The Effects And Mechanism Of LASS2 Expression To Bladder Cancer EJ Cells Tumorigenicity In Vivo

Purpose:To constructe Orthotopic bladder cancer metastasis model and Bladder cancer cell injected subcutaneously metastasis model in nude mice. By observing the location and number of tumor, RNA and protein was extracted, fixed tumor to make paraffin sections, Real-time quantitative PCR, western blot and immunohistochemistry to detect the variation of LASS2 and the indicators of proliferation or apoptosis (Bcl-2、 Bcl-xL、Ba、 Bim、caspase3、Ki-67), to investigate the effects and mechanism of LASS2 expression to bladder cancer EJcells tumorigenicity in vivo.Methods:1.Culturing the bladder cancer EJ cells, constructing orthotopic bladder cancer metastasis model and bladder cancer cell injected subcutaneously metastasis model in nude mice.(1)Orthotopic bladder cancer metastasis model:Intraperitoneal injection anaesthetize nude by 0.1% Pelltobarbitalum Natricum (50mg/kg), disinfected the vulva and lower abdomen, emptied nude bladder, inserted catheter (Epidural catheter) into bladder, rinsed the bladder by 100μl PBS and injected into 100μl trypsin, retained 30 minutes, rinsed the bladder by PBS again. 100μl EJ cells (1×107/ml cells) were instilled after emptying the bladder, retained 1 hour. The control group was injected by DMEM medium of bladder cancer cells.(2)Bladder cancer cell injected subcutaneously metastasis model:Disinfected the skin, after a conventional count of EJ cell lines, digest by EDTA/trypsin.5×106/0.1ml bladder cancer cells suspension were subcutaneously injected into the left subaxillary of each nude mice.2.SPF level feeding mice. Recorded the living conditions of nude twice of every week, touched the lower abdomen and squeezed the bladder to observe if there was hematuria, four and eight weeks later, executed and dissected mice to observe whether organ metastasis. Measured tumor volume and made tumor growth curve, removed the bladder and the tumor, RNA and protein was extracted, fixed tumor to make paraffin sections, Real-time quantitative PCR, western blot and immunohistochemistry to detect the variation of LASS2 and the indicators of proliferation or apoptosis (Bcl-2、Bcl-xL、Bax、Bim、caspase3、Ki-67).Result:1.Constructed Orthotopic bladder cancer metastasis model and bladder cancer cell injected subcutaneously metastasis model in nude mice. Simulating of Human bladder cancer growth and metastasis.2.Constructed orthotopic bladder cancer metastasis model tumorigenic result is not ideal, bladder cancer cell injected subcutaneously metastasis model tumor formation rate of 100%. The two models were not found transfer phenomenon in vivo. Pathological examination the two models tumor cells appeared atypia, nuclear was lager and deeply stained, parts of nuclear burst.3.1mmunohistochemical analysis confirmed LASS2 expressed mainly in the cytoplasm and cell membrane, few nuclei may also found transfection. Expression of LASS2:control group> Orthotopic bladder cancer group> Subcutaneously metastasis group.4.Immunohistochemical analysis to detect Ki67 protein expression,the result is: control group< Orthotopic bladder cancer group< Subcutaneously metastasis group.5.Quantitative PCR detect the family of Bcl2, the results show that Bcl-2 and Bcl-x are highly expression, Bcl-2 expressed in normal bladder tissue is significantly higher than that in orthotopic bladder cancer tissue and subcutaneously metastasis group. No significant difference in the expression level of Bax in each group. Bim expressed in orthotopic bladder cancer tissue is significantly lower than that in the other two groups.6.For caspase3, LASS2 and ki67 mRNA and protein levels were detected, the results showed:no significant difference in the expression of caspase3 in each group organization. LASS2 significantly increased expression in normal bladder tissue than the other two tumor tissues, and Ki67 expressed in orthotopic bladder cancer tissue is significantly higher than that in the other two groups.Conclusion:1.Constructed orthotopic bladder cancer metastasis model tumorigenic result is not ideal, bladder cancer cell injected subcutaneously metastasis model tumor formation rate of 100%. The two model showed no significant organ metastasis, the metastasis ability was inaccurate, whether LASS2 effected the metastasis ability of bladder cancer metastasis cells in vivo need further research.2.1n situ bladder tissue implant group, the expression of LASS2 lower than the normal bladder tissue group, comparison with subcutaneous tumor tissue, the level of LASS2 is improved. Orthotopic implantation tumor and subcutaneous tumor tissue, Ki67 expressed higher than those in the control group. This suggests that the expression of LASS2 may affect the tumorigenicity of bladder cancer cells in vivo, LASS2 influence on tumor proliferation may be related to altered the expression of ki67.