The Wild Type P53 Gene Transfered Into The Ovarian Cancer Cells A2780/DDP By The Ultrasound Mediated LHRHa-targeted Microbubbles Destruction In Vitro

PARTⅠPreparation of LHRHa-targeted microbubble contrast agent and targeting study in vitroObjective To prepare targeted microbubbles targeting ovarian cancer cells, and to assess its targeting ability in vitro.Methods The biotinylated luteinizing hormone releasing hormone analog (LHRHa) peptide was conjugated to the biotinylated microbubbles via streptavidin, and then were observed by fluorescence microscope. The LHRHa targeted microbubbles were added into the A2780/DDP cells to observe the adherence of the targeted microbubbles to the cells and normal microbubbles were used as control.Results The fluorescence was expressed on the targeted microbubbles shell in vitro. The LHRHa-targeted microbubles could adhere to the A2780/DDP cells specifically, whereas there was no adherence of normal microbubbles to the A2780/DDP cells. Conclusion The LHRHa-targeted microbubbles can be successfully constructed via biotin-streptavidin binding, and it has the targeting ability in vitro.PARTⅡThe influence of the different ultrasound exposure and microbubbles concentration to the cells A2780/DDPObjective To choose the optimal combination between the microbubbles and ultrasound for the gene transfection.Methods Cells in 9 groups were exposed to a ultrasound with different intensity of (0.5 W/cm~2,1.0 W/cm~2 and 1.5W/cm~2) and different time(30s, 50s and 70s) respectively, and then cells in 3 groups were exposed to the the optimal parameter combined with different microbubbles concentration which was 10 times,15 times,20 times more than cells concentration.then chose the optimal combination between the microbubbles and ultrasound .Results The cell survival rate was>90%,when exposed to a ultrasound 0.5W/cm~2,30s;And the cell survival rate was>90% , When exosed to the microbubbles concentration which is 10 times more than cells concentration combined with the ultrasound 0.5W/cm~2,30s.Conclusion The cell growth activity was in a decreasing with the growing ultrasound intensity ,exposure time and microbubbles concentration ;and the microbubbles concentration which is 10 times more than cells concentration combined with the ultrasound 0.5W/cm~2,30s was chosen to be the optimal parameter for the further gene transfection. PARTⅢPlasmid encoding a wide type P53 gene transfered into A2780/DDP cells by the ultrasound mediated LHRHa-targeted microbubbles destruction and the influence of the cell apoptosis after treatment.Objective To investigate the feasibility and efficiency of a plasmid DNA encoding wtp53 transfered into A2780/DDP cells via the Ultrasound -targeted microbubbles destruction (UTMD),and observe the cells apoptosis after the gene transfection.Methods1.the A2780/DDP cells were divided into 6 groups: A: pEGFP-N1-wtp53 alone;B: MBN+pEGFP-N1-wtp53; C: MBT+pEGFP-N1- wtp53; D :US+ pEGFP-N1-wtp53 E:US+MBN+ pEGFP-N1-wtp53;F: US+ MBT+ pEGFP-N1-wtp53; and every group were exposed to the US chosen in the PartⅡ,then the cells were observed via the inverted fluorescence microscope ,the transfection efficiency was analysed by FCM and the wtp53 mRNA expression was analysed via RT-PCR.2. the A2780/DDP cells were divided into 7 groups: A: Control; B:US alone;C:US+ MBN ;D:US+ MBT ;E: US+pEGFP-N1-wtp53:F: US+MBN+ pEGFP-N1-wtp53;G: US+ MBT+ pEGFP-N1-wtp53; every group were exposed to the US chosen in the PartⅡ,then the cell apoptosis efficiency was analysed via FCM.3. the A2780/DDP cells were divided into 2 groups: A: US+ MBT+ pEGFP-N1-wtp53; B: Control, then the cell cycle were analysed via FCM after 24h treatment.and the cell apoptosis was observed via TEMResults1.the cells expressed EGFP The EGFP was obviously observed in the US+MBT+pEGFP-N1-wtp53 group and US+ MBN+pEGFP-N1-wtp53 group, and a slight EGFP was expressed in the US+pEGFP-N1-wtp53 group;However, there was no EGFP expressed in other groups.2.the mean transfection efficiency The mean transfection efficiency in US+MBT+ pEGFP-N1-wtp53 group and US+ MBN+ pEGFP-N1-wtp53 group reached to (43.90±6.19)% and(25.33±4.44)% respectively ,There was a statistically significant differece between these two groups(p<0.05),and it showed a slight elevation(7.24±5.15)% in the group US+ pEGFP -N1- wtp53 ;the other groups exhibited a very low transfection efficiency(<1%)3.wtp53mRNA The wtp53mRNA levels were significantly increased in the US+MBT+pEGFP-N1-wtp53 group and US+ MBN+ pEGFP-N1-wtp53 group,the level of the former was higher than the latter, There was a statistically significant difference between these two groups(p<0.05),there was a very lower wtp53mRNA level in the US+ pEGFP-N1-wtp53 group, but there was no wtp53mRNA expressed in other groups.4.the cells apoptosis It demonstrated when the wtp53 gene was incorporated in to the cells by the US+MBT and US+MBN,The cell apoptosis efficiency reached to (39.67±5.95)% and (24.54±3.68)%, There was a statistically significant difference between these two groups(p<0.05).5.the cell cycle It demonstrated when the wtp53 was incorporated in to the cells by the UTMB , (62.79±4.65)% of cells was arrested in the cell cycle G1. Comparing with the control group, There was a statistically significant difference between these two groups(p<0.05).6.the apoptosis obversed via TEM It demonstrated when the wtp53 was incorporated by the UTMB, the apoptotic body appeared in the cells.Conclusion Ultrasound-targeted microbubbles destruction (UTMD)is an promising method for gene delivery。And the cells were induced to apoptosis after the incorporation of the wtp53.