MMR蛋白在雌激素致結(jié)腸癌細(xì)胞凋亡中的作用及SEPT9檢測結(jié)直腸癌的研究
發(fā)布時間：2018-12-08 10:35
【摘要】：背景與目的:近30年來,我國結(jié)直腸癌的發(fā)生率和死亡率呈明顯上升趨勢,結(jié)直腸癌已成為我國的重大公眾衛(wèi)生問題。隨著我國腫瘤防治戰(zhàn)略的整體前移,通過預(yù)防和早診早治來降低結(jié)直腸癌的發(fā)生率和死亡率越來越受重視�；瘜W(xué)預(yù)防是一種較有前景的結(jié)直腸癌一級預(yù)防方法。流行病學(xué)研究表明,雌激素具有預(yù)防結(jié)直腸癌發(fā)生的作用,但雌激素預(yù)防結(jié)直腸癌的具體機(jī)制尚不明確,錯配修復(fù)(mismatch repair,MMR)蛋白可能是其中較關(guān)鍵的因素之一。本文第一部分研究了MMR蛋白在雌激素引起的結(jié)腸癌細(xì)胞凋亡中的作用。已有充足的證據(jù)表明,篩查和早期診斷能降低結(jié)直腸癌的死亡率。糞便隱血試驗(yàn)是目前最常用的結(jié)直腸癌篩查方法,但是其敏感性欠佳。外周血septin9基因甲基化檢測(SEPT9)是一種新的結(jié)直腸癌診斷標(biāo)志物,但尚未在國內(nèi)開展使用。本文第二部分研究了新一代SEPT9在中國人群中檢測結(jié)直腸腫瘤的性能,并與免疫糞隱血試驗(yàn)(fecal immunochemical test,FIT)進(jìn)行了比較。方法:我們使用了兩種MMR蛋白缺陷的人結(jié)腸癌細(xì)胞株:hMLH1缺陷的HCT116細(xì)胞,和hMSH2缺陷的LoVo細(xì)胞。通過轉(zhuǎn)染hMLH1和hMSH2野生型cDNA,又構(gòu)建了相應(yīng)的MMR蛋白正常的細(xì)胞株。在給予雌二醇(17β-estradiol,E2)處理后,觀察不同MMR狀態(tài)的細(xì)胞活性和凋亡率的改變,以及凋亡相關(guān)蛋白Bax,caspase-9和caspase-3表達(dá)的變化。同時,也研究了過表達(dá)雌激素受體β(estrogen receptorβ,ERβ)蛋白對結(jié)腸癌細(xì)胞凋亡的影響。選取經(jīng)結(jié)腸鏡檢查和病理確診的135例結(jié)直腸癌患者,169例腺瘤性息肉患者(包括84例進(jìn)展期腺瘤和85例非進(jìn)展期腺瘤),81例增生性息肉患者,和91例健康對照者(結(jié)腸鏡檢查未見異常)。所有476名患者均在結(jié)腸鏡檢查前抽取外周血,以Epi proColon 2.0試劑盒行septin9基因甲基化檢測。其中177名患者同時接受了SEPT9和FIT檢查。以結(jié)腸鏡檢查為金標(biāo)準(zhǔn),計算SEPT9和FIT檢測結(jié)直腸腫瘤的敏感性和特異性,并以McNemar檢驗(yàn)比較SEPT9和FIT對結(jié)直腸癌和進(jìn)展期腺瘤的檢出率。結(jié)果:E2僅在hMLH1蛋白正常表達(dá)的結(jié)腸癌細(xì)胞中引起凋亡;而在hMLH1蛋白表達(dá)缺失的結(jié)腸癌細(xì)胞中,E2并不能引起明顯的凋亡反應(yīng)。過表達(dá)hMSH2或ERβ也能誘導(dǎo)結(jié)腸癌細(xì)胞凋亡,但這種效應(yīng)與E2處理無關(guān)。即使在hMSH2缺陷的LoVo細(xì)胞中,過表達(dá)hMLH1蛋白也增強(qiáng)了E2誘導(dǎo)的細(xì)胞凋亡反應(yīng)。SEPT9對結(jié)直腸癌的敏感性為74.8%(95%CI:67.0%-81.6%),特異性為87.4%(與非結(jié)直腸癌對比,95%CI:83.5%-90.6%)。SEPT9總的假陽性率為4.7%(95%CI:2.2%-8.6%)。SEPT9對I期結(jié)直腸癌的檢出率為66.7%,II期為82.6%,III期為84.1%,IV期為100%。SEPT9對進(jìn)展期腺瘤的敏感性為27.4%(95%CI:18.7%-37.6%)。FIT對結(jié)直腸癌的敏感性為58.0%(95%CI:46.1%-69.2%),特異性為82.4%(95%CI:74.4%-88.7%)。69例同時接受SEPT9和FIT檢查的結(jié)直腸癌患者中,SEPT9檢測出了25例FIT為陰性的結(jié)直腸癌,而FIT檢測出了12例SEPT9為陰性的結(jié)直腸癌。SEPT9對結(jié)直腸癌的檢測性能優(yōu)于FIT,但是對進(jìn)展期腺瘤的檢出率與FIT相似。結(jié)論:E2導(dǎo)致的結(jié)腸癌凋亡是依賴于錯配修復(fù)蛋白hMLH1的,hMLH1蛋白能增強(qiáng)E2誘導(dǎo)的結(jié)腸癌細(xì)胞凋亡,但這一過程可能獨(dú)立于錯配修復(fù)系統(tǒng)的錯配識別和修復(fù)能力。該結(jié)果提示,在接受雌激素替代治療前、或使用雌激素類似物預(yù)防結(jié)直腸癌前,應(yīng)該評估患者錯配修復(fù)蛋白狀態(tài),以使效益/風(fēng)險比最大化。SEPT9檢測結(jié)直腸癌的敏感性和特異性均高于FIT,可用于結(jié)直腸癌的篩查和早期診斷。但其對進(jìn)展期腺瘤的敏感性較差。SEPT9和FIT兩種方法在檢測結(jié)直腸癌上具有互補(bǔ)性,聯(lián)合使用這兩種方法檢測結(jié)直腸癌可以提高檢出率。
[Abstract]:BACKGROUND & OBJECTIVE: In the past 30 years, the incidence and mortality of colorectal cancer in China have increased significantly, and colorectal cancer has become a major public health problem in our country. With the overall advancement of our country's tumor control strategy, it is more and more important to reduce the incidence and mortality of colorectal cancer by prevention and early diagnosis. Chemical prevention is a promising method for colorectal cancer. Epidemiological studies have shown that estrogen has a role in the prevention of colorectal cancer, but the specific mechanism of estrogen to prevent colorectal cancer is not clear, and mismatch repair (MMR) proteins may be one of the most critical factors. The first part of this paper studies the role of MMR proteins in the apoptosis of colon cancer cells induced