The Relationship Between Content Of EMA~-CD44v6~+ Cells Of CSC And Prognosis Of Bladder Cancer And Research Of Relate Mechanism

Objective:The study based on the relevant previous scholars’ study which aim at verify EMACD44v6 + subsets cells which in bladder cancer tissue with stem cell properties by immunohistochemical double staining technique and establish a microarray method,and analyzing the relationship between the content of EMA~-CD44v6~+subsets in bladder cancer cell and the prognosis of patients with bladder cancer through the epidemiological investigation and statistical methods.In order to explore the characteristics of bladder cancer stem cells and how it does to generate the tumorigenesis mechanisms,we will be on the original generation of bladder tumor tissue culture experiments,establish tumor cell lines in the original generation.Immune magnetic bead separation technology and high throughput sequencing technical will be used to analysis the most closely associated signal pathway of bladder cancer incidence,find out the most obvious differences than the expression of mutated genes,and then through a series of molecular biology techniques analyzed the gene specific expression in bladder cancer stem cells.Thus provide a new direction for bladder cancer stem cell research,and provide a new target for clinical treatment.Methods:Collect and filter out each of the 15 cases of low-grade and high-grade samples from all urothelial carcinoma cases,processing immunohistochemical staining and immunohistochemical double staining for EMA and CD44v6 antibody respectively.Exploring different dyeing method,observation of double staining effect,choose the best double staining method.and using the epidemiological statistical analysis to explore a certain relationship between the content of EMA~-CD44v6~+cells and the prognosis of different grade bladder cancer.Collection and screening of 50 cases of low-grade(pTa)urothelial carcinoma and 50 cases of high-grade(pT2)urothelial carcinoma and 20 cases of normal bladder mucous membrane tissues specimens,those 120 wax blocks will be choose to make the tissue microarray and making the immunohistochemical double staining of EMA and CD44v6 antibody.Estimate thecontent of EMA~-CD44v6~+ cells in each sample.Investigate epidemiological of the prognosis of patients’ clinical data with status,recurrence time,the time of death.And use of statistical methods chi-square and Fisher’s exact probability method,with P <0.05 for the difference is statistically significant,the statistical analysis of the content of bladder cancer stem cells in patients with neoplasm(that is,the the content of EMA~-CD44v6~+cells)and its prognosis condition.Using the method of cell suspension culture and tissue block cultivation method to cultivat the original bladder tumor cells,according to the characteristic of difference attaching time between difference cells to remove fibroblasts impurities,observing the tumor cell growth.Using immune magnetic bead separation technology,sorting out the bladder EMA~-CD44v6~+ cells in primary cell,a part of them are continue training,another of them are being extraction of RNA,and high-throughput gene sequencing.Result:1.The study found that EMA+cells on bellows is located close to the position of the surface cells are relatively mature,the EMA-the basal parts located close to the epithelial cells and gradually upward migration.The expression of CD44v6 and EMA are on the contrary,CD44v6+cells located in the basal cell layer,and CD44v6-cells near the surface of bellow.And the location of the bladder cancer stem cells may exist as the basal cell layer,namely the EMA~-CD44v6~+location.2.The immunohistochemical staining expression positive parts of EMA and CD44v6 antibody are the cytomembrane/cytoplasma,EMA in bladder urothelial carcinoma in the expression positive rate of about 90%,the CD44v6 expression in bladder urothelial carcinoma rate is about 50%.To explore a variety of double staining method,found that the joint AEC DAB chromogenic markup approach works best.3.Statistical analysis found that the rate of EMA positive expression in bladder cancer tissue and tumor grade,stage,no statistical significance(p>0.05),and CD44v6 positive rate increase with bladder tumor staging classification negatively correlated(p<0.05).In each of the stages in the bladder urothelial carcinoma immunohistochemical double dyed EMA~-CD44v6~+cells with the content of different levels in patients with bladder cancer connection: as the bladder urothelial carcinoma the increase of histological grade and stage,EMA~-CD44v6~+cells were decreased(p <0.05).4.Compared with the tissue pieces culture method,using the method of cell suspension culture for bladder urothelial cancer cell primary culture more appropriate,tumor cells are more likely to stick wall,grow faster,cell growth pattern like epithelioid cells.Conclusion:1.According to analysis of the results of immunohistochemical staining show that EMA~-CD44v6~+ expression may be the bladder cancer stem cell surface markers.2.Using immunohistochemical double staining technique can intuitive and convenient reflects the bladder urothelial carcinoma in the area of the existence of cancer stem cells and content.3.The the content of EMA~-CD44v6~+cells reduce with the increase of the grade and stage of bladder urothelial carcinoma,namely that the content of EMA~-CD44v6~+ cells indirectly reflect the prognosis of patients with bladder tumor.4.Cell suspension culture method for bladder tumor cell primary culture method is better,the tumor cells are more pure and provide good foundation for late experiment.