【作者】 王研；

【导师】 王毅；

【作者基本信息】 吉林大学 ， 生物医学工程， 2009， 硕士

【摘要】 结肠癌是胃肠道常见的恶性肿瘤之一,每年有大量患者死于此病,至今仍缺乏有效的早期诊断方法。近20年我国尤其大城市的结肠癌发病率明显上升。由于结肠癌患者初期很少有症状,确诊时60%-70%的结肠癌已属晚期,故死亡率较高。早期结肠癌患者经治疗后的生存率可达90%。因此寻找早期诊断方法,探索有效的综合疗法是降低结肠癌死亡率的关键。自1938年起,前列腺酸性磷酸酶（prostatic acid phosphatase, PAP）便作为前列腺癌肿瘤标志物。多个报道提示在前列腺和前列腺癌之外的其他组织也有PAP的表达,因此它并不是绝对的组织特异性标志物。本实验利用RT-PCR法及Western blot法对PAP在结肠癌细胞中的表达水平进行检测,利用免疫组织化学方法对PAP在结肠癌组织中的PAP表达水平进行检测;利用ELISA法检测结肠癌患者和健康人血浆中抗PAP表位肽的抗体水平,利用ELISpot法检测结肠癌及健康人PBMCs中分泌PAP多肽特异性IFN-γ的细胞数量,利用⁵¹Cr释放法对PAP表位肽体外诱导产生的细胞毒性T淋巴细胞（cytotoxic T lymphocytes,CTLs）的细胞毒性进行测定,从而为结肠癌的诊断和治疗提供一些实验依据。更多还原

【Abstract】 Colon cancer is the most common malignant tumor of the gastrointestinal tract, which has been causing many people dead per year. In the recent 20 years in China, especially in the big cities, incidence has been obviously increased. Colon cancer almost has no early special symptoms. The developed symptoms are often changed bowel evacuation habit and excrement feature, abdominal pain and bowel obstruction symptoms. Due to the rapid progress, concealment, initial few symptoms, the colon cancer is still lack of effective early diagnostic methods. When diagnosed, 60%～70% of the colon cancer have been late process. Therefore the mortality rate is very high. While for the early colon cancer patients after treatment, the survival rate can reach 90%. Therefore the early diagnosis methods and effective combination therapy is the key to reduce colorectal cancer mortality. Improving the level of early diagnosis and performing the early treatment are important to improve the prognosis.Prostatic Acid Phosphatase （PAP） is an enzyme secreted by prostate cancer epithelial cells. It is the main source for the APC. It is an isozyme coming from the Cytolysosome of the prostate cancer epithelial cells. Human prostate is the main source for the enzyme. As the prostate cancer markers, concentration measurement for serum PAP is widely used in the prostate cancer diagnosis and the monitoring for the treatment. Compared with prostate-specific antigen （PSA）, PAP is a more accurate indicator for tumor micrometastases. In addition, the PAP is the immune therapy target for the metastatic non-androgen dependence prostatic carcinoma. PAP was once considered as the prostate specific tumor markers. Several articles report that PAP also exists in non-prostate organ. It seems that PAP is not an prostate-specific tumor marker. In previors study, We have confirmed that the positive expression of PAP in the stomach, ovarian and breast cancer. PAP is the new immune therapy target for the stomach, ovarian and breast epithelial tissue cancer. In this study, the PAP expression in colon cancer cell lines and colon cancer tissue and the possibility of PAP as a immunotherapy target of colon cancer were investigated .（1）The expression of PAP in colon cancer cell lines1)The expression of PAP mRNA in Colon carcinoma cell linesThe expression of PAP mRNA in various colon carcinoma cell lines was investigated by the RT-PCR method. Total RNA was isolated from 1×10⁷ colon carcinoma cells, cDNA was synthesized by RT-PCR. PAP cDNA was detected by PCR amplification using a set of oligonucleotide primers specific to PAP. The mRNA expression of PAP was detected in 3（colo201.colo205.colo320）of 5 colon adenocarcinoma cell lines（colo201, colo 205, colo 320, HCT116 and LoVo ）.2)The expression of PAP protein in colon carcinoma cell linesThe expression of the PAP protein in colon carcinoma cell lines was examined by Western blot analysis. The cytoplasmic protein of different colon carcinoma cell lines were extracted according to the manual. The protein was separated by SDS-PAGE. The proteins in the acrylamide gel were blotted to a Hybond-polyvinylidene difluoride membraneand probed with Anti-PAP mouse monoclonal antibody. The PAP protein was found to be expressed in colo201,colo205 and colo320.3)Expression of PAP in colon cancer tissuesThe PAP expression of colon adenocarcinoma tissues were examined by immunohistochemical staining. As a result, colon cancer tissues were positive for PAP.（2）Induction of Peptide-Specific CTLs and Assay of Cytotoxicity1)HLA-A2-restricted PAP epitope peptide18 PAP epitope peptides which have the binding motif to HLA-A2 molecules, were required from the amino acid sequence of PAP through software analysis. All peptides were dissolved with dimethyl sulfoxide at a concen-tration of 10 mg/mL.2)Detection of anti- PAP peptide antibodyAnti- PAP peptide antibody（IgG） in the plasma of colon cancer patients and healthy donors were examined by ELISA analysis. High level anti- PAP_135-143、PAP_112-120、PAP_33-41、PAP_201-210 or PAP_196-205 IgG could be found in the plasma of most colon cancer patients.3)Induction of PAP peptide-Specific IFN-γsecreting CTLsPBMCs of HLA-A2⁺ cancer patients or HLA-A2⁺ healthy donors were incubated with PAP_135-143、PAP_112-120、PAP_33-41、PAP_201-210 or PAP_196-205 peptide respectively. The IFN-γsecretion were examined by the ELISPOT test. It is showed that peptide-specific IFN-γsecretion could be induced by PAP_112-120 or PAP_201-210 peptide.4)⁵¹Cr release assay of CytotoxicityThe cytotoxicity of PAP_112-120 or PAP_201-210 peptide-specific CTLs was examined by ⁵¹Cr-release assay. As a result, the PAP_112-120-specific CTLs lysed the PAP+/HLA-A2+ colon cancer cell colo 205 specificly.In conclusion, PAP mRNA and PAP protein were revealed to be expressed in colon cancer cell lines. PAP protein were positive in colon cancer tissue. PAP112-120 peptide-specific CTLs induced from colon cancer patients lysed the PAP+/HLA-A2+ colon cancer cell colo 205 specificly. HLA-A2-restricted PAP112-120 peptide could be used in the immunotherapy of colon cancer.更多还原