【摘要】：研究背景:結(jié)直腸癌(CRC)是全球范圍內(nèi)最常見的惡性腫瘤之一,目前我國結(jié)直腸癌患者發(fā)現(xiàn)多屬晚期,五年生存率低,而結(jié)直腸進(jìn)展期腺瘤(CRA)是結(jié)直腸癌最重要的癌前病變。大量數(shù)據(jù)顯示,TNM I期結(jié)直腸癌可通過外科手術(shù)治愈,II期率的五年生存率為78%,III期和IV期的五年生存率不足40%,探索TNM I、II期CRC和CRA的最佳篩查方法具有較高的臨床意義。最近研究發(fā)現(xiàn),糞便脫落細(xì)胞mi R-135b在結(jié)直腸癌、結(jié)直腸腺瘤的表達(dá)量高于正常對照,差異具有統(tǒng)計(jì)學(xué)意義,但診斷意義較低,可能與當(dāng)前采用的糞便脫落細(xì)胞富集方法導(dǎo)致的細(xì)胞富集率低有關(guān)。我們首次采用了h NOK/h Sef單克隆抗體-葉酸磁珠法,富集清腸液中的腸道上皮脫落細(xì)胞,并評估該方法的富集效率。同時(shí)以TNM I、II期CRC、進(jìn)展期腺瘤和正常對照為研究對象,評估糞便脫落細(xì)胞mi R-135b診斷結(jié)直腸進(jìn)展期腺瘤和TNM I、II期癌的診斷意義及其與臨床病理特點(diǎn)的相關(guān)性。目的:1.驗(yàn)證mi R-135b在結(jié)直腸癌癌組織與癌旁正常粘膜組織的表達(dá)量差異,及其與TNM分期的關(guān)系。2.評估hNOK/hSef單克隆抗體-葉酸納米磁珠法富集糞便脫落細(xì)胞的效率。3.評估糞便脫落細(xì)胞mi R-135b診斷TNM I、II期CRC和CRA的診斷意義及其與臨床病理特點(diǎn)的相關(guān)性。方法:1.抽取北京軍區(qū)總醫(yī)院消化內(nèi)窺鏡室2014年9月至2015年12月期間行結(jié)腸鏡檢查并經(jīng)病理學(xué)確診的CRC患者18例,不伴有其他惡性腫瘤,未進(jìn)行手術(shù)、放化療等任何治療。在行結(jié)腸鏡檢查時(shí)留取癌組織及癌旁正常黏膜組織,離體后立刻放入盛有RNALater RNA stablization reagent的凍存管中,置于㧟80℃冰箱中保存。提取結(jié)直腸癌癌組織、癌旁正常組織的總RNA,電泳檢測提取到的總RNA完整性,實(shí)時(shí)定量PCR(real-time PCR,q RT-PCR)測定其中mi R-135b的相對表達(dá)量。驗(yàn)證mi R-135b在結(jié)直腸癌患者癌組織表達(dá)量與癌旁正常粘膜組織的差異,及其與TNM分期的關(guān)系。2.抽取北京軍區(qū)總醫(yī)院消化內(nèi)窺鏡室2014年9月至2015年12月期間行結(jié)腸鏡檢查的患者102例,其中TNM I、II期結(jié)直腸癌患者52例;進(jìn)展期腺瘤患者34例(腺瘤直徑大于1cm);正常人16例作為對照。以上病例均經(jīng)病理學(xué)診斷證實(shí),不伴有其他惡性腫瘤,患者未進(jìn)行手術(shù)、放化療等任何治療。收集患者口服導(dǎo)瀉藥后的清腸液送至實(shí)驗(yàn)室,采用h NOK/h Sef單克隆抗體-葉酸磁珠法富集糞便脫落細(xì)胞,制得細(xì)胞懸液,分為2份。一份用于細(xì)胞計(jì)數(shù)和提取DNA后通過q RT-PCR檢測細(xì)胞內(nèi)參基因(β-actin)信號,評價(jià)富集細(xì)胞的效率;另一份用于提取RNA,q RT-PCR測定其中mi R-135b的相對表達(dá)量。評估糞便脫落細(xì)胞mi R-135b表達(dá)量對TNM I、II期CRC、CRA和正常對照的診斷意義及其與臨床病理特點(diǎn)的相關(guān)性。結(jié)果:1.所有樣本提取的總RNA OD值在1.8-2.0之間,說明樣本未受污染,電泳圖可見28s、18s、5s三條帶,說明RNA具有較高的完整性。2.miR-135b在CRC患者癌組織中的表達(dá)高于癌旁正常組織(P=0.021),且與TNM分期進(jìn)展呈正相關(guān)。3.糞便脫落細(xì)胞懸液反應(yīng)前平均細(xì)胞計(jì)數(shù)為1.0×106/ml,反應(yīng)后平均細(xì)胞計(jì)數(shù)為0.97×106/ml,所有樣本內(nèi)參基因(β-actin)信號均為陽性。4.糞便脫落細(xì)胞mi R-135b在進(jìn)展期腺瘤的表達(dá)水平高于正常對照(P=0.029);在TNM I、II期結(jié)直腸癌糞便脫落細(xì)胞中的表達(dá)水平高于正常對照(P=0.038);TNM I、II期的表達(dá)水平無明顯區(qū)別(P=0.290)。根據(jù)ROC曲線結(jié)果分析,截?cái)帱c(diǎn)(cut off值)=1.63時(shí),糞便脫落細(xì)胞mi R-135b在進(jìn)展期腺瘤、TNM I、II期結(jié)直腸癌的敏感性分別為76.92%、78.24%,在正常對照的特異性為87.50%,AUC分別為0.70(P=0.02)、0.80(P0.001)。5.早期結(jié)直腸癌患者糞便脫落細(xì)胞中miR-135b的表達(dá)與患者年齡、性別、腫瘤分化水平、TNM分期和所在部位無關(guān)。結(jié)論:1.miR-135b在CRC患者癌組織中的表達(dá)量高于癌旁組織,且與TNM分期進(jìn)展呈正相關(guān)。2.h NOK/h Sef單克隆抗體-葉酸納米磁珠法富集糞便中脫落腸道上皮細(xì)胞具有較高的效率。3.糞便脫落細(xì)胞mi R-135b具有較好的TNM I、II期結(jié)直腸癌和進(jìn)展期腺瘤診斷價(jià)值,其表達(dá)水平與年齡、性別、腫瘤部位、分化程度無關(guān),TNM I和II期的表達(dá)量無明顯差異。
[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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R735.34

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