Establishment Of Bladder Carcinoma Cells Intraluminal Implantation Model System And Preliminary Study On The Relative Factors

Objective: To screen a high invasive and intraluminal seeding potential offspring from an invasive bladder carcinoma cell line EJ , and establish a consecutive visible human bladder carcinoma orthotopic animal model , then preliminary study on the relative factors of intraluminal implantation metastasis of bladder carcinoma, provided an ideal models and platform for the basic and clinical study on bladder carcinoma.Methods: Use the human bladder carcinoma cell line EJ as parental cell line, according to its ability penetrating the matrigel covering the bottom of special constructed in vitro invasive chamber, to screen a high invasive potential offspring cell line; to compare the basic biologic behavior of offspring with that of parental. The parental and offspring cell lines were transfected with pEGFP-C1 by Lipofectamine?2000 reagent, and screened by the culture medium containing geneticin (G418) and limited dilution in 96 well flat-bottom culture plates. The cells with enhanced expression of EGFP were selected and cultured ; the offspring cell lines with high invasive ability and stably high expression of EGFP were instilled into the bladders of athymic mouse, and proliferate the tumor cell in the implantation metastasis bladder by primary culture, then repeat the in vivo selection for several round and establish offspring cell line with high intraluminal implantation metastasis, then to detect its basic biological characteristics; on that basis, to establish a modal of human bladder cancer cell intraluminal implantation metastasis in urinary bladder of athymic mouse. By scanning electron microscope, whole body optical imaging system, laser scan Confocal microscopy and flow cytometry to observed the process of human bladder cancer cell implantation metastasis in urinary bladder of athymic mouse. Bladder cancer cell EJ were cultured in filtrated urine, and the cell survival and growth were observed by inverted microscope. EJ, HepG2 and Ecv304 were cultured in urine, isotonic Na chloride and basal culture, and the OD values of cells of different times were detected by MTT. By using a method that gradually addition the urine into medium, the cells that had high adaptability for urine were selected and cultured. To detect the content of CXCL12 (SDF-1) of mouse bladder which were damaged by trypsin or not. We also investigate the relationship of the damage of mucosa of urinary bladder with the intraluminal implantation metastasis.Results: After over 15 generation, the morphous of the offspring cell lines (EJ-m3) was similar to that of their parental cell lines. The in vitro invasiveness was elevated significantly (p<0.001), and the propagation speed was obviously accelerated compared with their origins (p<0.05). The neoplasm emerging of the offspring cells that were produced in athymic mice by intravesical instillation was higher than their parental cell lines. A high invasive ability human bladder cancer cell lines with enhanced expression of GFP gene was established. The expressing rate of GFP gene was 99.9%. The proliferation index, auxodrome and the invasive ability of transfected cells showed no significant difference to cells with not. The expression of CXCR4 was detected in the cells of bladder tumor, and the number of EJ-im3-GFP is higher than EJ. Then the cell line with high implantation metastasis was selected and cultured, and the model of human bladder cancer cell intraluminal implantation metastasis in urinary bladder of athymic mouse was accepted. By using the procedure scanning electron microscope, whole body optical imaging system, laser scan Confocal microscopy and flow cytometry the process of human bladder cancer cell implantation metastasis in urinary bladder of athymic mouse was observed which was the type of upside down parabola. The bladder cancer cells that were cultured in urine can adhere on culture flask at about 12h, and a few cells that grew about 24h could be observed. The OD value of cells of different times were detected by MTT, and multivariate analysis authentication show that when the culture time was identical, the survival number of different cells were different (P=0.001), and Q analysis authentication showed that the survival number of EJ were greater than HepG2 (P<0.001) and Ecv304 (P=0.014), and the survival numbers of HepG2 and Ecv304 had no significant difference (P=0.062). The cells that have high adaptability to urine have multiplicity survival when cultured in urine for 24h, and there were survival cells when cultural for 48h. The mucosa of urinary bladder that was damaged by trypsin was engorgement, dropsy and inflammatory cells infiltrating. The content of CXCL12 (SDF-1) of mouse bladder which were damaged by trypsin was increaser than which was not, and the neoplasm emerging rate of athymic mouse which were damaged by trypsin was higher than which were not.Conclusion: The in vitro invasiveness was direct correlation with intraluminal implantation metastasis. The process of transfection pEGFP-C1 into badder cancer cells cannot affect the basic biological behavior of these cells. The expression of CXCR4 was detected in the cells of bladder tumor, and the number of EJ-im3-GFP is higher than EJ. This model of human bladder cancer cell implantation metastasis in urinary bladder of athymic mouse have the advantages of high rate of neoplasm formation and can measure the tumor progression over time in each mouse, but the mice haven't to be sacrificed, and the process of implantation metastasis was the type of upside down parabola. Bladder cancer cells can survival in urine (at least 12 hour). The mucosa of urinary bladder which was damaged by trypsin was engorgement, dropsy and inflammatory cells infiltrate, and the content of CXCL12 (SDF-1) of mouse' bladder was increaser, and the neoplasm emerging of athymic mouse was higher than which were not.