CA215及其單克隆抗體RP215在結(jié)直腸癌中的研究
發(fā)布時間：2018-08-18 19:43
【摘要】：[研究背景與目的]CA215是一個糖蛋白中的糖類相關(guān)抗原表位,與人免疫球蛋白的重鏈?zhǔn)峭吹?在正常人的免疫球蛋白和人體組織中幾乎不表達(dá),但在大多數(shù)腫瘤組織中表達(dá)�，F(xiàn)有的文獻(xiàn)提示,CA125及其單克隆抗體RP215可作為多種腫瘤的免疫診斷標(biāo)志物,并開發(fā)靶向抗體的免疫治療藥物。本課題旨在研究CA215在結(jié)直腸癌患者組織以及血清中的表達(dá)情況,結(jié)合患者的臨床病理特征資料,分析CA215與結(jié)直腸癌侵襲、轉(zhuǎn)移和預(yù)后的關(guān)系,評估CA215能否作為一個合適的腫瘤標(biāo)志物用于結(jié)直腸癌的診斷和療效監(jiān)測。初步研究RP215對結(jié)直腸癌細(xì)胞系的免疫治療的效果。[方法]1.采用免疫組織化學(xué)方法檢測CA215在132例結(jié)直腸癌患者腫瘤組織中的表達(dá)情況,分選出CA215陽性染色和陰性染色的患者,研究CA215的表達(dá)與臨床病理資料之間的關(guān)系。2.采用ELISA方法分別檢測83例健康人、15例IBD、20例結(jié)腸息肉患者和103例結(jié)直腸癌患者血清中CA215的水平(血清CA215水平11.35 AU/ml定義為陽性),評估其作為結(jié)直腸癌患者血清標(biāo)志物的可行性。比較其與臨床上常用的血清腫瘤標(biāo)志物CEA、CA199在結(jié)直腸癌診療中的特異性和敏感性。3.采用蛋白質(zhì)印跡法(Western Blot)檢測腸癌細(xì)胞系SW480、Caco2、HCT116和Lovo以及正常腸上皮細(xì)胞NCM460中CA215的表達(dá)情況,采用細(xì)胞涂片免疫化學(xué)方法檢測CA215在NCM460、SW480及HCT116中的表達(dá)情況。4.采用CCK8細(xì)胞增殖實(shí)驗(yàn)和流式細(xì)胞術(shù)分別測定RP215在不同濃度、不同作用時間下,對結(jié)直腸癌細(xì)胞SW480、HCT116及人正常結(jié)腸上皮細(xì)胞系NCM460增殖及凋亡中的影響。[結(jié)果]1. 132例結(jié)直腸腺癌患者中CA215陽性(免疫組化評分≥1分)患者有68例,陽性率為51.5%。CA215陰性(免疫組化評分1分)的患者共有64例,占48.5%。CA215的表達(dá)與結(jié)直腸癌患者的局部浸潤、腫瘤的組織學(xué)分化程度、有無淋巴結(jié)轉(zhuǎn)移和遠(yuǎn)處轉(zhuǎn)移相關(guān)(P0.05)。而與結(jié)直腸癌患者的年齡、性別和腫瘤大小無關(guān)(P0.05)。2.血清ELISA結(jié)果顯示:克羅恩組及結(jié)腸息肉組與健康對照組相比,均沒有顯著的差異(p0.05),而結(jié)直腸癌組與健康對照組相比,二者之間有顯著差異(P0.01)。此外,對于結(jié)直腸癌不同分期:早期(Ⅰ、Ⅱ期)相比于晚期(Ⅲ、IV期)的患者,其血清CA215的水平之間有顯著差異(P0.01),分期越高的患者其血清CA215水平越高。但是在早期中(I期與Ⅱ期之間)以及晚期(Ⅲ期與IV期之間)無明顯差異(p0.05)。同時在結(jié)直腸癌I期也能檢測到顯著升高的CA215水平。CA215在結(jié)直腸癌中的檢出率將近50%,與CEA (38%)和CA199 (16%)相比,均有所提高。同時CA215聯(lián)合CEA檢測可以將結(jié)直腸癌的檢出率提高到56-94%。3.Western Blot結(jié)果顯示CA215在正常結(jié)腸上皮細(xì)胞NCM460中呈現(xiàn)低表達(dá),在腸癌細(xì)胞系SW480、Caco2、HCT116、Lovo中均呈陽性表達(dá),其中SW480表達(dá)最高。細(xì)胞免疫化學(xué)結(jié)果顯示CA215在正常結(jié)腸上皮細(xì)胞NCM460中呈現(xiàn)低表達(dá),在SW480和HCT116中呈陽性表達(dá),其中細(xì)胞膜和細(xì)胞質(zhì)均表達(dá),但以細(xì)胞膜表達(dá)為主。4. CCK8細(xì)胞增殖實(shí)驗(yàn)結(jié)果顯示,3種濃度(0.1μg/ml, 1μg/ml, 1Oμg/ml)的RP215均能抑制結(jié)直腸癌細(xì)胞系SW480與HCT116的增殖,并且具有一定的劑量依賴性和時間依賴性,濃度越高、作用時間越長其對腸癌細(xì)胞增殖的抑制效應(yīng)越強(qiáng)。而對于正常腸上皮細(xì)胞系NCM460, 3種濃度的RP215均不能抑制其增值。流式細(xì)胞術(shù)凋亡檢測結(jié)果顯示,3種濃度(0.1μg/ml,1μg/ml,10μg/ml)的 RP215 均能誘導(dǎo)結(jié)直腸癌細(xì)胞系 SW480與HCT116的凋亡,RP215濃度越高,誘導(dǎo)細(xì)胞凋亡的能力越強(qiáng)。但是不具有統(tǒng)計(jì)學(xué)差異(P0.05)。而在正常腸上皮細(xì)胞系NCM460中RP215不能誘導(dǎo)細(xì)胞凋亡。[結(jié)論]1、 結(jié)直腸癌患者組織中CA215的表達(dá)與患者局部浸潤、腫瘤的組織學(xué)分化程度、有無淋巴結(jié)轉(zhuǎn)移和遠(yuǎn)處轉(zhuǎn)移相關(guān),同時結(jié)直腸癌患者血清中CA215水平與腫瘤的分期(早期和晚期)相關(guān),這些都提示CA215可能作為結(jié)直腸癌患者侵襲、轉(zhuǎn)移和不良預(yù)后的生物標(biāo)志物。2、 與CEA、CA199相比,CA215具有更高的檢出率,而且CA215聯(lián)合CEA能進(jìn)一步提高結(jié)直腸癌患者的診斷率。由于CA215在早期檢出率高于CEA和CA199,而在晚期結(jié)直腸癌中CEA的檢出率高于CA215,所以CA215更適用于結(jié)直腸癌的早期診斷。3、 CA215的單克隆抗體RP215能抑制結(jié)直腸癌細(xì)胞的增值,誘導(dǎo)腫瘤凋亡,因此基于CA215的單克隆抗體也許可以成為結(jié)直腸癌免疫治療的新藥物。
[Abstract]:[BACKGROUND AND OBJECTIVE] CA215 is a glycoprotein epitope associated with carbohydrates. It is homologous to the heavy chain of human immunoglobulin. It is hardly expressed in normal human immunoglobulin and human tissues, but it is expressed in most tumor tissues. The aim of this study is to investigate the expression of CA215 in colorectal cancer tissues and serum, to analyze the relationship between CA215 and invasion, metastasis and prognosis of colorectal cancer, and to evaluate whether CA215 can be used as a suitable