基于过表达羧酸酯酶的人骨髓间充质干细胞联合LY2334737对膀胱癌的抑制作用研究OACSTPCD

Objective:To investigate the effect of human bone mesenchymal stromal cells(hBMSCs)overexpressing car-boxylesterases(CES)combined with LY2334737 on the treatment of bladder cancer in nude mice.Method:Adenovirus-me-diated overexpressed human CES2 transfected hBMSCs were assigned to the blank group(without any treatment),the empty carrier group(transfected with empty carrier)and the transfection group(transfected with CES2 overexpressed adenovirus).The transfection efficiency was detected by flow cytometry,and the expression of CES2 was observed by fluorescence micro-scope.Western blot and reverse transcription polymerase chain reaction(RT-PCR)were used to detect the transfection effect of hBMSCs.After transfection for 48 h,hBMSCs were treated with LY2334737(1 μmol/L)for 72 h.The treated cells were assigned to the blank group(hBMSCs),the control group(Ad-hBMSCs+LY2334737)and the experimental group(CES2-hBMSCs+LY2334737).The proliferation and apoptosis of hBMSCs were detected by CCK-8 method and flow cytometry.Human bladder cancer cells TCCSUP and hBMSCs were co-cultured using Transwell chamber and divided into 5 groups:The blank group,the empty carrier group,the transfection group,the Ad-hBMSCs+LY2334737 group and the CES2-hBMSCs+LY2334737 group.The proliferation and apoptosis of TCCSUP cells were determined by CCK-8 method and flow cytometry.Sixty female nude mice were randomly divided into 6 groups:The blank group(injected with TCCSUP cells only),the con-trol group(TCCSUP cells and Ad-hBMSCs injected),the experimental group(TCCSUP cells and CES2-hBMSCs injected),the LY2334737 group(TCCSUP cells injected+oral LY2334737),the Ad-hBMSCs+LY2334737 group(injected TCCSUP cells and Ad-hBMSCs+oral LY2334737),the CES2-hBMSCs+LY2334737 group(injected TCCSUP cells and CES2-hBM-SCs+oral LY2334737).After nude mice were anaesthetized,TCCSUP cells were injected subcutaneously into the dorsal midline.Ad-hBMSCs or CES2-hBMSCs were injected around the tumor 1 week and 2 weeks after the injection of TCCSUP cells.1 h after BMSCs was initially injected,LY2334737 was orally administered once a day for 14 days.One day after the final treatment with LY2334737,the animals were killed before weighing the tumor,the tumor volume was measured,and HE staining was performed on the tumor tissue.Results:The transfection rate of 99.56％in the transfection group was deter-mined by flow cytometry.RT-PCR results showed that compared with the blank group and the empty carrier group,CES2 mRNA expression in the transfection group increased,and the difference was statistically significant(t=27.642,P＜0.000 1;t=30.300,P＜0.000 1).Western blot results showed that compared with the blank group and the empty carrier group,the expression of CES2 protein in the transfection group increased(t=9.678,P=0.000 6;t=9.516,P=0.000 7),indicating that adenovirus overexpressing CES2 could successfully transfect hBMSCs.The cytotoxicity of LY2334737 on hBMSCs was detected by CCK-8 method and flow cytometry.Pairwise comparison between the the blank group,the control group and the experimental group showed that there was no statistically significant difference in the cytotox-icity of LY2334737 on hBMSCs(F=1.631,P=0.236 3).These results indicated that LY2334737 was not cytotoxic to hBMSCs.Compared with the empty carrier group,the Ad-hBMSCs+LY2334737 group could inhibit the proliferation of TCCSUP cells(t=5.421,P=0.000 6).Compared with the transfection group,the CES2-hBMSCs+LY2334737 group could inhibit the growth of TCCSUP cells(t=7.044,P=0.000 1),indicating that CES2-hBMSCs combined with LY2334737 can promote the apoptosis of TCCSUP cells.In animal experiments,the results of the blank group comparing to other 5 groups found that CES2-hBMSCs combined with LY2334737 could effectively inhibit the tumor growth in nude mice with bladder cancer(t=29.360,P＜0.000 1).Conclusion:CES2-BMSCs combined with LY2334737 can inhibit bladder tumor to a certain extent,which provides a certain evidence for the treatment of bladder cancer.