by estrogen. There is ample evidence that screening and early diagnosis can reduce the mortality of colorectal cancer. The fecal occult blood test is the most commonly used screening method for colorectal cancer, but its sensitivity is not good. Peripheral blood septin9 gene methylation detection (SEPT9) is a new diagnostic marker for colorectal cancer, but has not been used in the country. The second part of this paper studies the performance of the new generation of SEPT9 in the detection of colorectal tumors in Chinese population, and compared with the faecal occult blood test (FIT). Methods: We used two types of MMR protein-deficient human colon cancer cell lines: hMLH1-deficient HCT116 cells and hMSH2-deficient LoVo cells. By transfecting hMLH1 and hMSH2 wild-type cDNA, the normal cell line of MMR protein was constructed. The changes of cell activity and apoptosis rate in different MMR states, as well as the changes of the expression of Bax, caspase-9 and caspase-3, were observed after the treatment with estradiol (17)-estadiol, E2). The effect of estrogen receptor antagonist (ER) on the apoptosis of colon cancer cells was also studied. Of the 135 patients with colorectal cancer who were diagnosed by colonoscopy and pathology, 169 patients with adenomatous polyposis (including 84 progressive and 85 non-progressive adenomas), 81 hyperplastic polyps, and 91 healthy controls (no abnormalities in colonoscopy) were selected. Peripheral blood was extracted from all 476 patients prior to the colonoscopy and the septin9 gene methylation was detected with the Epi proColon 2.0 kit. Of these, 177 patients received both the SEPT9 and FIT tests. The sensitivity and specificity of SEPT9 and FIT in the detection of colorectal tumors were calculated using colonoscopy as the gold standard, and the detection rates of SEPT9 and FIT for colorectal cancer and advanced adenoma were compared with McNemar test. Results: E2 induced apoptosis only in colon cancer cells that were normally expressed in hMLH1 protein. In colon cancer cells with hMLH1 protein expression, E2 could not cause a significant apoptosis reaction. Overexpression of hMSH2 or ER antigen can also induce apoptosis of colon cancer cells, but this effect is not related to E2 treatment. Even in the hMSH2-deficient LoVo cells, the overexpression of hMLH1 protein also enhances the E2-induced apoptosis reaction. The sensitivity of SEPT9 to colorectal cancer was 74.8% (95% CI: 67. 0%-81.6%), and the specificity was 87.4% (compared with non-colorectal cancer, 95% CI: 83.5%-90.6%). The total false positive rate of SEPT9 was 4.7% (95% CI: 2.2%-80.6%). The detection rate of SEPT9 in stage I colorectal cancer was 65.7%, 82.6% in phase II, 84.1% in phase III and 100% in stage IV. The sensitivity of SEPT9 to the progression of colorectal cancer was 27. 4% (95% CI: 18. 7%-37. 6%). The sensitivity of FIT to colorectal cancer was 58. 0% (95% CI: 46. 1%-69.2%), and the specificity was 82.4% (95% CI: 74. 4%-85.7%). 