tumor marker. [Methods] 1. Immunohistochemical method was used to detect the expression of CA215 in 132 colorectal cancer tissues, and CA215 positive staining and negative staining were selected to study the expression and prognosis of CA215. Serum CA215 levels were measured by ELISA in 83 healthy subjects, 15 IBD patients, 20 colorectal polyps patients and 103 colorectal cancer patients (serum CA215 levels 11.35 AU/ml were defined as positive) to evaluate the feasibility of using CA215 as a serum marker in colorectal cancer patients. Specificity and sensitivity of tumor markers CEA and CA199 in colorectal cancer diagnosis and treatment. 3. Expression of CA215 in colorectal cancer cell lines SW480, Caco2, HCT116, Lovo and normal intestinal epithelial cells NCM460 was detected by Western Blot, and the expression of CA215 in NCM460, SW480 and HCT116 was detected by cell smear immunochemistry. The effects of RP215 on proliferation and apoptosis of colorectal cancer cells SW480, HCT116 and human normal colon epithelial cell line NCM460 were measured by CCK8 cell proliferation assay and flow cytometry respectively at different concentrations and at different time of action. [Results] CA215 was positive in 1.132 patients with colorectal adenocarcinoma (immunohistochemical score < 1 point) The expression of CA215 was associated with local invasion, histological differentiation, lymph node metastasis and distant metastasis in colorectal cancer patients (P 0.05). There was no correlation between the expression of CA215 and the age, sex and tumor size of colorectal cancer patients (P 0.05). The results of serum ELISA showed that there was no significant difference between Crohn group and colon polyp group and healthy control group (p0.05), but there was significant difference between colorectal cancer group and healthy control group (p0.01). There was a significant difference between the two groups (P 0.01). The higher the stage, the higher the serum CA215 level. However, there was no significant difference between the early stage (between stage I and stage I I) and the late stage (between stage I I I and stage IV) (P 0.05). At the same time, the significantly elevated CA215 level was also detected in colorectal cancer stage I. The detection rate of CA215 in colorectal cancer was nearly 50%, and CEA (38%). Compared with CA199 (16%), CA215 and CEA could increase the detection rate of colorectal cancer to 56-94%. 3. Western Blot results showed that CA215 was low expressed in normal colon epithelial cells NCM460, and was positive in colorectal cancer cell lines SW480, Caco2, HCT116 and Lovo, with the highest expression of SW480. The results showed that CA215 was low expressed in normal colon epithelial cells NCM460 and positive in SW480 and HCT116. Cell membrane and cytoplasm were both expressed, but mainly in cell membrane. 