The Inhibitory Effect Of Gene HepaCAM Transfection On The Proliferation In Human Bladder Carcinoma T24 Cell In Vitro And Vivo

PART ONEObjective:To construct separately human bladder carcinoma T24 cell line stably expressing GFP and hepaCAM proteins.Methods:T24 cells were transfected with plasmid pEGFP-N2 and pEGFP-N2-hepaCAM by lipofectamine.Then we screened the transfected cell by G418.Individual G418-resistant colonies was isolated by limited dilution and some of the monocolonal cell line T24/GFP and T24/hepaCAM-GFP were obtained.We observed the expression of GFP in T24/GFP and T24/hepaCAM-GFP cell lines under microscopy. We identified the moloclonal transfected cell lines through detcting hepaCAM mRNA ,GFP and hepaCAM proteins by RT-PCR and Western blot.Rusult:A lots of green fluorescence was found out in T24/GFP cell and T24/hepaCAM-GFP cell. We also found 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 high expression of hepaCAM mRNA was detected by RT-PCR assay in T24/hepaCAM-GFP cell. The expression of GFP and hepaCAM proteins were detected by Western blot assay in T24/GFP and T24/hepaCAM-GFP cells.Conclusion:We constructed successfully the stable transfected cell lines T24/GFP and T24/hepaCAM-GFP ,which were expression of GFP and hepaCAM proteins.It layed the foundaton for the effect of gene hepaCAM transfection on the proliferation in T24 cell in vitro and vivo.PART TWOObjective : To explore the effect of gene hepaCAM on the proliferation in T24 cell in vitro.Methods: The T24/GFP, T24/hepaCAM-GFP and T24 cell lines were named empty vector group,experiment group and control group cells .The effects of hepaCAM on cell proliferation were measured by MTT and colony formation assays. The cell cycle progression of each group were measured by flow cytometry. The expression of CyclinD1 mRNA of all groups were detected by RT-PCR.Rusult: The MTT assay showed that cell growth rate at 120h in experiment group (54.01±12.06 %) was significantly slow when compared with control group(161.67±12.17 %) and empty vetor group(160.63±13.95 %)(P<0.01);The clonal formation assay showed that the rate of clonal formation in experiment group(27.67±4.53%) were greatly decreased when compared with control group(88.06±7.69%) and empty vetor group(86.83±5.89%).(P<0.01); Flow cytometric data showed that the proportion of G0/G1 phase (78.70±1.96%) of experiment group was significantly higher than control group(61.59±2.14%) and empty vetor group(59.25±3.17%)(P<0.05). The expression of CyclinD1 mRNA of experiment group was lower than control group and empty vetor group by RT-PCR assay.Conclusion: HepaCAM gene might suppress the proliferation of T24 cells in vitro through arresting cell cycle in the G0/G1 phase and inhibitting the expression of CyclinD1 mRNA.PART THREEObjective : To explore the effect of gene hepaCAM transfection in human bladder carcinoma T24 cell line in nude mice.Methods:The T24,T24/GFP and T24/hepaCAM-GFP cells were transplanted subcutaneous1y in nude mice,and then they were named separately control group, empty vector group and experiment group .The tumor growth was observed about each group.Rusult: The tumor growth rate in experiment group was significantly slow when compared with control group and empty vetor group .The volume of tumor at 23 day in experiment group was significantly small than control group and empty vetor group [(0.188±0.039)cm3 vs(1.002±0.060)cm3,(0.989±0.093)cm3, P<0.01],whereas the weight of tumor was greatly decreased[(0.340±0.114)g vs (1.120±0.259)g,( 0.880±0.239)g, P<0.01].Conclusion: HepaCAM gene might suppress the proliferation of T24 cell in nude mice.