肺癌功能性抗原的分离鉴定及其与肺癌临床相关性研究

【作者】 王翠红；

【导师】 吴翠娇；

【作者基本信息】 青岛大学 ， 细胞生物学， 2010， 硕士

【摘要】 目的：采用功能性抗体库技术已筛选出的抗肺癌单抗13C9,确定其识别的抗原在肺癌细胞和组织中的表达,测定该单抗抑制肺癌生长的能力,进一步鉴定其识别的抗原蛋白。研究该抗原蛋白的表达与肺癌临床参数的关系,以期为肺癌靶向治疗提供有价值的分子靶位和治疗剂。方法：采用细胞免疫荧光、免疫组织化学检测13C9单抗识别的抗原蛋白在肺癌细胞及组织中的表达和定位；ELISA测定13C9单抗的产量和亚型；裸鼠体内移植瘤治疗实验和体外肺癌细胞增殖实验,评价13C9单抗对肺癌细胞生长的作用；采用斑点杂交优化、RIPA法提取13C9单抗识别的抗原蛋白,行蛋白印迹(Western blotting)、免疫沉淀、MALDI-TOF-PMF质谱技术,鉴定该抗原蛋白。收集临床肺癌标本,应用免疫组织化学检测M蛋白的表达,分析其与肺癌临床病理参数的相关性。结果：细胞免疫荧光检测13C9单抗识别的抗原在肺癌12种固定细胞中表达阳性率达90%以上,强度为强阳性(+++)；在活细胞中,其识别的抗原表达于细胞膜上。免疫组织化学检测结果：174例配对组织中,13C9单抗识别的抗原在肺癌组织中阳性表达率87.4%,在配对癌旁组织中阳性表达率16.7%,差异非常显著(t=3.5555E-48,P<0.01)。培养杂交瘤细胞产生13C9单抗,ELISA测定其产量为4.38ug/ml,亚型为小鼠IgM、k型。裸鼠体内移植瘤抑制实验,13C9单抗抑瘤率达63.29%,与对照组比较,差异显著(P=0.01,P<0.05)。斑点杂交优化13C9单抗识别的抗原蛋白主要定位于细胞膜,经RIPA法提取、免疫沉淀、电泳后切胶行MALDI-TOF-PMF质谱技术鉴定,结果13C9单抗识别的抗原蛋白为M蛋白,分子量为226KD。商业化抗体进行质谱结果回证,证明13C9单抗与商业化的抗体识别的抗原蛋白同为M蛋白。对于肺癌临床标本的免疫组织化学检测及其与肺癌临床病理参数的相关性的分析结果显示,在331例肺癌组织中,M蛋白的表达在鳞癌组织高于在腺癌组织(P=0.000,P<0.01),在T3+T4期肺组织高于T1+T2期肺组织(P=0.001,P<0.01)。在161例鳞癌中M蛋白的表达随T分期的增加而增强；在137例腺癌中M蛋白的表达随分化程度的减低而增强。结论：本实验结果证明：①13C9单抗为功能性单抗,其识别的抗原在肺癌多种细胞和组织中表达,对裸鼠体内移植瘤具有明显的抑制作用。②13C9单抗识别的抗原M蛋白为肺癌功能性抗原蛋白,分子量为226KD,主要定位于肺癌细胞膜。③M蛋白的表达与肺癌的类型及临床病理参数存在相关性：在肺鳞癌中的表达高于肺腺癌；在肺鳞癌中,M蛋白的表达与T分期呈正相关；在肺腺癌中,M蛋白的表达与分化程度呈负相关。本研究有望为研制新的肺癌靶向治疗剂和治疗靶标提供新的实验依据。更多还原

