Preliminary Establishment Of Detection Method On Hormone Independent Prostate Cancer

Objective:To make a kit of Elisa this can detect 7 kinds of protein in prostate cancer tissues at the same time.With a preliminary screening of CRPC related protein indicators in order to achieve early diagnosis of CRPC,and to differentiate subtype of prostate cancer may exist and to test the potential therapeutic targets.Providing a reference for the early diagnosis of CRPC and individualized therapy of prostate cancer.Methods:There are three parts in this study1.Using Affymetrix expression profile Gene chip to detect androgen dependent and androgen independent prostatic carcinoma in nude mouse model which established in our lab before.Screening two types of differentially expressed genes in ADPC and CRPC.Clustering analysis method is used to analyze the correlation of gene expression profile,Use of molecular function Annotation System(Molecule Annotation System,MAS)for data analysis.Using SILAC high-throughput detection phosphorylated proteomics and bioinfonnatics analysis in ADPC and CRPC tissues.Preliminarily determines the Hippo-Yapl-mTOR and Raf/pAK2/Erk signaling pathway play key roles in the conversion of CRPC.In comparisons with gene chip and proteomics results,we screened several protein which overexpression in CRPC than ADPC.Further selection of BPH,ADPC and CRPC tissue for validation.By comparing the gene chip,proteomics screening results and our investigation before,with refer to reference and the prostate protein database we preliminary selected 7 kinds of protein as indicator of early diagnosis of CRPC and individualized treatment.2.Preparation and screening high specificity and affinity antibodies is the key to antibody microarray technology.First,we take advantage of GeneBank database to find seven kinds of gene sequence which code protein accordingly,Using NCBI BLAST analysis system to determine the specific sequence.Next we adopted the method of genetic recombination for expression of 7 proteins.The purified protein as antigen was used to immune Balb/c mice for 5 times,after meet the requirement of antibody titer by Elisa,then mice spleen celles and myeloma celles were fused together to form hybridoma.After screening for positive monoclonal cell lines,we choosed the best clone to inoculate the mice abdominal cavity for manufacturing lots of monoclonal antibody.Seven kinds of recombinant protein as antigen were used to immune rabbit for 5 times to make polyclonal antibody,and then test antibody titer in the blood by Elisa,when met the requirements then purified antibody for further study.3.Indirect Elisa kit which we explored was used to detect 20 cases of human prostate tissue samples including 5 cases of BPH,10 cases of ADPC and 5 cases of CRPC.To verify the sensitivity and specificity of homemade antibody,we tested FKBP5 protein with double antibody sandwich method to quantify the amount of the FKBP5 in prostatic tissue.For further development of quantitative antibody microarray experiment,preliminary exploration and optimization of antigen-antibody reaction conditions were detected in our primary test.To study the feasibility of united detection of seven kinds of target protein,preliminary screening in the early diagnosis of CRPC,discussing the clinical value inguiding individualized treatment.Results:1.Affymetrix expression profile gene chip screening revealed 651,gene with obvious difference among which raised 475 genes,down-regulated176 gene,high-throughput proteomics screening revealed phosphorylation protein between ADPC and CRPC were more than 200.By comparison of gene chip and proteomics results,and according to our previous studies,literature review and prostate cancer proteomics databases,we screen out 7 kinds of protein such as ACACA、FKBP5、PIM-1、NT、COX-2、Lyn and AKT1 as indicators of hormone independence prostate cancer screening or potential therapeutic targets proteins.2.Using genetic engineering methods seven protein was successfully recombined among which three were expressed fully and four partly.By use of recombinant protein to immune animals,wegot a total of 7 pair of high specificity and high titer antibodies which detected by Elisa showed all antibody titer were>1:200000.Western blot and IHC test showed high specificity and sensitivity,and can be used for the Elisa kit and antibody microarrays.3.After repeated and optimized conditions experiments,we preliminary developed the indirect Elisa method for detection of multiple indicators of CRPC synchronous.And then we selected FKBP5 by use of double antibody and sandwich method to quantify concentration of protein in diffrent prostate tissue.Preliminary test results showed that the Elisa has high coincidence rate with IHC staining about 85.7%,the quantitative detection of prostate cancer FKBP5 protein concentrations can facilitate the development of antibody microarrays with providing the preliminary experimental data.Conclusions:1.Self-made seven protein antibody can be used for diagnostic prostate cancer molecular classification of Elisa kit,and further development of antibody microarrays,indirect Elisa can be used to detect the different proteins between ADPC and CRPC,it established a quick screening technology platform in the early diagnosis of CRPC and guided individualized treatment of prostate cancer,and meanwhile,it laid the foundation for the next step of quantitative antibody chip.2.The preliminary results showed that FKBP5,AKT,NT,PIM-1 can be used as CRPC screening markers,combination of multiple markers can improve the detection sensitivity.Seven proteins involved in different signaling pathways,and expressed in prostate cancer were significantly higher than that of benign prostatic hyperplasia,they can be used as potential targets of individualized treatment,and testing different markers may help to choose individualized medicine.