4. CCK8 cell proliferation test showed that RP115 at three concentrations (0.1 ug/ml, 1 ug/ml, 1 Oug/ml) could inhibit the proliferation of colorectal cancer cell line SW480. The higher the concentration, the longer the action time, the stronger the inhibitory effect on the proliferation of colorectal cancer cells. However, for the normal colorectal epithelial cell line NCM460, RP215 of three concentrations could not inhibit the proliferation of HCT116. Flow cytometry showed that the three concentrations (0.1 ug/ml, 1 ug/ml) of apoptosis detection. RP215 could induce apoptosis of colorectal cancer cell lines SW480 and HCT116. The higher the concentration of RP215, the stronger the ability to induce apoptosis. However, there was no statistical difference (P 0.05). RP215 could not induce apoptosis in the normal intestinal epithelial cell line NCM460. [Conclusion]1. The expression of CA215 in colorectal cancer tissues was associated with the disease. Local infiltration, histological differentiation, lymph node metastasis and distant metastasis were correlated, and serum CA215 levels were correlated with tumor staging (early and late), suggesting that CA215 may be a biomarker of invasion, metastasis and poor prognosis in patients with colorectal cancer. The detection rate of CA215 in early stage was higher than that of CEA and CA199, and that of CEA in advanced stage was higher than that of CA215. Therefore, CA215 is more suitable for early diagnosis of colorectal cancer. 3. The monoclonal antibody RP215 of CA215 can inhibit colorectal cancer. Cell proliferation induces tumor apoptosis, so monoclonal antibodies based on CA215 may be a new drug for immunotherapy of colorectal cancer.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.34
本文編號：2190487
[Abstract]:[BACKGROUND AND OBJECTIVE] CA215 is a glycoprotein epitope associated with carbohydrates. It is homologous to the heavy chain of human immunoglobulin. It is hardly expressed in normal human immunoglobulin and human tissues, but it is expressed in most tumor tissues. The aim of this study is to investigate the expression of CA215 in colorectal cancer tissues and serum, to analyze the relationship between CA215 and invasion, metastasis and prognosis of colorectal cancer, and to evaluate whether CA215 can be used as a suitable tumor marker. [Methods] 1. Immunohistochemical method was used to detect the expression of CA215 in 132 colorectal cancer tissues, and CA215 positive staining and negative staining were selected to study the expression and prognosis of CA215. Serum CA215 levels were measured by ELISA in 83 healthy subjects, 15 IBD patients, 20 colorectal polyps patients and 103 colorectal cancer patients (serum CA215 levels 11.35 AU/ml were defined as positive) to evaluate the feasibility of using CA215 as a serum marker in colorectal cancer patients. Specificity and sensitivity of tumor markers CEA and CA199 in colorectal cancer diagnosis and treatment. 3. Expression of CA215 in colorectal cancer cell lines SW480, Caco2, HCT116, Lovo and normal intestinal epithelial cells NCM460 was detected by Western Blot, and the expression of CA215 in NCM460, SW480 and HCT116 was detected by cell smear immunochemistry. The effects of RP215 on proliferation and apoptosis of colorectal cancer cells SW480, HCT116 and human normal colon epithelial cell line NCM460 were measured by CCK8 cell proliferation assay and flow cytometry respectively at different concentrations and at different time of action. [Results] CA215 was positive in 1.132 patients with colorectal adenocarcinoma (immunohistochemical score < 1 point) The expression of CA215 was associated with local invasion, histological differentiation, lymph node metastasis and distant metastasis in colorectal cancer patients (P 0.05). There