摘要
本研究通过免疫组织化学染色方法,进一步证明TSP50的肿瘤-睾丸抗原特性;应用CpG岛甲基化位点的直接测序法,探讨启动子甲基化水平对TSP50表达的作用机制;甲基化敏感代表性差异分析法与CpG岛基因芯片技术相结合,分离、鉴定新的肿瘤-睾丸抗原。
本研究通过免疫组织化学染色发现肺癌TSP50蛋白表达的阳性率89%;胃癌TSP50蛋白表达的阳性率88%;肝癌TSP50蛋白表达的阳性率87 %;结肠腺癌TSP50蛋白表达的阳性率94 %;前列腺癌TSP50蛋白表达的阳性率71%。同时还发现少部分肿瘤组织边缘或远隔的正常组织中也有少许的TSP50蛋白表达。通过CpG岛甲基化位点的直接测序法,检测了人正常睾丸组织、正常乳腺组织、乳腺癌组织、激光显微切割的乳腺癌组织和不表达TSP50蛋白的人精原细胞瘤细胞系细胞以及分离的鼠精原细胞、精母细胞和精子细胞的TSP50启动子区域CpG岛甲基化位点的甲基化水平。结果表明TSP50蛋白阳性的组织,其启动子区域CpG岛甲基化位点的甲基化状态处于较低的水平。激光显微切割的乳腺癌肿瘤细胞较单纯的乳腺癌组织其甲基化水平要更低一些。
本研究将甲基敏感差异分析技术与CpG岛基因芯片技术结合起来,将正常乳腺组织、乳腺癌组织和睾丸组织的基因组DNA用甲基敏感的限制性内切酶HapⅡ酶切,正常乳腺组织和乳腺癌组织为一组,正常乳腺组织和睾丸组织为一组,分别进行CpG岛基因芯片杂交。在结果中选择了具有显著差异的103-E-5(CpG岛),发现103-E-5的附近存在有Pax6。通过RT-PCR检测了pax6的mRNA在正常组织中的表达情况,结果发现Pax6的mRNA仅表达在正常的睾丸组织、脑组织和神经组织中。进一步检测发现Pax6的mRNA在乳腺癌细胞系有表达。通过免疫组织化学染色发现在正常乳腺组织中不表达Pax6蛋白,正常睾丸组织中表达Pax6蛋白,乳腺癌组织中也表达Pax6蛋白。荧光素酶报告载体检测103-E-5(CpG岛)处的基因序列具有启动子活性。
综上所述,本研究从蛋白质水平进一步证明了TSP50是多种肿瘤组织都表达的一类肿瘤-睾丸抗原;TSP50启动子区域CpG岛甲基化位点的甲基化水平对TSP50的表达具有重要的作用;Pax6是一种新的肿瘤-睾丸抗原,深入研究其功能将会为肿瘤的免疫治疗或靶向治疗提供新的思路。
Cancer is one of the diseases which difficult to cure, and the incidence gradually increased. Although the research of tumor acheived great progress, but effective prevention and treatment of these deadly disease is not very many. With the development of immunology, molecular biology and genetic engineering technology, tumor immunotherapy become the fourth treatment model as following surgery, radiotherapy and chemotherapy treatment. Research and development of new tumor vaccines become an imporment tumor immunotherapy. An ideal tumor antigen for immunotherapy must be specific, the stable expression in tumor cells, does not exist in normal tissue, It is essential to the growth, invasion and metastasis of tumor cells. Therefore, the search for such a specific tumo antigen which expressed in the tumor tissue, and did not express in the normal tissue is very important.
Cancer - testis antigen consistent with the requirements of the manufacturing of tumor vaccines. CTA must have the following characteristics: 1)CTA must should expressed Limited in gamete development system and tumor tissues; 2)Its coding gene mainly located on the X chromosome; 3)Most CTA antigen belong to multigene family; 4)In cancer patients has immunogenicity; 5) its expression is different In different tumor tissues; 6) its expression associated with Tumor metastasis and aggravation. Testes has a special characteristics in in the body's immune. Many of the testes antigens did not recognize by the immune system cells,not expressed in normal tissues, only xpressed in normal testis.And they are also highly expressed in human tumor tissues. As the germ cells did not express human leukocyte antigen (HLA) molecules, and the existence of blood-testis barrier,so in the production of anti-tumor immunity CTA would not damage normal tissues and germ cell. We consider CTA is a promising cancer vaccine candidate antigens.
Promoter of the CTA and the site of the first exon there is generally a high degree of duplication of CpG structure,this we call it CpG island. 5 - methyl cytosine concentration present in the dinucleotide cytosine (CpG) in the Mammalian Genome,
other locations has little of 5 - methyl cytosine, In different organizations and the same organization of different developmental stages, CpG island methylation status is different, Normal tissues usually do not occur methylation. When the tumor occurs, compared with normal tissue origin ,the promoter of CTA and first exon regions of CpG island is often to Demethylation, According to this characteristic, By methylation-sensitive representation difference analysis of human breast cancer organizations, the study found that the methylation status of a testis-specific protein TSP50. detected by RT-PCR we found TSP50 was not expressed in 15 kinds of normal tissues, Unique expression in human testis, at the same time also expressed in some types of breast cancer. Immunohistochemical staining showed TSP50 protein expressed in breast cancer,but not expressed at the normal breast epithelial tissue; testis also expressed TSP50, however, no TSP50 expression of spermatogonia, spermatocytes expressed TSP50 protein when Spermatocytes differentiate to sperm cells,it did not express TSP50. Research shows that mice TSP50 has homology with the human TSP50,so we did further research on human and mouse TSP50.
