表达量与癌旁正常粘膜组织的差异,及其与TNM分期的关系。2.抽取北京军区总医院消化内窥镜室2014年9月至2015年12月期间行结肠镜检查的患者102例,其中TNM I、II期结直肠癌患者52例;进展期腺瘤患者34例(腺瘤直径大于1cm);正常人16例作为对照。以上病例均经病理学诊断证实,不伴有其他恶性肿瘤,患者未进行手术、放化疗等任何治疗。收集患者口服导泻药后的清肠液送至实验室,采用h NOK/h Sef单克隆抗体-叶酸磁珠法富集粪便脱落细胞,制得细胞悬液,分为2份。一份用于细胞计数和提取DNA后通过q RT-PCR检测细胞内参基因(β-actin)信号,评价富集细胞的效率;另一份用于提取RNA,q RT-PCR测定其中mi R-135b的相对表达量。评估粪便脱落细胞mi R-135b表达量对TNM I、II期CRC、CRA和正常对照的诊断意义及其与临床病理特点的相关性。结果:1.所有样本提取的总RNA OD值在1.8-2.0之间,说明样本未受污染,电泳图可见28s、18s、5s三条带,说明RNA具有较高的完整性。2.miR-135b在CRC患者癌组织中的表达高于癌旁正常组织(P=0.021),且与TNM分期进展呈正相关。3.粪便脱落细胞悬液反应前平均细胞计数为1.0×106/ml,反应后平均细胞计数为0.97×106/ml,所有样本内参基因(β-actin)信号均为阳性。4.粪便脱落细胞mi R-135b在进展期腺瘤的表达水平高于正常对照(P=0.029);在TNM I、II期结直肠癌粪便脱落细胞中的表达水平高于正常对照(P=0.038);TNM I、II期的表达水平无明显区别(P=0.290)。根据ROC曲线结果分析,截断点(cut off值)=1.63时,粪便脱落细胞mi R-135b在进展期腺瘤、TNM I、II期结直肠癌的敏感性分别为76.92%、78.24%,在正常对照的特异性为87.50%,AUC分别为0.70(P=0.02)、0.80(P0.001)。5.早期结直肠癌患者粪便脱落细胞中miR-135b的表达与患者年龄、性别、肿瘤分化水平、TNM分期和所在部位无关。结论:1.miR-135b在CRC患者癌组织中的表达量高于癌旁组织,且与TNM分期进展呈正相关。2.h NOK/h Sef单克隆抗体-叶酸纳米磁珠法富集粪便中脱落肠道上皮细胞具有较高的效率。3.粪便脱落细胞mi R-135b具有较好的TNM I、II期结直肠癌和进展期腺瘤诊断价值,其表达水平与年龄、性别、肿瘤部位、分化程度无关,TNM I和II期的表达量无明显差异。
[Abstract]:Background: colorectal cancer (CRC) is one of the most common malignant tumors in the world. At present, colorectal cancer patients in China are found to be late, with a low five year survival rate, and a progressive colorectal adenoma (CRA) is the most important precancerous lesion of colorectal cancer. A large number of data show that TNM I colorectal cancer can be cured by surgery and the II stage rate The five year survival rate was 78%, the five year survival rate of III and IV was less than 40%. The best screening method for TNM I and II CRC and CRA was of high clinical significance. The recent study found that the expression of MI R-135b in the fecal cells was higher than that of normal exposure, and the difference was statistically significant but the diagnostic significance was low. It may be related to the low cell enrichment rate caused by the method of fecal exfoliative cell enrichment. We first used the H NOK/h Sef monoclonal antibody - folic acid bead method to enrich the intestinal epithelial exfoliative cells in the intestinal liquid and evaluate the enrichment efficiency of this method. At the same time, TNM I, II phase CRC, progressing adenoma and normal control were studied. To assess the diagnostic significance of MI R-135b in the diagnosis of advanced colorectal adenomas and TNM I, II phase and its correlation with clinicopathological features. Objective: 1. to verify the difference in the expression of MI R-135b in colorectal cancer tissues and normal mucosa adjacent to the carcinoma, and the relationship with TNM staging and.2. assessment of hNOK/hSef monoclonal antibodies - leaf. Efficiency of acid nanomagnetic beads enrichment of fecal shedding cells.3. to evaluate the diagnostic significance of MI R-135b in decedal cells for the diagnosis of TNM I, II CRC and CRA and its correlation with the clinicopathological features. Methods: 1. extraction of colonoscopy from September 2014 to December 2015 in the digestive endoscope room of General Hospital of Beijing Military Region and the pathological diagnosis of CRC There were 18 patients with no other malignant tumor, without operation, radiotherapy and chemotherapy. In the case of colonoscopy, the cancer tissue and the normal mucosa adjacent to the cancer were left in the cryopreservation tube with RNALater RNA stablization reagent, and stored in the refrigerator at 80 degrees centigrade. The cancer tissue of colorectal cancer and the normal tissue adjacent to the cancer were extracted. Total RNA, total RNA integrity, real-time quantitative PCR (real-time PCR, Q RT-PCR) determination of the relative expression of MI R-135b in real-time quantitative PCR (real-time PCR, Q RT-PCR). Verify the difference between the expression of MI R-135b in the cancer tissues of the patients with colorectal cancer and the normal mucosa adjacent to the carcinoma, and the relationship with the TNM stage in the endoscope room of General Hospital of Beijing Military Region, 2014 From September to December 2015, 102 patients underwent colonoscopy, including TNM I, 52 cases of II stage colorectal cancer, 34 patients with progressing adenoma (adenoma diameter greater than 1cm) and 16 normal persons as control. All cases were confirmed by pathological diagnosis without other malignant tumors. The