CA215及其单克隆抗体RP215在结直肠癌中的研究
发布时间：2018-08-18 19:43
【摘要】：[研究背景与目的]CA215是一个糖蛋白中的糖类相关抗原表位,与人免疫球蛋白的重链是同源的,在正常人的免疫球蛋白和人体组织中几乎不表达,但在大多数肿瘤组织中表达。现有的文献提示,CA125及其单克隆抗体RP215可作为多种肿瘤的免疫诊断标志物,并开发靶向抗体的免疫治疗药物。本课题旨在研究CA215在结直肠癌患者组织以及血清中的表达情况,结合患者的临床病理特征资料,分析CA215与结直肠癌侵袭、转移和预后的关系,评估CA215能否作为一个合适的肿瘤标志物用于结直肠癌的诊断和疗效监测。初步研究RP215对结直肠癌细胞系的免疫治疗的效果。[方法]1.采用免疫组织化学方法检测CA215在132例结直肠癌患者肿瘤组织中的表达情况,分选出CA215阳性染色和阴性染色的患者,研究CA215的表达与临床病理资料之间的关系。2.采用ELISA方法分别检测83例健康人、15例IBD、20例结肠息肉患者和103例结直肠癌患者血清中CA215的水平(血清CA215水平11.35 AU/ml定义为阳性),评估其作为结直肠癌患者血清标志物的可行性。比较其与临床上常用的血清肿瘤标志物CEA、CA199在结直肠癌诊疗中的特异性和敏感性。3.采用蛋白质印迹法(Western Blot)检测肠癌细胞系SW480、Caco2、HCT116和Lovo以及正常肠上皮细胞NCM460中CA215的表达情况,采用细胞涂片免疫化学方法检测CA215在NCM460、SW480及HCT116中的表达情况。4.采用CCK8细胞增殖实验和流式细胞术分别测定RP215在不同浓度、不同作用时间下,对结直肠癌细胞SW480、HCT116及人正常结肠上皮细胞系NCM460增殖及凋亡中的影响。[结果]1. 132例结直肠腺癌患者中CA215阳性(免疫组化评分≥1分)患者有68例,阳性率为51.5%。CA215阴性(免疫组化评分1分)的患者共有64例,占48.5%。CA215的表达与结直肠癌患者的局部浸润、肿瘤的组织学分化程度、有无淋巴结转移和远处转移相关(P0.05)。而与结直肠癌患者的年龄、性别和肿瘤大小无关(P0.05)。2.血清ELISA结果显示:克罗恩组及结肠息肉组与健康对照组相比,均没有显著的差异(p0.05),而结直肠癌组与健康对照组相比,二者之间有显著差异(P0.01)。此外,对于结直肠癌不同分期:早期(Ⅰ、Ⅱ期)相比于晚期(Ⅲ、IV期)的患者,其血清CA215的水平之间有显著差异(P0.01),分期越高的患者其血清CA215水平越高。但是在早期中(I期与Ⅱ期之间)以及晚期(Ⅲ期与IV期之间)无明显差异(p0.05)。同时在结直肠癌I期也能检测到显著升高的CA215水平。CA215在结直肠癌中的检出率将近50%,与CEA (38%)和CA199 (16%)相比,均有所提高。同时CA215联合CEA检测可以将结直肠癌的检出率提高到56-94%。3.Western Blot结果显示CA215在正常结肠上皮细胞NCM460中呈现低表达,在肠癌细胞系SW480、Caco2、HCT116、Lovo中均呈阳性表达,其中SW480表达最高。细胞免疫化学结果显示CA215在正常结肠上皮细胞NCM460中呈现低表达,在SW480和HCT116中呈阳性表达,其中细胞膜和细胞质均表达,但以细胞膜表达为主。4. CCK8细胞增殖实验结果显示,3种浓度(0.1μg/ml, 1μg/ml, 1Oμg/ml)的RP215均能抑制结直肠癌细胞系SW480与HCT116的增殖,并且具有一定的剂量依赖性和时间依赖性,浓度越高、作用时间越长其对肠癌细胞增殖的抑制效应越强。而对于正常肠上皮细胞系NCM460, 3种浓度的RP215均不能抑制其增值。流式细胞术凋亡检测结果显示,3种浓度(0.1μg/ml,1μg/ml,10μg/ml)的 RP215 均能诱导结直肠癌细胞系 SW480与HCT116的凋亡,RP215浓度越高,诱导细胞凋亡的能力越强。但是不具有统计学差异(P0.05)。而在正常肠上皮细胞系NCM460中RP215不能诱导细胞凋亡。[结论]1、 结直肠癌患者组织中CA215的表达与患者局部浸润、肿瘤的组织学分化程度、有无淋巴结转移和远处转移相关,同时结直肠癌患者血清中CA215水平与肿瘤的分期(早期和晚期)相关,这些都提示CA215可能作为结直肠癌患者侵袭、转移和不良预后的生物标志物。2、 与CEA、CA199相比,CA215具有更高的检出率,而且CA215联合CEA能进一步提高结直肠癌患者的诊断率。由于CA215在早期检出率高于CEA和CA199,而在晚期结直肠癌中CEA的检出率高于CA215,所以CA215更适用于结直肠癌的早期诊断。3、 CA215的单克隆抗体RP215能抑制结直肠癌细胞的增值,诱导肿瘤凋亡,因此基于CA215的单克隆抗体也许可以成为结直肠癌免疫治疗的新药物。
[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.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
本文编号：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.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
【参考文献】
相关期刊论文 前1条
1 韩建蓉;吴丽娜;刘蕊;;探讨肿瘤标志物在结直肠癌患者中的诊断价值[J];实用检验医师杂志;2013年03期
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