Experimental Study On Killing Bladder Cancer Cells By Carrying YCD-Connexin26 Gene Nanomicrobubbles

Objective:A cation nanomicrobubble capable of carrying a double gene was prepared.Analyze the effectiveness of the gene transfection with dual-gene cationic nanomicrobubbles combined with ultrasound-targeted microbubble destruction.To explore whether upregulating the expression of connexin26(Cx26)in bladder cancer cells can enhance the bystander effect of yeast cytosine deaminase/5-fluorocytosine(YCD/5-FC)and increase the efficiency of killing tumor cells.Methods:Cationic nanomicrobubbles were prepared by membrane hydration and mechanical shaking method.Electrostatic adsorption and complexation carried plasmids.Human bladder cancer T24 cells were transfected by cationic nanomicrobubbles combined with ultrasound irradiation(US).The transfection efficiency was observed by fluorescence microscopy and flow cytometry;RT-PCR and Western blot were used to detect the relative expression of mRNA or protein after plasmid transfection.CCK-8 method was used to compare the effects of 5-FC prodrugs on the survival rate of T24 cells transfected with different gene microbubbles;flow cytometry was used to observe the apoptosis of bladder cancer cells after connexin26(Cx26)expression.Results:Cation nanomicrobubbles(CMB)with good stability(472.5±48.57)nm were prepared and the average surface potential was+(28.32±2.78)mv.The maximum gene binding and binding rates of CMB were 4?g/10~8microbubble and 66.7%,respectively.Under certain ultrasound irradiation conditions,the transfection efficiency of the plasmid was(30.28±2.04)%.RT-PCR and Western blot showed that the target gene was successfully transfected and effectively expressed by nanomicrobubble-binding ultrasound irradiation.When the plasmid YCD:CX26 was transfected with5:5,after prodrug 5-FC(300?mol/L)induction for 24H:the survival rate of T24 cells was(42.3±4.6)%;recovery of Cx26 After the expression,the apoptosis rate of the YCD-Cx26 nanomicrobubbles group was(60.68±2.61)%,which was significantly higher than that of the YCD single vacuole group(46.42±2.13)%.The difference was statistically significant(P<0.01).Conclutions:CMB can enhance the carrying capacity of plasmids.Combined with ultrasound irradiation,transfection efficiency can be effectively improved and the target gene can be successfully expressed.connexin26(Cx26)can improve the communication between cells,strengthen the bystander effect of suicide gene system YCD/5-FC,promote tumor cell apoptosis,and increase the efficiency of killing bladder cancer cells.