Ultrasound Microbubble-mediated Targeted Transfection Of Simdr1Improve Chemotherapy Of Testicular Tumor

Testicular cancer is the common cancer diagnosis in genitourinarysystem of children. However, the poor effect of chemotherapy of malignanttesticular tumors is a big problem. Tumor existed in testis tissues with theprotection of blood-testis barrier, so the chemotherapy drug is difficult toreach and play role. Our previous study have found that P-glycoprotein(P-gp) is associated with the multidrug resistance of tumor, P-gp exists incapillary endothelial cells of the blood-testis barrier to form biologicalbarrier, which play important roles in drug resistance. Therefore, how tobreak through the barrier and reversal of drug resistance, has become veryimportant in testicular tumor treatment. In this study, we try to construct aeukaryotic expression plasmid pSEB-siMDR1carrying small interferenceRNA fragment for MDR1. By using gene silencing method, P-gpexpression in endothelial cell was inhibited, so chemotherapy drugs wentthrough the blood-testis barrier and entered into the testicular tissue.However, the efficacy of in vivo targeted gene transfection is still low.Herein the application of ultrasound microbubbles provides a new method for in vivo targeting siMDR1gene transfection and chemotherapyorientation. With intravenous injection, microbubbles carryingpSEB-siMDR1plasmid arrived at the testis through circulation, under theeffect of ultrasound targeted microbubble destruction, siMDR1waseffectively transfected esticular capillary endothelial cells so that inhibitedthe expression of P-gp, providing a good condition for tumor chemotherapy.This method can improve the cure rate of testicular tumor. Based on theabove hypothesis, we set up this research at four parts as follows.PART ONE Construction and functional verification of eukaryoticexpression plasmid carrying simdr1Objective: To construct the eukaryotic expression plasmid carryingsiRNA for MDR1gene and detect inhibition efficiency to screen out theeffective siRNA sequence for later research.Methods: Four pairs of siRNA sequence for rat MDR1gene weredesigned and annealed in vitro, then cloned in pSEB-HUS vector to obtaineukaryotic expression plasmid pSEB-siMDR1, which was verified by usingPCR, endonuclease cutting and gene sequencing.Then pSEB-siMDR1weretransfected into L2ryc dells by Lipfectamine2000, we set up individualfour pSEB-siMDR1transfection, equimolar amounts of three effectivepSEB-siMDR1transfection as pool group and pSEB-HUS vectortransfection as negative control. Results: Specific300bp fragment was amplified by PCR frompSEB-siMDR1, compared with no PCR product from pSEB-HUS vector.By using NotⅠendonuclease cutting, pSEB-HUS wasdigested into1321bpand5000bp of DNA fragment,while only7000bp of DNA fragment wasdigested from recombinant pSEB-siMDR1.Four siRNA specific for MDR1were clarified to be correctly cloned in adenovirus vector by genesequencing. GFP was positive in L2ryc cells with different pSEB-siMDR1transfection groups. The mRNA and protein expression of MDR1inL2RYC were significantly inhibited by three of four pairs of siMDR1srespectively(p<0.05), especially by pool siMDR1s.Conclusion: Successfully construct the recombinant eukaryoticexpression plasmid pSEB-siMDR1, which can effectively inhibit the geneand protein expression of MDR1. The highest silencing efficiency wasobserved in the pooled plasmids group. Therefore, for the followingexperiment, we chose to use the pooled plasmids to transfect cells.PART TWO Ultrasound-microbubble mediated pseb-simdr1transfection reverse drug resistance in yolk sac carcinoma L2cellin vitroSECTION ONE Optimized condition of Ultrasound-microbubbledestruction for gene delivery in vitroObjective: To investigate the main factors in Ultrasound-microbubble destruction for gene delivery and screen out the best condition ofpSEB-siMDR1transfection in L2RYC cells.Methods: lipid microbubbles were prepared with mechanical oscillationtion,pSEB-siMDR1plasmid DNA was packaged on the surface of microbubbleby using polylysine adsorption to made siMDR1-loaded lipid microbubble.Cells were exposed to ultrasound with different radiation frequency andsound intensity at different time period. Trypan blue staining was used todetect cell survival, GFP expression was observed under fluorescentmicroscoped, Transfection efficiency was checked by using flowcytometry.Result: Transfection conditions were different in different cells.Transfection efficiency was improved with increase ultrasound intensityand exposure time, however, exposed to ultrasound intensity of more than0.75W/cm2, cell suvival rates was too low to be used in the study. We foundout ultrasound with intensity of0.5W/cm2for30s could effectivelytransfect plasmids into cells with26%efficiency and90%cell survival.Conclusion: Suitable ultrasonic intensity and exposure time cansignificantly promote plasmid transfection, meanwhile not do obviouseffect on cell survival. SECTION TWO Ultrasound-microbubble destruction promotespSEB-siMDR1transfection for reversing Drug Resistance in Yolk SacCarcinoma L2CellObjective: To study transfection efficiency of siMDR1mediated byultrasound-microbubble in vitro and the inhibition of MDR1geneexpression, P-gp expression and its function.Methods: pSEB-siMDR1or siMDR1-loaded lipid microbubble were addedon cultured cells. Experimental groups were set up as follows:①Plasmid(group Ⅰ),②P lasmid and microbubble (group Ⅱ),③Plasmid andultrasound (group Ⅲ),④P lasmid, microbubble and ultrasound (groupⅣ),⑤negtive control (groupⅤ),⑥Liposome contro(lgroup Lipo), cells wereexposed on ultrasound intensity of0.5W/cm2for30s. transfectionefficiency was detected with flow cytometry. Real-time PCR andWestern-blot were carried out to evaluate mRNA expression of MDR1andprotein expression of