The Expression Of Hepacam In TCCB And The Biology Functions In T24 Cells

PART ONEObjective: To investigate the expression of hepaCAM in transitional cell carcinoma of bladder(TCCB) and its clinical significance.Methods: The expression of hepaCAM mRNA in 28 normal bladder tissues and 34 TCCB specimens were detected by RT-PCR,and its relationship with tumor clinicopathotogical characteristics were analyzed.Rusult: The expression of hepaCAM mRNA in 34 TCCB specimens were significantly decreased than in normal bladder tissues(p<0.01).The sub-expression of hepaCAM gene was correlated with tumor pathotogical grade(p<0.05),but it were not closely associated with clinical stage,sex,age and occurence(p>0.05).Conclusion: hepaCAM gene was sub-expression in TCCB tissues which play an important role in bladder carcinogenesis development.It may act as a new diagnosis index of TCCB. PART TWOObjective: The identification of eukaryotic expression plasmid of pEGFP-N2-hepaCAM and its mutant pEGFP-N2-hepaCAM-mt.To observe the localization of hepaCAM and hepaCAM-mt protein in T24 cells and establish a stable and efficient transfection method of bladder cancer cell line.Methods: The plasmids were digested by HindⅢand Bam HⅠ, the sequences of the recombinant plasmids were also verified by sequencing.The transfection efficiency was calculated by fluorenscence microscope. The localization of hepaCAM and hepaCAM-mt protein in T24 cells were observed by confocal microscopy.Western bolt analysis were taken to verified the stable expressions of proteins.Rusult: The recombinant plasmids were verified by endonuclease digesting and DNA sequencing.The stable and efficient transfection method was established, the best transfection time was 48h and the most suitable liposome-plasmid proportion was 2:1.Confocal microscopy shows hepaCAM protein was localized on the perinuclear membrane, cytoplasm and at the tip of cell surface protrusions that was about to make contact with adjacent cell surfaces in well spread cells;whereas in the cells which contacts, hepaCAM was predominantly accumulated at the sites of cell-cell contacts on cell membrane.The hepaCAM-mt protein has also the same localization in well spread cells,however,when the cells contacted ,the hepaCAM-mt protein was not like the hepaCAM,but located on the cell non-specificity.And the stable expression cell was also established,which could express the hepaCAM and hepaCAM-mt proteins.Conclusion: The recombinant plasmids could transfect the T24 cells and express the hepaCAM and hepaCAM-mt proteins stably,which lay the foundation for the roles of hepaCAM in T24 cells for further studies.PART THREEObjective: To study the relationship between transfection of hepaCAM or its mutation gene and the abilities of cell adhesion,motility and proliferation of T24 cells.Methods: T24 cells were transfected with mock plasmid pEGFP-N2,pEGFP-N2-hepaCAM and pEGFP-N2-hepaCAM-mt with lipofectamine. The stable colonies were obtained by G418 screening. Cell adhesion and motility ability were tested by cell spreading /detachment assay and wound healing assay/matrigel invasion assay。The proliferative ability of T24 cells were investigated by MTT assay.Rusult: In vitro experiments as cell spreading /detachment assays and wound healing / matrigel invasion assays showed the cell adhesion and cell motility properties of hepaCAM group were apparently enhanced when compared with the cells which transfected with mock-vehicle groups or non-transfection cells(p<0.01),but when the cells transfected with hepaCAM-mt compared with non-transfection and mock-vehicle cells,it show not significance(P>0.05). The MTT assay showed cell proliferation ability in the hepaCAM group were notablly decreased when compared with the the cells which transfected with mock-vehiclecells or non-transfection cells(P<0.01),but it show not significance when the cells transfected with hepaCAM-mt compared with non-transfection and mock-vehicle cells(P>0.05).Conclusion: The hepaCAM gene can restrain malignant phenotypes of the human TCCB cells in vitro ,and could inhibit the TCCB invasion and metastasis by influencing the function of cell adherence, motility properties and cell proliferative factors , hepaCAM gene maybe a new tumor-supperssor gene ; the extracellular domain-truncated mutant hepaCAM gene have not impact on the fuctions of T24 cells'proliferative ability,adhesion and motility abilities。The extracellular domain of hepaCAM is very important for whole functions of hepaCAM.