Expression Research Of The CXCL1 And The Different Subtype IL-8 In Malignant Ovarian Tumor

Chapter 1 THE STATUS OF PROTEOMICS IN OVARIAN TUMOR RESEARCH(OVERVIEW)Malignant ovarian tumor is one of the most threatening malignant tumors in female reproductive system.As the occult clinical symptoms and the inefficient diagnosis methods,more than 80%of patients become late when they seek medical treatment.Normal tumor marker CA125 is not so strong that can be the separate screening biomarker for ovarian cancer.However,the high-throughput proteomic technologies and bioinformatic tools provide large-scale powerful weapons for exploring new ovarian cancer biomarkers.The melanoma activating factor CXCL1(GRO-a) and the IL-8(CXCL8) belong to the chemokine factors,playing important roles in various malignant tumors.A number of studies show that the melanoma activator CXCL1 and the IL-8 effect the occurrence of ovarian malignancies,and the important process of the signal transduction.In our early research,we found that the CXCL1 and the different subtype IL-8 may be well used in the early diagnosis of ovarian cancer.To identify the above result and to promote tumor marker from a single application to the changes of comprehensive evaluation,this research worked.Chapter 2 PROKARYOTIC EXPRESSION OF THE CXCL1 AND THE DIFFERENT SUBTYPE IL-8Objective:Through genetic engineering technology,amplified coding genes of the CXCL1 and the different subtype IL-8 mature peptides,constructing the prokaryotic expression vectors,expressing the mature peptides of the CXCL1 and the different subtype IL-8.Methods:Using ovarian cancer tissue,extracting the total mRNA,RT-PCR amplified the coding genes of the CXCL1 and the different subtypes IL-8(1-77aa),IL-8(6-77aa) and IL-8(9-77aa) mature peptides.Connecting the amplified products with the prokaryotic expression vectors(PET SUMO),cloning and sequencing.Results:The coding genes were successfully amplified,and the recombinant plasmids were 100%verified by the sequencing.Translating the expression E.coil,the expression products were testified by the MALDI-TOF-MS. Conclusion:The CXCL1 and the different subtypes IL-8(1-77aa),IL-8(6-77aa) and IL-8(9-77aa) were successfully expressed trough prokaryotic expression.The prokaryotic expressing products can be well used for the future reseach as solid foundation.Namely,as the high-purity antigens inducing the specific monoclonal antibodies,or the standards in MS detection,.Chapter 3 THE LCM COMBINING THE IMMUNOMAGETIC BEADS IN THE MS DETECTION AND THE EXPRESSION RESEARCH OF THE CXCL1 AND THE DIFFERENT SUBTYPE IL-8 IN MALIGNANT OVARIAN TUMOR Objective:To establish the MS detecting method for reseaching the expression of the CXCL1 and the IL-8(1-77aa),IL-8(6-77aa) and IL-8(9-77aa) in ovarian epithelial cells.To explore the expression of the CXCL1 and the different subtype IL-8 and its relationship with the early diagnosis of ovarian cancer.Methods:Through the LCM,collecting the epithelial cells of ovarian cancer,benign and normal ovarian tissue.Cracking such cells,the cracked samples reacted with immunomagnetic beads which were coated the specific antibodies.Concentrated by the micro-chromatography column,then the concentrated liquid was detected by the MALDI-TOF-MS.Results:â‘ Through the LCM,epithelial cells were accurately captured to 10~5-10~6for each sample.The MALDI-TOF-MS detection showed that the immunomagnetic beads combined well with the CXCL1 and the different subtype IL-8,and the various subtypes of IL-8 can be well distinguished trough this method.â‘¡The CXCL1 expressed highly in malignant cells(90%),but none in benign or normal controls.â‘¢The IL-8(1-77aa) expressed in ovarian malignant,benign and normal cells(40%,100%,66.67%respectively);The IL-8(6-77aa) expressed in malignant cells(30%),but none in benign or normal controls;The IL-8(9-77aa) expressed none in all the cell samples.CONCLUSION:â‘ The method for reseaching the expression of the CXCL1 and the IL-8(1-77aa),IL-8(6-77aa) and IL-8(9-77aa) in ovarian epithelial cells was successfully established.â‘¡The CXCL1 is excreted by the ovarian malignant cells and may be the available tumor marker.â‘¢The expression of different subtype IL-8 in malignant,benign and normal tissue cells with respective situation,suggesting that the reseach of different subtype IL-8 may has significant value in the study of early ovarian cancer diagnosis.