P-gp, respectively. Daunorubicin accumulation assaywas used to detect function of P-gp. MTT assay was also performed todetermine cell viability of L2-RYC cells response to Vincristine andDactinomycin.Results: GFP fluorescence was observed only in the fourth group and Lipogroup. Compared with other experimental groups, transfection efficiencyof pSEB-MDR1was30%in the fourth group, the mRNA expression ofMDR1and protein expression of P-gp were inhibited significantly.Intracellular accumulation of Daunorubicin in L2-RYC cells of fourthgroup was much more than other groups. The IC50of Vincristine and Dactinomycin were1.34μg/ml and0.11μg/ml in group Ⅳ which werestatistically different from other groups (P<0.05).Conclusion: Ultrasound-microbubble contrast agent can promote the invitro siMDR1gene transfection in L2RYC cells, and siMDR1caneffectively inhibit the endogenous MDR1mRNA expression, P-gpexpression and its function, while reverse chemical drug resistance ofL2-RYC cells.PART THREE Ultrasound-microbubble destruction promotespSEB-siMDR1targeting transfection in vivoSECTION ONE An animal model of rat testis tumorsObjective: Take yolk sac tumor as an example to establish thepreparation methods for an animal model of rat testicular tumors.Method: Three-week and3-months old male SD rats were selected,cultured rat L2RYC cells were prepared into a cell suspension of1×10~6,1×10~7and1×10~8/ml. In an aseptic condition,10μl cell suspension wasinjected into the right rete testis, normal left rete testis was self-control.Each group contains10rats. Per week measurement of Testicular diameterwas measured per postoperative week, B-ultrasound was performed toobserve the growth of testicular cancer, and testis was extracted for HEstaining and immunohistochemical examination.Results: With injection of cell suspension at the concentration of1×10~8/ml, the testis of both3-week and3-month old rats rapidly growth within1-2weeks. The testis was partly ruptured, internal liquefaction and pathologicalnecrosis. When the injected cell suspension at the concentration of1×107/ml, no testis tumor formed in3-month old rats whereas inoculatedtumors occurred gradually in3-week old rats, after three weeks, the tumorrate was100%. With tumor growth, the body function of rats deterioratedprogressively and died at fourth week.Conclusions: The cell suspension at concentration of1×10~7/ml wasappropriate to construct animal model of rat testicular tumors. Too manytumor cells injection resulted in short-term necrosis in testis, while notumor formation with few cells injection. Prepared rat model of testicularyolk sac tumor has similar histological phenotype compared with clinicaltesticular tumor, which can be used for subsequent experiments.SECTION TWO Ultrasound microbubble-mediated targetedtransfection of pSEB-siMDR1in vivoObjective: To investigate the application of Ultrasound-microbubbledestruction in in vivo transfection of pSEB-siMDR1into rat testis, anddetect the effect of siMDR1on inhibition of MDR1expression in testicularcapillary endothelial cells.Methods: The pSEB-siMDR1-loaded lipid microbubble was injected intorat tail vein, after5min, the testis area was continuously exposed to ultrasound for10mins. Experimental groups were set up as follows:①Plasmid alone (group Ⅰ),②Plasmid and microbubble (group Ⅱ),③Plasmid and ultrasound (group Ⅲ),④Plasmid, microbubble andultrasound (group Ⅳ),⑤negtive control (group Ⅴ). Testis was preparedinto frozen section, immunofluoresence was carried out to detectcolonization site of pSEB-siMDR1plasmid transfection, Western blot andimmunohistochemical were used to detect P-gp localization and semi-quantitative expression. Daunorubicin accumulation test was performed toinvestigate the intracellular accumulation of Daunorubicin in testiculartissue of different treatd groups.Results: After exposed to ultrasound, green fluorescent protein expressionwas visible only in the walls of the capillaries of groupⅣ. MDR1gene andP-gp expression was significantly decreased (P <0.05), the red fluorescenceof daunorubicin in testicular tissue of group Ⅳ accumulated significantlymore than that of other experimental groups.Conclusion: Ultrasound-microbubble destruction could enhancetransfection of pSEB-siMDR1in rat testis capillaries, inhibit P-gpexpression and its drug pump function, providing a more accessible way tocarry chemotherapy drugs into testicular tissue. PART FOUR Ultrasound microbubbles-mediated targetedtransfection of pSEB-siMDR1promote testicular tumorchemotherapyObjective: To investigate whether it can enhance the chemotherapy effectof testis tumor that ultrasonic microbubble-mediated transfection ofpSEB-siMDR1to the testis of tumor-bearing rats.Methods: the modeling rats were separated to four groups:①a singlechemotherapy drug (group A),②microbubbles+ultrasound+chemotherapy drug (group B),③gene contained microbubbles+ultrasound+chemotherapy drugs (Group C),④control group (group D).Tumor growth and survival of tumor-bearing rats were monitoredcontinuously. The rats were sacrificed one week post administration. Testistumor size and weight were measured. Tumor pathological changes wereassessed with H.E. staining. Apoptotic genes expression of Fas and p53were detected by RT-PCR.Results: With the addition of chemotherapy drugs, in group C, tumorgrowth was inhibited and relative testicular volume was significantlysmaller than the other groups. At the same time point, survival was alsohigher than other groups and the tumor cells decreased in group C. Part oftesticular tissues were retained. Apoptotic genes expression of Fas and p53were increased.Conclusion: Ultrasound microbubble-mediated targeted transfection ofpSEB-siMDR1can enhance the chemotherapy effect of testicular tumor,increase death of tumor cells and improved survival of tumor-bearing rats.