| Colorectal cancer,also known as colorectal cancer(colorectal cancer,CRC),is a common gastrointestinal disease.It is the second largest malignancy in the United States.In China,CRC ranks fifth in malignant tumors.The prognosis of this tumor is closely related to the extent of tumor differentiation and whether the invasion and metastasis are involved.The five-year survival rate of in situ carcinoma(stage I)exceeds 90%,and the five-year survival rate is only about 10%.In this regard,there is an urgent need to explore more specific and sensitive biomarkers to improve the screening and diagnostic effects of CRC.In recent years,the rapid development of proteomics technology in a variety of malignant tumor research has also been widely used for us to further understand the pathogenesis of cancer and to carry out individual treatment of cancer.The aim of this study is to examine the differences in protein expression profiles between colorectal cancer tissues and adjacent tissues by proteomics,and to establish a differential expression profile of colorectal cancer in order to screen out the biology of colorectal cancer mark.In this study,ATP5 B is selected as the target protein from the differentially expressed protein bands to construct monoclonal antibody,and the relationship between the expression of ATP5 B and the clinicopathological parameters is analyzed.There are four parts in this research:Part I: Establishment of differential protein profile of TNM stage ? colorectal cancer and adjacent tissuesMethod:1.The specimens were 20 pairs of colorectal adenocarcinoma and matched adjacent tissues,(adjacent tissues from the cancer tissue> 10 cm.)Specimens were not receiving any treatment,and confirmed by pathology for TNM stage ? adenocarcinoma.2.The total tumor tissue and adjacent tissues are extracted,and the protein concentration of the tissue is analyzed.3.Preparation of a proteolytic polypeptide mixture.4.The use of two-dimensional liquid chromatography for sample separation.5.Through the LTQXL ion hydrazine mass spectrometry on the two-dimensional chromatography elution of the peptide detection.6.Analyze the bioinformatics of the mass spectrometry data to find proteins that differ in the expression of cancer tissue from adjacent tissues.Result:1.579 protein spots are obtained in colorectal cancer specimens by twodimensional chromatography and mass spectrometry.492 protein spots are obtained in adjacent tissues.These protein spots are compared and retrieved by the database.It is found that there were 35 proteins differentially expressed in cancer tissues and adjacent tissues.2.20 proteins expressed in tumor tissue were FBLN1 ? ATP5 B ? TPM4 ?FGA?GSTP1?HIST1H3J?IGHA1?SERPINH1?TUBB3?IGHA1?TPM3?CKAP4?IGHA2? POSTN?FGB ?FGG? PKM2?HIST1H2AE? MYH10 and TUBB.3.The 10 proteins down-regulated in tumor tissue include: SYNM?MYH11?PKM2?KRT10?FLNC?H2AFX?CAP1?MYL6?TUBB2A ?TLN1.4.ATP5 B is selected as the target protein from differentially expressed proteins.Part II: ATP5 B gene amplification,vector construction and induction of expression of fusion proteinMethod:1.The gene sequence is derived from Gen Bank-nucletide,which is designed to amplify the target gene.2.The recombinant plasmid BL21(DE3)(p ET28a: ATP5B)is inoculated into LB medium and transformed into BL21 cells.3.Ultrasonic disintegration of cells,nickel agarose affinity chromatography purification of the target protein.4.SDS-PAGE purified protein.Result:1.The p2828a: ATP5B(prokaryotic expression plasmid)is successfully constructed and sequenced by DNA sequencing and Gen Bank-nucletide.2.The purified protein is identified by SDS-PAGE and purified.Part ? Preparation,purification and identification of ATP5 B monoclonal antibodyMethod:1.Prepare of ATP5 B protein immunogen,and injected into mice,and immunized mice.Surgery to take the mouse spleen,preparation of cells,fusion of myeloma cells.2.ELISA is used to select the positive cell lines3.Culture,selection of positive cell lines.4.Preparation of ascites in positive hybridoma cell lines and detection of ascites antibody titer.5.The purified antibody protein is purified by affinity chromatography to obtain ATP5 B monoclonal antibody.6.Antibody concentration,purity,titer and IHC detectionResult:1.To obtain a strain capable of stabilizing the anti-human ATP5 B monoclonal antibody.2.Antibody titer reached 1: 640000,antibody purity of 95% or more.Part ?: Expression of ATP5 B in colorectal cancer and its relationship with clinicopathological parametersMethod:1.Collected 30 pairs of colorectal cancer tissues and adjacent tissues.2.The expression of ATP5 B in colorectal cancer tissues and adjacent tissues is detected by immunohistochemical method.3.The relationship between the expression of ATP5 B protein and the clinicopathological parameters of colorectal cancer is analyzed by statistical software SPSS17.0.Result:1.Immunohistochemistry is used to detect the expression of ATP5 B in cancer tissues and adjacent tissues.After comparing the two,the expression rate in cancer tissue is higher.2.According to TNM staging,the expression of ATP5 B protein was lower in stage ? and ? tissues.The positive expression rate of ATP5 B protein in tumor tissue with lymph node metastasis or distant metastasis is higher.Conclusions1.The differential protein profile of TNM stage ? colorectal cancer tissues and adjacent tissues is successfully established in this study.2.The hybridoma cell lines of ATP5 B protein are successfully established and purified.The monoclonal antibody of ATP5 B protein is prepared as the basis for the study of the function of ATP5 B protein and the clinical application of monoclonal antibody.3.The expression of ATP5 B in colorectal cancer tissues is significantly higher than that in adjacent tissues.4.The relationship between ATP5 B protein and clinicopathological parameters of colorectal cancer patients is analyzed,which provids a potential basis for judging the development of colorectal cancer. |
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