The Study Of Anti-tumor Effect Of Ultrasound Mediated Microbubbles Carrying Suicide Gene Against Ovarian Carcinoma Cell

The ovarian carcinoma's case fatality is more higher than other gynecology tumors, in the latest 20 years, although it made much progress in the therapy for this carcinoma, the 5-year survival rate of patient in advanced stage remained in 15%-30%. The ovarian carcinoma is a neoplasm which formed by the cells lost the normal control from the gene level and lead to the paraplasm induced by many kinds of high risk factors. Its etiopathogenisis is still unknown. Usually the growth of organism cell is regulated dually by the proto-oncogene and antioncogene. Under the normal conditions, the growth of cells keeps balance synergistically regulated by these two kinds of gene. This balance is broken if the function of antioncogene is weaken or the proto-oncogene is activated, then the carcinomas occur. With the development of molecular biology and gene technology, the gene therapy has provided one kind of new treatment strategy for the ovarian carcinoma patient. But it is limited greatly to apply the gene therapy on clinical because still lacked one kind of safe,stable,high effective gene transfection method at present. Recently, study suggested that ultrasound-targeted microbubble destruction (UTMD) is a new type of non-invasive gene transfection technology. The profound study on UTMD mediated gene target theropy is expected to supply a new,non-invasive,high effective,targeted and safe method.Firstly, we construct a co-expression vector of enhanced green fluorescent protein(EGFP)and HSV1TK;then, we analyzed the possibility of microbubble carrying suicide gene to transfect ovarian carcinoma cell lines SKOV3 and screened the optimal ultrasound parameters;finally, the vitro expriment is performed to study inhibition the proliferation and induction the apoptosis of microbubble carrying suicide gene to transfect ovarian carcinoma cell,the expression of HSV1TK gene after transfection in ovary carcinoma cells was obsered and the casualty effect of suicide gene on ovary carcinoma cells was investigated, which can provide the reliable experiment basis for studying and evaluating the application of UTMD induced suicide gene to transfect ovarian carcinoma cells, Thus providing a new way and method for the ovarian carcinoma gene therapy。Three parts are included in the present study. Part one Construction co-expression vector of enhanced green fluorescent protein and HSV1TK and expression in ovarian carcinoma cells SKOV3Objective:To construct EGFP and HSV1TK co-expression vector and to detect its expression and function in eukaryocyte ovarian carcinoma cells SKOV3.Methods:The PCR products HSV1TK were inserted into cloning vector pMD18-T to construct the plasmid of pMD18/ HSV1TK.The HSV1TK gene fragment obtained from pMD18T/ HSV1TK that was digested,and then inserted into pcDNA3.1-EGFP. The recombinant pcDNA3.1-EGFP/ HSV1TK was identified with restriction analysis and DNA sequence. The expression plasmid pcDNA3.1-EGFP/ HSV1TK was transfected into ovarian carcinoma cells SKOV3 mediated by liposome reagent, then the expressions of EGFP in cells were observed by fluorescence microscopy and HSV1TK was detected with RT-PCR. The killing SKOV3 effect and bystander effect of HSV1TK/GCV is observed by MTT and light microscope.Results:The sequence of the cloned DNA fragment was identical to HSV1TK that was reported on Gene bank, and the HSV1TK gene was inserted into eukaryotic expression vector pcDNA3.1-EGFP correctly. The recombinant expression plasmid was successfully transferred into ovarian carcinoma cells SKOV3,then observed by fluorescent microscope and effective expression of HSV1TK was also testified by RT-PCR. We can observe the killing SKOV3 effect and bystander effect of HSV1TK/GCV obviously by MTT and light microscope.Conclusion:The recombinant eukaryotic co-expression vector of EGFP and HSV1TK was successfully constructed and effectively expressed in ovarian carcinoma cells SKOV3. HSV1TK /GCV system has better killing effects and bystander effect in SKOV3 cells.Part two Ultrasound parameters and transfection efficiency of microbubble carrying suicide gene for transfecting ovarian carcinoma cell linesObjective:To investigate the ultrasound parameters and transfection efficiency of microbubble carrying herpes simplex virus thymidine kinase suicide gene(HSV1TK)for transfecting SKOV3 cells mediated by the ultrasound.Methods:Under the combination conditions of different ultrasound exposure times (8,15,30,and 60s with the interval as 1s) and microbubbles at different concentrations, SKOV3 cells were exposed to the ultrasound so as to screen out the optimal ultrasound exposure time and microbubble concentration by MTT assays. The SKOV3 cells were divided into 6 groups and treated by ultrasound, ultrasound combined with liposome, ultrasound combined with microbubbles and liposome, and ultrasound combined with microbubbles respectively. The groups of negative control and positive contro1 were treated by naked plasmid and liposome respectively, and then they were used to transfect plasmids pcDNA3.1-EGFP/ HSV1TK. The transfection efficiencies were observed qualitatively by fluorescence microscope and quantitatively by flow cytometry (FCM) .The expression of HSV1TK was detected by reverse transcription polymerase chain reaction (RT-PCR).Results:MTT assays showed that the transfection had no significant inhibition to cell viability under the conditions of the microbubble concentration (0.5 W/cm2), frequency (1 MHz), microbubble concentration (5.6×107/ml), and the interval of every 8 s ultrasound exposure time (1s). Under the fluorescence microscope, the green fluorescence intensity for the group of ultrasound combined with microbubbles and liposome was the greatest. The analysis of FCM showed that the transfection efficiency in group of ultrasound and microbubbles was higher than that in group of ultrasound〔(11.74±0.19)% vs(2.19±0.22)%〕.Furthermore, the transfection efficiency in group of ultrasound combined with microbubbles and liposome was(25.62±0.08)%, which was the highest among all groups (P<0.05). The detection of RT-PCR showed the expression of HSV1TK in group of ultrasound combined with microbubbles and liposome was the highest among the groups (P<0.05).Conclusion: Under the conditions of the optimal ultrasound parameters,ultrasound- mediated microbubbles can not only independently promote the hsv1tk gene transfection, but also strengthen the transfection efficiency of liposome. Meanwhile it can also be effectively expressed in SKOV3.PART three Study of anti-tumor effects in ultrasound microbubbles mediated suicide gene system on ovarian carcinoma cellsObjective:To study the anti-tumor effects in ultrasound microbubbles mediated suicide gene system on ovarian carcinoma cells.Methods:under the conditions of optimal ultrasound transfection parameters,single ultrasound or ultrasound combined with liposome mediated carrying suicide gene microbubbles,pORF-HSV1TK plasmid was transfected into the SKOV3 cells. RT-PCR was used to detect the expression of HSV1TK mRNA in SKOV3; After transfected SKOV3 cells co-culture with different concentrations of the prodrug ganciclovir (GCV), MTT was used to assay inhibition rates of proliferation in transfected cells,and the inhibition rate changes of proliferation over time at optimal concentration of GCV. Light microscope was used to observe changes of quantity and morphology of transfected cells at optimal concentration of GCV for each group; Flow cytometry (FCM) was used to detect the rate of early apoptosis and cell cycle of SKOV3 cells in each group.Results:The expression of HSV1TK mRNA was found in each transfected group by RT-PCR (except the negative control group), which is most in group of the ultrasound irradiation microbubbles combined with liposome; The inhibition rates of proliferation induced by HSV1TK/GCV to SKOV3 cells is stronger in group of the ultrasound irradiation microbubbles combined with liposome than that in other groups between the concentration of 10-100μg/ml of GCV;In each group, the larger GCV dosage was, the higher inhibition rates of proliferation was. After administrating GCV of 100μg/ml, during the course of 24-48h the inhibition rates of proliferation increased with the increase of time in each group,which is stronger in group of the ultrasound irradiation microbubbles combined with liposome than others; Compare with other groups, SKOV3 cells in group of the ultrasound irradiation microbubbles combined with liposome decreased significantly and abnomal cells are more by light microscope. The rate of early apoptosis of SKOV3 cells in group of the ultrasound irradiation microbubbles combined with liposome is (49.13±0.82)%, most of their cells cycle are in G1 phase.Conclusion : Ultrasound-mediated microbubbles can carry out anti-tumor effect on transfected SKOV3 cells in HSV1TK / GCV system by promoting to suppress the proliferation and induce early apoptosis of the cells, and the cells cycle was blocked in the G1 phase.