Immunohistochemistry analysis of Lung cancer, stomach cancer, liver cancer, colon cancer, prostate cancer show , total of 18 cases of lung cancer specimens, including lung squamous cell carcinoma, lung adenocarcinoma, mucinous carcinoma of the lung and small cell lung cancer, 16 cases of TSP50 protein was positive, the positive rate is 89%. the only two cases of small cell lung cancer were negative; A total of 51 cases of gastric cancer, including squamous cell carcinoma of the stomach, gastric cancer and gastric cancer mucinous,45 cases of TSP50 protein was positive, the positive rate is 88%,a total of 30 cases of hepatoma, including hepatocellular carcinoma and bile duct cell carcinoma,26 cases of TSP50 protein was positive, the positive rate is 87%. 1 case of the tumor surrounding normal liver tissue also found TSP50 expression, the expression of TSP50 were negative at the distant parts of liver tissue; Colon adenocarcinoma specimenwere obtained from 48 patients, 45 cases of TSP50 was positive, the positive rate is 94%. Among them, 3 cases from normal colon tumor tissues have TSP50 expression; 14 cases of Prostate cancer specimens, including a prostate cancer, two prostate cancer and three prostate cancer,10 cases of TSP50 protein was positive, the positive rate is 71%. TSP50 protein are expression in these more common tumors, From the level of protein proofed that TSP50 is a cncer-testis antigens. In immunohistochemical staining showed in normal tissues of the colon also express TSP50 protein, normal liver tissue adjacent tumor tissue also express of TSP50. It Suppose to be that in some normal tissues also express TSP50 ,this is likely to affect the specificity of immunotherapy.
TSP50 was expressed in the testis and breast cancer tissues,but In normal breast tissues and human seminoma does not express TSP50. so we studied each of these organizations TSP50 promoter region CpG island methylation status. The results showed that normal breast tissue and human seminoma does not express TSP50, 12 methylated sites are at relatively high levels of methylation, breast tissue expressed TSP50, it's 12 methylated sites are at lower level methylation. By laser microdissection, the breast cancer tumor cells was cut down alone, the results showed 12 sites are at more lower methylation level, we consider this is the discharge of the tumor stroma hypermethylated genes.
Immunohistochemical staining showed that in human testis and mouse testis spermato- gonia did not express TSP50 protein, spermatocytes were expressed TSP50 protein, sperm cells did not express TSP50 protein. Separation of spermatogonia, spermatocytes and sperm of mouse testis,detection of rat TSP50 promoter region CpG island methylation status, four methylation sites of 12 methylation sites in the spermatogonia, spermatocytes and sperm cells,are all of demethylation, It supposed that these four sites and the promoter activity does not have relatedness, the remaining eight sites spermatogonia methylation levels are higher, in spermatocytes of the methylation level is relatively lower, methylation level of sperm cells is high. When the promoter of spermatogonia is hypermethylation, TSP50 gene silence,no expression of TSP50 protein. When spermatogonia differentiate into spermatocytes enter meiosis, promoter of CpG island methylation sites demethylation, expression of TSP50 protein. When spermatocytes gradually differentiate into sperm cells, promoter of CpG island methylation sites have started to methylation, expression level of TSP50 protein started to decrease, and finally do not express.
The level of promoter methylation experimental results of organizations which expression of TSP50 protein and organizations which did not express TSP50 protein are consistent with expression of TSP50 protein in these tissues. Thus proving that the methylation level of TSP50 p promoter CpG island determined the promoter activity, Also determines the expression of TSP50 protein in tissues.
When using traditional Methylation Sensitive-Representational Difference Analysis (MS-RDA) technique separate Cancer - testis antigen,it has many defects.such as the heavy workload, the process cumbersome and complex, long experimental period, the result uncertain, the high cost . With the progress of biological research, the completion of the human genome, various gene chip was produced and already on the market, gene chip of CpG island of the human genome is complete. In this study, Methylation Sensitive-Representational Difference Analysis (MS-RDA) technique combined with CpG island microarray technique, rapid, high throughput to discover, isolation and identification of new Cancer - testis antigen. In this study, genomic DNA which from the normal breast tissue, breast cancer and testis was digest by Methyl-sensitive restriction enzyme HapⅡ, Normal breast tissue and breast cancer tissue、normal breast tissue and testicular tissue in one group to CpG island microarray hybridization. in the results the 103-E-5 (CpG islands) with significant differences was selected, near103-E-5 Pax6 was found. By RT-PCR, expression of the pax6 mRNA was detected in normal tissues using RT-PCR technique, The results showed that the expression of the Pax6 mRNA only in normal testis tissue and nerve tissue. Further testing found that the Pax6 mRNA expressed in breast cancer cell lines. immunohistochemistry (IHC) technique demonstrated that in normal breast tissue did not express Pax6 protein, in normal testis Pax6 protein was expressed, in breast cancer tissue also expressed Pax6 protein. 103-E-5 is a length of 286bp of the gene sequence, from Pax6 Start site there has 4333bp, cloning of this fragment as the center of the 1732bp sequence, insert pGL4 vector, the results show that the gene sequences paragraph have activity,it may be the second Pax6 promoter. Based on the above preliminary studies suggested Pax6 is a new cancer-testis antigens.
引文
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