69 of 69 patients with colorectal cancer who received both SEPT9 and FIT examination showed 25 FIT-negative colorectal cancer, and FIT detected 12 SEPT9-negative colorectal cancer. The detection performance of SEPT9 for colorectal cancer is superior to that of FIT, but the detection rate of the progression adenoma is similar to that of FIT. Conclusion: The apoptosis of colon cancer cells induced by E2 is dependent on the mismatch repair protein hMLH1, and the hMLH1 protein can enhance the apoptosis of colon cancer cells induced by E2, but this process may be independent of mismatch identification and repair capacity of the mismatch repair system. The results suggest that the patient's mismatch repair protein status should be assessed before the estrogen replacement therapy or the use of an estrogen analog to prevent colorectal cancer to maximize the benefit/ risk ratio. The sensitivity and specificity of SEPT9 in the detection of colorectal cancer are higher than that of FIT, which can be used for screening and early diagnosis of colorectal cancer. but its sensitivity to the progression of the adenoma is poor. The two methods of SEPT9 and FIT are complementary in the detection of colorectal cancer, and the detection of colorectal cancer by combination of these two methods can improve the detection rate.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735.3
本文編號：2368205
[Abstract]:BACKGROUND & OBJECTIVE: In the past 30 years, the incidence and mortality of colorectal cancer in China have increased significantly, and colorectal cancer has become a major public health problem in our country. With the overall advancement of our country's tumor control strategy, it is more and more important to reduce the incidence and mortality of colorectal cancer by prevention and early diagnosis. Chemical prevention is a promising method for colorectal cancer. Epidemiological studies have shown that estrogen has a role in the prevention of colorectal cancer, but the specific mechanism of estrogen to prevent colorectal cancer is not clear, and mismatch repair (MMR) proteins may be one of the most critical factors. The first part of this paper studies the role of MMR proteins in the apoptosis of colon cancer cells induced by estrogen. There is ample evidence that screening and early diagnosis can reduce the mortality of colorectal cancer. The fecal occult blood test is the most commonly used screening method for colorectal cancer, but its sensitivity is not good. Peripheral blood septin9 gene methylation detection (SEPT9) is a new diagnostic marker for colorectal cancer, but has not been used in the country. The second part of this paper studies the performance of the new generation of SEPT9 in the detection of colorectal tumors in Chinese population, and compared with the faecal occult blood test (FIT). Methods: We used two types of MMR protein-deficient human colon cancer cell lines: hMLH1-deficient HCT116 cells and hMSH2-deficient LoVo cells. By transfecting hMLH1 and hMSH2 wild-type cDNA, the normal cell line of MMR protein was constructed. The changes of cell activity and apoptosis rate in different MMR states, as well as the changes of the expression of Bax, caspase-9 and caspase-3, were observed after the treatment with estradiol (17)-estadiol, E2). The effect of estrogen receptor antagonist (ER) on the apoptosis of colon cancer cells was also studied. Of the 135 patients with colorectal cancer who