patients were not operated on, radiotherapy and chemotherapy and any other treatment. The intestinal liquid after oral catharsis was sent to the laboratory, and the H NOK/h Sef monoclonal antibody - folic acid beads were used to enrich the fecal exfoliative cells, and the cell suspension was obtained. The cell suspension was divided into 2 parts. One portion was used for cell count and DNA to detect the cell parameter (Beta -actin) by Q RT-PCR, and the other was used to extract the cells. The relative expression of MI R-135b was measured by RNA and Q RT-PCR. The diagnostic significance of MI R-135b expression in the exfoliated cells of feces to TNM I, II CRC, CRA and normal controls and its correlation with the clinicopathological features. The three bands showed that the expression of RNA with high integrity.2.miR-135b was higher in the cancer tissues of CRC patients than that in the normal tissue (P=0.021), and the average cell count was 1 x 106/ml before the.3. stool exfoliative cell suspension, and the average cell count was 0.97 x 106/ml after the reaction, and all the sample internal reference genes (beta -actin) The expression level of MI R-135b in the positive.4. fecal shedding cells was higher than that in the normal control (P=0.029); in TNM I, the expression level of the excrement exfoliated cells in II stage colorectal cancer was higher than that of the normal control (P=0.038); there was no significant difference between TNM I, II stage (P=0.290). At =1.63, the sensitivity of MI R-135b in fecal exfoliative cells in progressing adenomas, TNM I and II stage colorectal cancer was 76.92%, 78.24%, respectively, 87.50% in the normal control, 0.70 (P=0.02) in AUC and 0.80 (P0.001).5. in the early colorectal cancer patients' fecal abscission cell miR-135b expression with the patient's age, sex, the level of tumor differentiation, TNM. Conclusion: the expression of staging and location is independent of location. Conclusion: the expression of 1.miR-135b in the cancer tissues of CRC patients is higher than that in the paracancerous tissue, and it is positively correlated with the progression of TNM staging, and there is a positive correlation with the progress of the TNM staging..2.h NOK/h Sef monoclonal antibody - folic acid nanomagnetic bead method enriches the exfoliated intestinal epithelial cells in the feces and has a higher efficiency..3. fecal excretion cells mi R-135b has a better TNM I, II The diagnostic value of colorectal cancer and progressing adenoma was not related to age, sex, tumor location, and degree of differentiation, and there was no significant difference in the expression of TNM I and II.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.34
本文编号：2126977
[Abstract]:Background: colorectal cancer (CRC) is one of the most common malignant tumors in the world. At present, colorectal cancer patients in China are found to be late, with a low five year survival rate, and a progressive colorectal adenoma (CRA) is the most important precancerous lesion of colorectal cancer. A large number of data show that TNM I colorectal cancer can be cured by surgery and the II stage rate The five year survival rate was 78%, the five year survival rate of III and IV was less than 40%. The best screening method for TNM I and II CRC and CRA was of high clinical significance. The recent study found that the expression of MI R-135b in the fecal cells was higher than that of normal exposure, and the difference was statistically significant but the diagnostic significance was low. It may be related to the low cell enrichment rate caused by the method of fecal exfoliative cell enrichment. We first used the H NOK/h Sef monoclonal antibody - folic acid bead method to enrich the intestinal epithelial exfoliative cells in the intestinal liquid and evaluate the enrichment efficiency of this method. At the same time, TNM I, II phase CRC, progressing adenoma and normal control were studied. To assess the diagnostic significance of MI R-135b in the diagnosis of advanced colorectal adenomas and TNM I, II phase and its correlation with clinicopathological features. Objective: 1. to verify the difference in the expression of MI R-135b in colorectal cancer tissues and normal mucosa adjacent to the carcinoma, and the relationship with TNM staging and.2. assessment of hNOK/hSef monoclonal antibodies - leaf. Efficiency of acid nanomagnetic beads