1.PLC Epsilon Antiserum Preparation And Its Clinical Application In Bladder Cancer2.The Function And Mechanism Of HepaCAM In Prostate Cancer Cells

Objective：1. To construct a prokaryotic expression system of the PLCε, to express,purify and identify the recombinant PLCε protein.2. To prepare the PLCε-specific rabbit antiserum, to purify and identifythe specificity of antiserum, and its clinical application in bladder cancer.Method：1. The part of PLCε gene was obtained by RT-PCR from the cDNA ofhuman bladder cancer cells, and recombined into pGEX-6P-1vector toconstruct a recombinant expression vector pGEX-6P-1/PLCε.2. The recombinant plasmid was transformed into E.coli BL21(DE3)and then induced with IPTG for expression. The fused GST-PLCε proteinwas purified by affinity GST chromatography system, and identified byWestern blotting.3. Rabbit were immunized with the pure recombinant proteinGST-PLCε to prepare antiserum. The antiserum was purified throughProtein A antibody purification system and identified. The titer andspecificity of rabbit antiserum were evaluated by ELISA and Western blot.4. Coating the ELISA plate by PLCε antiserum, and preparation of simple rapid PLC epsilon protein ELISA kit. To detect the PLC epsilonprotein expression in30cases bladder cancer and6cases normal humanserum, and preliminary explore the PLC epsilon antiserum application inbladder cancerResults：1. The PLCε gene was partly amplified and the prokaryotic expressionvector of pGEX-6P-1/PLCε was successfully constructed by RT-PCR, generecombination, restriction endonuclease. The recombinant protein GST-PLCε was purified through GST affinity chromatography.2. We obtained the antiserum after immunizing rabbit with the purifiedGST-PLCε fused protein. The ELISA titer of the rabbit antiserum was1:16000approximately. Western blot analysis showed that antiserum couldbind the purified GST-PLCε fused protein and the PLCε protein extractedfrom human bladder cancer cells specifically.3. The PLC epsilon protein ELISA kit detected the PLCε proteinexpression in bladder cancer and normal human serum. PLC epsilonexpression in bladder cancer patients serum was5.62±2.79ng/ml is higherthan normal control group2.53±0.42ng/ml (P <0.05), the difference isstatistically significant.Conclusion：The GST-PLCε protein with high purity was successfully expressed inE.coli and the PLCε-specific rabbit antiserum was prepared. The antiserum was preliminary applied in serological test of bladder cancer. PLC epsilonantiserum for further research of protein function mechanism laids a solidfoundation, but also for the development of rapid detection of bladder cancerkit provides experimental data. Objective：1. To explore the HepaCAM gene expression in prostate tissue andcell lines and its clinical significance.2. To investigate the function and mechanism of HepaCAM in prostatecancer after cells treated with adenovirus vector, including growth,migration, invasion and survival.Method：1. HepaCAM expression has been detected by RT-PCR, Westernblotting and immunohistochemistry staining in prostate cell lines RWPE-1,LNCap, DU145, PC3and in75human prostate tissue specimens,respectively. Statistical analysis of the correlation of the expression andclinical pathological parameters.2. The cell proliferation ability was detected by WST-8assay. The roleof HepaCAM in prostate cancer cell migration and invasion was examinedby wound healing and transwell assay. And flow cytometry was used toobserve the apoptosis of prostate cancer cells. Western blot was used tomeasure the protein level of HepaCAM, Bax, Bcl-2, MMP2, MMP9andGAPDH. 3. We detected the mRNA and protein expression levels of AR inprostate cancer LNCap cells by immunofluorescence staining, RT-PCR andWestern blot after overexpression of HepaCAM. Western blot and ELISAwas used to examine the expression of cytoplasm and secreted PSA.Meanwhile, P-ERK and t-ERK protein expression were detected byWestern blot after cells treated with EGF and Ad-GFP-HepaCAM.Results：1. The HepaCAM expression was significantly down-regulated inprostate cancer tissues and undetected in prostate cancer cells. Moreover,the low HepaCAM expression was not statistically associated withclinicopathological characteristics of prostate cancer.2. Overexpression of HepaCAM in prostate cancer cells decreased thecell proliferation, migration and invasion, and induced the cell apoptosis.Meanwhile, overexpression of HepaCAM downregulated the expression ofBcl-2, MMP2and MMP9, and upregulated Bax.3. Overexpression of HepaCAM prevented the Androgen Receptortranslocation from the cytoplasm to the nucleus and down-regulated theMAPK/ERK signaling. And HepaCAM also inhibited the cytoplasm andsecreted PSA expression levels.Conclusion：Our results suggested that HepaCAM acted as a tumor suppressor inprostate cancer. HepaCAM inhibited cell viability and motility which might be through suppressing the nuclear translocation of Androgen Receptor anddown-regulating the ERK signaling. Therefore, it was indicated thatHepaCAM may be a potential therapeutic target for prostate cancer.