| 1?Background Bladder cancer is the most common malignancy of the urinary system,it is also one of the ten most common cancers of human being.Bladder cancer accounted for the first incidence of Genitourinary tumors as well.Urothelial carcinoma was previously known as transitional cell carcinoma,which is the most common type of bladder cancer,accounting for more than 80% of patients with bladder cancer.At present,bladder urothelial carcinoma presented with the characteristics of the the overall incidence are younger and higher than ever,the recurrence rate of non invasive bladder cancer after surgery is high while the quality of life after radical cystectomy decline rapidly and the radiation and chemotherapy sensitivity is poor,the prognosis of patients is poor in case of metastasis.New therapeutic target is needed for the sake of treatment effect.Tumor is organ-like structure which composed of different cell types whose interactions are required to develope and promote their growth and metastasis.Carcinogenic cells recruit nontumorigenic cells both from the neighboring tissues and from the circulation as well,the cells construct the tumor microenvironment which promote cancer progression through paracrine signaling and physical interactions.The tumor microenvironment including cancer-associated fibroblasts,endothelial cells,immune cells,adipocytes and cancer stem cells which can differentiate into metastatic epithelial cells,mesenchymal stem cells which differentiate into fibroblasts and other kinds of cells as well as types of extracellular matrix proteins that are needed for messaging and stimulation of tumor growth.These different kinds of tumor stroma,especially MSCs and stromal cells deriving from MSCs,has recently been recognized as playing an important role in carcinogenesis,tumor growth,development,and progression at the early steps of tumorigenesis and influencing the construction of the microenvironment of tumors,that is essential for tumor maintenance and metastasis to other tissues.The presence of stromal cells is found in bladder mucosa and underlying base layer.It is hypothesized that the existence of bladder tissue mesenchymal stem cells(mesenchymal stem cell,MSC)in bladder tissues,for the reason that the MSC has currently been isolated from a variety of tissues including bone marrow,muscle,fat,umbilical cord and so on.MSC embraces the potential ability of differentiating into multiple tissue cells,and producing a variety of cytokines and growth factors involved in inflammation and tumorigenesis as well.2? research purposes This thesis intends to explore the methods of isolating and culturing mesenchymal stem cells(human bladder derived Mesenchymal stem cells,h BSC)from human bladder tissue,and then analyze the differences between h BSC and other tissue-derived mesenchymal stem cells,the last step of the research is probing into the effection and mechanism of h BSC in bladder cancer cell growth migration and autophagy.3?Research contents(1)Isolattion and cultivation of human bladder mesenchymal stem cells(h BSC)from excised normal tissues in radical cystectomy specimens.Its cell surface antigen molecules are identified by flow cytometry,then the h BSC is induced to adipogenic,osteogenic and three cartilage catigaries differentiation,followed by inducing h BSC to other types of bladder tissue cells;(2)Differences between Hbsc and bone marrow mesenchymal and umbilical cord sources of human mesenchymal stem cells are drawed in terms of the cell proliferation,telomerase activity and cytokine secretion;(3)The h BSC is cultured with bladder cancer cell number 5637 and J82 by Transwell co-culture cells,then conditioned broth medium is used for the cultivation of bladder cancer number 5637 and J82,cell proliferation and cell migration are observed and documented,Western blot detection of p70,AKT and phosphorylation of STAT3 protein level are observed,EMT related genetic expression is detected with fluorogenic quantitative PCR.(4)HBSC conditioned broth medium for bladder cancer cell number5637 and J82 cultivation,LAMP2 is detected withcell immunofluorescence test,Western blot for the detection of autophagy gene LC3 B,plasmids transfected with EGFP-LC3 B fluorescent protein distribution was observed by processed,Annexin V-FITC labeled adoption rate of apoptosis by flow cytometry nutritional deficiencies when assessing the impact h BSC of autophagy cell bladder cancer cell lines.4?The results(1)The human bladder mesenchymal stem cells(h BSC)are isolated and collected successfully and an appropriate h BSC culture system is established.T he h BSC character is identified by flow cytometry by expression of CD13,CD29,CD73,CD90 and CD105 molecules without expression of CD14,CD19,CD31,CD34,CD45 and HLA-DR,low expression of HLA-ABC is observed as well.HBSC can be induced of differentiation into fat,bone and cartilage cells,with a special broth medium h BSC can be differentiated into urothelium-like endothelial and smooth muscle-like cells too.(2)Compared with human bone marrow-derived mesenchymal stem cells(h BM-MSC)and human umbilical cord sources mesenchymal stem cells(h UC-MSC),h BSC possess the same growth rate,there is no significant difference between h BSC and h BM-MSC in terms of telomerase activity,while in terms of cytokine secretion,h BSC secrets slightly more EGF,IGF1,IL-1?,and IL-6 than h BM-MSC and h UC-MSC.(3)To cultivation h BSC with bladder cancer cell lines 5637 and J82,promotion of the growth and migration are observed in bladder cancer cell number 5637,and not obvious in J82 line.The mechanism is believed that the intracellular p70 and STAT3 proteins are induced in bladder cancer cell number 5637 phosphorylation is increased cell growth rate,and promotion of h BSC in line 5637 EMT transformed cells,thereby contributing to line 5637 migration.(4)In the circumstance of low magnesium serum or serum-free starvation,h BSC conditioned broth medium can promotes the autophagy of line 5637 cells,the intracellular LC3B?level increased and punctate aggregates LC3 B and LAMP2 observed in line 5637 cells.(5)Conclusion In the study,it is found that the bladder mesenchymal stem cells(h BSC)can be isolated and collected from bladder submucosa and muscle tissues.Human bladder mesenchymal stem cells(h BSC)possess the potential capacity of differentiation into 3 department of blood cells and expression of antigen molecules compared with three other source-derived MSC cells.HBSC cell differed from other MSC cells in proliferation,telomerase activity and cytokine secretion.HBSC can also promote bladder cancer cell lines in vitro co-cultivation of 5637 growth,m TOR and STAT3 pathways play an important role in it;HBSC can also induce line 5627 cells to EMT transformation,therefore promoting cell migration.The capacity of autophagy and survive in serum starvation are promoted in line5637 cells.Conclusively,the study increased our knowledge about bladder tissues and cells construction,it is proposed in this study a new chemical therapy target for the bladder cancer treatment. |
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