The Inhibition Effect Of Ultrasound Microbubble Carrying HSV1-TK Gene Combined With Ganciclovir On Prostate Cancer Cell

Objective: To observe the transfection of ultrasonic microbubble withherpes simplex virus thymidine kinase(HSV1-TK)gene and the inhibitingeffect on prostate cancer cell line PC-3combined with ganciclovir.Methods: low-frequency ultrasonic with sound intensity of1.0W/cm2, frequency of1MHz the irradiation time for0,30,60,90,180s isused to Irradiae prostate cancer PC-3cells by MTT. Ultrasoundmicrobubble surface was adsorbed with HSV1-TK gene, and transfectunder optimal ultrasound irradiation, and to observe the expression andgene transfection of TK gene by fluorescence microscopy and Western blot.The experiment was divided into4groups: control, pure TK group,ultrasound+TK group, ultrasound+microbubble+TK group.Western blotmethod includes: Extracting total protein,the concentration were measured,adding buffer and boiled, gel loading, electrophoresis, transferred to a membrane, closed, and dropping the first antibody, incubating overnight at4°C, washing by TBST, secondary antibody labeled HRP incubating at37℃for1h, washing by TBST, imaging, taking photos.MTT assay is used toobserve the inhibition of cell proliferation after using differentconcentrations of the prodrug ganciclovir (GCV). Flow cytometry (FCM)is used to test the cycle impact of Prostate cancer cells(PC-3) after usingthe optimal concentration of the prodrug ganciclovir (GCV) againsttransfected(HSV1-TK). MTT assay can observe the killing effect oftransfected and HSV1-TK/GCV suicide gene therapy system on Prostatecancer cells(PC-3).Results: MTT assay shows when the ultrasonic intensity is1.0W/cm2,frequency is1MHz and irradiation time is30s, ultrasonic irradiation has nosignificant inhibitory effect on cancer cell, so select this parameter as theoptimal irradiation conditions for follow-up of microbubble portabilitygene transfection. This could significantly improve the transfectionefficiency of HSV1-TK gene against the PC-3cells and get stableexpression. The experiment of ultrasonic irradiation+plasmid TK group,we found TK gene can transfer to PC-3cell and have stable expression, theexpression is higher than the other samples(P<0.05). MTT assay showswhtn GCV concentration≤100ug/ml, there is no apparent toxic effectson prostate cancer cell growth, cell survival is≥91％, comparing withconcentration10000and1000ug/ml, there are significant difference in cell survival rate, this sample is Statistically significant(P<0.05). So we choosethe optimal concentration of GCV is100ug/ml; When adding GCV as theprodrug, the survival rate of ultrasonic irradiation+plasmid TK group isabout32%, cell killing rate is significantly higher than that in the controlgroup and experimental group(P<0.05); Flow cytometry testing shows inultrasonic irradiation+plasmid TK group, most of the cell growth isstopped in the period of S.Conclusions: Irradiation of ultrasound microbubble contrast as acarrier can significantly improve the transfection efficiency of suicide gene(HSV1-TK), combining with prodrug GCV, has significant killing effect onprostate cancer. This research can provide some experimental basis for thegene therapy of prostate cancer...