enrichment of fecal shedding cells.3. to evaluate the diagnostic significance of MI R-135b in decedal cells for the diagnosis of TNM I, II CRC and CRA and its correlation with the clinicopathological features. Methods: 1. extraction of colonoscopy from September 2014 to December 2015 in the digestive endoscope room of General Hospital of Beijing Military Region and the pathological diagnosis of CRC There were 18 patients with no other malignant tumor, without operation, radiotherapy and chemotherapy. In the case of colonoscopy, the cancer tissue and the normal mucosa adjacent to the cancer were left in the cryopreservation tube with RNALater RNA stablization reagent, and stored in the refrigerator at 80 degrees centigrade. The cancer tissue of colorectal cancer and the normal tissue adjacent to the cancer were extracted. Total RNA, total RNA integrity, real-time quantitative PCR (real-time PCR, Q RT-PCR) determination of the relative expression of MI R-135b in real-time quantitative PCR (real-time PCR, Q RT-PCR). Verify the difference between the expression of MI R-135b in the cancer tissues of the patients with colorectal cancer and the normal mucosa adjacent to the carcinoma, and the relationship with the TNM stage in the endoscope room of General Hospital of Beijing Military Region, 2014 From September to December 2015, 102 patients underwent colonoscopy, including TNM I, 52 cases of II stage colorectal cancer, 34 patients with progressing adenoma (adenoma diameter greater than 1cm) and 16 normal persons as control. All cases were confirmed by pathological diagnosis without other malignant tumors. The patients were not operated on, radiotherapy and chemotherapy and any other treatment. The intestinal liquid after oral catharsis was sent to the laboratory, and the H NOK/h Sef monoclonal antibody - folic acid beads were used to enrich the fecal exfoliative cells, and the cell suspension was obtained. The cell suspension was divided into 2 parts. One portion was used for cell count and DNA to detect the cell parameter (Beta -actin) by Q RT-PCR, and the other was used to extract the cells. The relative expression of MI R-135b was measured by RNA and Q RT-PCR. The diagnostic significance of MI R-135b expression in the exfoliated cells of feces to TNM I, II CRC, CRA and normal controls and its correlation with the clinicopathological features. The three bands showed that the expression of RNA with high integrity.2.miR-135b was higher in the cancer tissues of CRC patients than that in the normal tissue (P=0.021), and the average cell count was 1 x 106/ml before the.3. stool exfoliative cell suspension, and the average cell count was 0.97 x 106/ml after the reaction, and all the sample internal reference genes (beta -actin) The expression level of MI R-135b in the positive.4. fecal shedding cells was higher than that in the normal control (P=0.029); in TNM I, the expression level of the excrement exfoliated cells in II stage colorectal cancer was higher than that of the normal control (P=0.038); there was no significant difference between TNM I, II stage (P=0.290). At =1.63, the sensitivity of MI R-135b in fecal exfoliative cells in progressing adenomas, TNM I and II stage colorectal cancer was 76.92%, 78.24%, respectively, 87.50% in the normal control, 0.70 (P=0.02) in AUC and 0.80 (P0.001).5. in the early colorectal cancer patients' fecal abscission cell miR-135b expression with the patient's age, sex, the level of tumor differentiation, TNM. Conclusion: the expression of staging and location is independent of location. Conclusion: the expression of 1.miR-135b in the cancer tissues of CRC patients is higher than that in the paracancerous tissue, and it is positively correlated with the progression of TNM staging, and there is a positive correlation with the progress of the TNM staging..2.h NOK/h Sef monoclonal antibody - folic acid nanomagnetic bead method enriches the exfoliated intestinal epithelial cells in the feces and has a higher efficiency..3. fecal excretion cells mi R-135b has a better TNM I, II The diagnostic value of colorectal cancer and progressing adenoma was not related to age, sex, tumor location, and degree of differentiation, and there was no significant difference in the expression of TNM I and II.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.34
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