was no correlation between the expression of CA215 and the age, sex and tumor size of colorectal cancer patients (P 0.05). The results of serum ELISA showed that there was no significant difference between Crohn group and colon polyp group and healthy control group (p0.05), but there was significant difference between colorectal cancer group and healthy control group (p0.01). There was a significant difference between the two groups (P 0.01). The higher the stage, the higher the serum CA215 level. However, there was no significant difference between the early stage (between stage I and stage I I) and the late stage (between stage I I I and stage IV) (P 0.05). At the same time, the significantly elevated CA215 level was also detected in colorectal cancer stage I. The detection rate of CA215 in colorectal cancer was nearly 50%, and CEA (38%). Compared with CA199 (16%), CA215 and CEA could increase the detection rate of colorectal cancer to 56-94%. 3. Western Blot results showed that CA215 was low expressed in normal colon epithelial cells NCM460, and was positive in colorectal cancer cell lines SW480, Caco2, HCT116 and Lovo, with the highest expression of SW480. The results showed that CA215 was low expressed in normal colon epithelial cells NCM460 and positive in SW480 and HCT116. Cell membrane and cytoplasm were both expressed, but mainly in cell membrane. 4. CCK8 cell proliferation test showed that RP115 at three concentrations (0.1 ug/ml, 1 ug/ml, 1 Oug/ml) could inhibit the proliferation of colorectal cancer cell line SW480. The higher the concentration, the longer the action time, the stronger the inhibitory effect on the proliferation of colorectal cancer cells. However, for the normal colorectal epithelial cell line NCM460, RP215 of three concentrations could not inhibit the proliferation of HCT116. Flow cytometry showed that the three concentrations (0.1 ug/ml, 1 ug/ml) of apoptosis detection. RP215 could induce apoptosis of colorectal cancer cell lines SW480 and HCT116. The higher the concentration of RP215, the stronger the ability to induce apoptosis. However, there was no statistical difference (P 0.05). RP215 could not induce apoptosis in the normal intestinal epithelial cell line NCM460. [Conclusion]1. The expression of CA215 in colorectal cancer tissues was associated with the disease. Local infiltration, histological differentiation, lymph node metastasis and distant metastasis were correlated, and serum CA215 levels were correlated with tumor staging (early and late), suggesting that CA215 may be a biomarker of invasion, metastasis and poor prognosis in patients with colorectal cancer. The detection rate of CA215 in early stage was higher than that of CEA and CA199, and that of CEA in advanced stage was higher than that of CA215. Therefore, CA215 is more suitable for early diagnosis of colorectal cancer. 3. The monoclonal antibody RP215 of CA215 can inhibit colorectal cancer. Cell proliferation induces tumor apoptosis, so monoclonal antibodies based on CA215 may be a new drug for immunotherapy of colorectal cancer.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.34
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 韓建蓉;吳麗娜;劉蕊;;探討腫瘤標(biāo)志物在結(jié)直腸癌患者中的診斷價值[J];實(shí)用檢驗(yàn)醫(yī)師雜志;2013年03期
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