【Abstract】 Objective A monoclonal antibody 13C9, which had been targeted by screening of functional monoclonal antibody library against lung cancer, was adopted to recognize its specific antigen on multiple cultured-cell-types and tissues of lung cancer. Its function to the growth of lung cancer was detected both in vivo and in vitro. Furthermore the 13C9-specific antigen was identified. And the relationship between the expression of the antigen protein in lung cancer and clinical parameters of lung cancer was studied. This study may provide a clue for exploring valuable molecular targets and treatment agents for targeted therapy of lung cancer.Methods With immunofluorescence and immunohistochemistry techniques, the expression of 13C9-specific antigen was detected and localized on multiple cultured-cell-types and tissues of lung cancer. ELISA was applied to determine the production of 13C9 monoclonal antibody by culturing hybridoma cells and its subtype. Experiments of tumor treatment in vivo and vitro were carried to evaluate the inhibiting functions of the 13C9 antibody to the growth of lung cancer. Thereafter, the 13C9-specific antigen was optimized by dot blotting techque, extracted by RIPA technique. And with Western blotting, immunoprecipitation, MALDI-TOF-PMF mass spectrometry, the 13C9-specific antigen was identified. On the other hand, clinical specimens of lung cancer were collected and detected with immunohistochemistry. The correlation between the expression of the antigen protein in lung cancer and clinical parameters of lung cancer was studied.Results Results from immunofluorescence showed that among 12 fixed lung cancer cell lines, positive rate of the recognition of the antibody 13C9 to its antigen was above 90%, intensity was +++; with viable cells of lung cancer cell lines, the antibody 13C9-recognized antigen was demonstrated on the cell membrane. Immunohistochemical results showed that within 174 cases of paired tissues of lung cancer, positive rate of the 13C9-recognized antigen in lung cancer tissues was up to 87.4%, compared with the paired adjacent tissues which was just 16.7%,had a significant difference (t=3.5555E-48, P<0.01). The production of the antibody 13C9 by culturing hybridoma cells was 4.38ug/ml, belong to IgM, Kapa subtype, measured by ELISA. Transplant tumor inhibition experiment showed that the antibody 13C9 inhibit the lung tumor growth in mice by 63.29%, compared with control group (P=0.01, P<0.05). By dot blotting, the 13C9-recognized antigen protein was optimized and localized on the cell membrane. Then with RIPA extraction, immunoprecipitation and electrophoresis, the antigen protein band on the gel-line was excised and identified by MALDI-TOF-PMF. The molecular weight of the 13C9-recognized antigen protein was 226KD, and we named it as M protein. Conformation experiment was taken with commercial antibody, which demonstrated that M protein was recognized by both the antibody 13C9 and the commercial antibody. Investigation to the results from immunohistochemistry showed, that in 331 cases of lung cancer the expression of M protein in squamous cell carcinoma was higher than that in adenocarcinoma (P=0.000, P<0.01), the expression of M protein in T3+T4 stage was higher than in T1+T2 stage, (P=0.001, P<0.01). The expression of M protein in squamous cell carcinoma of 161 lung cancer patients was more intense with T stage advance, while in adenocarcinoma of 137 lung cancer patients, the expression of M protein was more intense with decreasion of tissue differentiation.Conclusion Results from this study demonstrated:①13C9 possesses characteristics of a functional monoclonal antibody,13C9-recognized antigen expressed in multiple cultured-cell-types and tissues of lung cancer, inhibitive effect of antibody 13C9 to the growth of the tumor in nude mice was obvious.②The antibody 13C9-recognized antigen which named M protein, may belong as a functional protein in lung cancer; its molecular weight was 226KD, localized on cell membrane.③There are possible correlation between the expression of M protein and the type as well as the clinical parameters of lung cancer:the expression of M protein in squamous cell carcinoma was higher than that in adenocarcinoma, the expression of M protein shows a positive correlation with T stage advance especially in lung squamous cell carcinoma, while in lung adenocarcinoma it shows a negative correlation with the advance of tissue differentiation. This study may provide new experimental data for further exploring and developing of new targets and new targeting agents in lung cancer treatment.更多还原