Basical Research Of P53 Plasmid Transfected Into HELA Cells By Ultrasound Microbubble Intensifier

Cervical cancer is one of the commonest malignant tumor in female.The incidence of cervical cancer is only lower than which of cervicalbreast cancer,holding Number 2.Nowadays,the therapy methods mainly includes: operation, radiotherapy,chemotherapy, immunotherapy ,thermotherapy et al. Gene therapy for cervical cancer is still at the starting research stage. As a new gene vector,ultrasound microbubbles are able to complete gene transfection securely,efficiently and targetably. In the study, we detected the transfection efficiency of wtp53 in HeLa cells that was mediated by ultrasound-microbubble and explored the effect of wtp53 in HeLa cells.PART ONE THE INFLUENCE OF THE DENSITY OF MICROBUBBLE TO HELA CELLSObjective To explore the influence of the density of microbubble to HeLa cells ,basing on fixed intensity of ultrasound,in order to get the optimal parameter for following research. Method Divided HeLa cells into 8 groups,four of which were given the density of microbubble 1%,5%,10%,20% respectively and ultrasound irradiation 0.5W/cm2,30s.The other groups were given the same densities of microbubble but different ultrasound irradiation 0.75W/cm2 ,30s. The inhibitory effects of different ultrasound microbubble condition on Hela cells was detected by MTT assay.Results The survival rates of HeLa cells,with the density of microbubble1%,5%,10%,20%,exposed to ultrasound 0.5W/cm2,30s,were respectively (94.25±0.76)%,(86.78±0.87)%,(82.41±1.81)%,(62.72±1.71)%. The survival rates of HeLa cells, with the same densities of microbubble,exposed to ultrasound 0.75W/cm2,30s,were respectively(92.59±1.32)%,(84.00±0.69)%,(75.74±0.44)%,(59.35±0.58)%.Conclusion Ultrasound-microbubble could inhibit Hela cells in vitro. To accompany ultrasound intensity enhancing and microbubble density increasing ,the inhibitory action to the HeLa cells would strengthen.we chose the optimal parameter(ultrasound intensity 0.5 W/cm2,30s, microbubble density10%)for following research.PART TWO THE DIFFERENCE OF P53 TRANSFECTION EFFICIENCY BY DIFFERENT GENE VECTORSObjective To transfer p53 gene into Hela cells by ultrasound microbubble .And then compare the transfection efficiency with that of passed method.Method HeLa cells were divided into five groups and each group was transfected by different means: (A) simple plasmid group,(B)plasmid-ultrasound group,(C)plasmid-ultrasound-microbubble group,(D) plasmid-liposome group,(E)blank control group.At 24-48h after transfection, positive expression rate of EGFP was detected by fluorescence microscopy and the mRNA expression of p53 was detected by semi-quantitative RT-PCR.Result1.Fluorescence microscopy shows that the expression of green fluorescent protein in C group was higher than that in B,D groups(P<0.05),and there were no green fluorescent cells in A group and E group.2.RT-PCR confirmed that the specific p53 electrophoresis strips were seen in C group and D group. The optical density of the strip from C group was higher than that of D group (P<0.05).There were no the specific strips in other groups .Conclusion The transfer of foreign p53 gene into HeLa cells could be enhanced by proper density of microbubble and optimal ultrasound action . As a new gene vector,ultrasound-microbubble can raise the transfection efficiency compared with traditional methods. PART THREE THE GROUTH ACTIVITY OF HELA CELLS AFTER P53 PLASMID TRANSFECTED BY DIFFERENT METHODSObjective To study the influencen to the grouth activity of HeLa cells,after p53 plasmid being transfected by different methods.Methods HeLa cells were divided into five groups and each group was transfected by different means: (A) simple plasmid group,(B) plasmid-ultrasound group,(C)plasmid-ultrasound-microbubble group,(D) plasmid-liposome group,(E)blank control group.At 24-48h after transfection, the inhibition rate of cells growth were determined by MTT assay and the cell cycle were detected by flow eytometry.Results1. MTT showed that cells of each group were inhibited ,at 24,48,72,96hours after transfection .The growth inhibition of plasmid-ultrasound-microbubble group cells was the most obvious.2. The cell cycles of transferred p53 cells was arrested from G1 to S phase in group C and group D.Compared with blank control group ,the differences have statistical significance. Compared group C with group D ,the difference was not obvious.Conclusion Wtp53 has obviously antiblastic effect towards HeLa cells and the inhibited effect was the most significant after transfection mediated by ultrasound-microbubble. The cell cycles of transferred wtp53 cells could be arrested in G1 stage, The two methods,mediated by ultrasound microbubbble and liposome,both were able to make the cell cycles of transferred wtp53 cells be arrested in G1 stage.