were diagnosed by colonoscopy and pathology, 169 patients with adenomatous polyposis (including 84 progressive and 85 non-progressive adenomas), 81 hyperplastic polyps, and 91 healthy controls (no abnormalities in colonoscopy) were selected. Peripheral blood was extracted from all 476 patients prior to the colonoscopy and the septin9 gene methylation was detected with the Epi proColon 2.0 kit. Of these, 177 patients received both the SEPT9 and FIT tests. The sensitivity and specificity of SEPT9 and FIT in the detection of colorectal tumors were calculated using colonoscopy as the gold standard, and the detection rates of SEPT9 and FIT for colorectal cancer and advanced adenoma were compared with McNemar test. Results: E2 induced apoptosis only in colon cancer cells that were normally expressed in hMLH1 protein. In colon cancer cells with hMLH1 protein expression, E2 could not cause a significant apoptosis reaction. Overexpression of hMSH2 or ER antigen can also induce apoptosis of colon cancer cells, but this effect is not related to E2 treatment. Even in the hMSH2-deficient LoVo cells, the overexpression of hMLH1 protein also enhances the E2-induced apoptosis reaction. The sensitivity of SEPT9 to colorectal cancer was 74.8% (95% CI: 67. 0%-81.6%), and the specificity was 87.4% (compared with non-colorectal cancer, 95% CI: 83.5%-90.6%). The total false positive rate of SEPT9 was 4.7% (95% CI: 2.2%-80.6%). The detection rate of SEPT9 in stage I colorectal cancer was 65.7%, 82.6% in phase II, 84.1% in phase III and 100% in stage IV. The sensitivity of SEPT9 to the progression of colorectal cancer was 27. 4% (95% CI: 18. 7%-37. 6%). The sensitivity of FIT to colorectal cancer was 58. 0% (95% CI: 46. 1%-69.2%), and the specificity was 82.4% (95% CI: 74. 4%-85.7%). 69 of 69 patients with colorectal cancer who received both SEPT9 and FIT examination showed 25 FIT-negative colorectal cancer, and FIT detected 12 SEPT9-negative colorectal cancer. The detection performance of SEPT9 for colorectal cancer is superior to that of FIT, but the detection rate of the progression adenoma is similar to that of FIT. Conclusion: The apoptosis of colon cancer cells induced by E2 is dependent on the mismatch repair protein hMLH1, and the hMLH1 protein can enhance the apoptosis of colon cancer cells induced by E2, but this process may be independent of mismatch identification and repair capacity of the mismatch repair system. The results suggest that the patient's mismatch repair protein status should be assessed before the estrogen replacement therapy or the use of an estrogen analog to prevent colorectal cancer to maximize the benefit/ risk ratio. The sensitivity and specificity of SEPT9 in the detection of colorectal cancer are higher than that of FIT, which can be used for screening and early diagnosis of colorectal cancer. but its sensitivity to the progression of the adenoma is poor. The two methods of SEPT9 and FIT are complementary in the detection of colorectal cancer, and the detection of colorectal cancer by combination of these two methods can improve the detection rate.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735.3
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