| Ischemic cardiomyopathy（ICM）is an common kind of heart disease and theleading cause of endangering the health and life of human. Nowadays conventionaltreatment for ICM includes medical therapy, surgical therapy and Interventionaltherapy. However, a proportion of patients have little curative effect because ofrefractory ischemic cardiomyopathy. With the development of gene therapy, thealternative approach of delivering potent angiogenic factors to stimulate new vesselgrowth has undergone intense investigation over the past decade.After many years research, gene therapy has good application prospect in diseasetreatment. However, successful gene therapy depends the development of safety andhigh efficiency gene carriers and gene delivery approaches. Virus vector is apromising strategy for gene delivery. Although viral vectors have been demonstratedhigh transfection efficiency, its safety is always the difficulties of gene therapy.Maybe non-virus vectors have a better application prospect than virus vectors becauseit is safe, vast and easily prepared. However, its transfection efficiency is low. Hence,safety and high efficiency gene transfection approaches is a hot area of research.Present study shows that ultrasound contrast agent which may be effective carrier ofgene have great potential in treatment. The effects of ultrasound contrast agent intreatment of diseases have made gradual improvement. Ultrasound microbubble-mediated gene transfection become more widely available. However, ultrasound microbubble-mediated gene transfection into stem cells, epidermal cells, myocardialcells and different kinds of tissues and organs has no nice effect. PEI is a targetingno-virus vector and can increase transfection efficiency. Hence, we choice the genetransfection mediated by ultrasound-targeted microbubble destruction and PEIsilences the expression of PHD2and induces angiogenesis factor expression.Although a lot of research demonstrate the feasibility and effectiveness ofultrasound microbubble-mediated gene transfection, ultrasound microbubble-mediate-d gene transfection can induce cells and tissues injury in the transient cavitation effect.Therefore, ultrasound microbubble-mediated gene transfection need optimize parame-ters in order to obtain high transfection efficiency with minimal loss in cell viability.The objective of this experiment is optimizing ultrasonic irradiation parameters, andultrasound microbubble-mediated PHD2-shRNA plasmid transfer normoxia andanoxic H9C2cells in this relative optimal transfection parameter to demonstrate thedown-regulation of the gene expression PHD2by PHD2-shRNA can enhance theexpression of HIF-1a and the expression of HIF-1a can affect the expression of thedownstream angiogenesis factors. It is prepared for the treatment of rat myocardialinfarction. The present study is divided into two parts as follows. Part1. In Vitro Studies on the optimal parameters of EGFP genetransfection in H9C2cells mediated by ultrasound-targetedmicrobubble destruction and PEIObjective To study the contribution of different parameters of EGFP genetransfection in H9C2cells mediated by ultrasound-targeted microbubble destructionand PEI，and to optimize ultrasonic irradiation parameters in order to obtain hightransfection efficiency with minimal loss in cell viability. Methods The H9C2cellswere cultured in24-well dishes and divided into6groups of blank control(C)，ultrasound and microbubbles group(U+M)，PEI group(P)，PEI and ultrasoundgroup(P+U)，PEI and microbubbles(P+M)，PEI plus ultrasound and microbubblesgroup(P+U+M)．At12hours,24hours,48hours and72hours after transfection，theEGFP gene expression in H9C2cells was detected by fluorescence microscopy. Findthe group that expresses fluorescence protein most. Then this group set differentultrasound parameters，including US intensity, microbubbles concentration, exposuretime and plasmid concentration that would affect transfection efficiency of EGFP andcell viability of H9C2cells. The trend of EGFP transfection efficiency was measuredby flow cytometer and cell viability was measured by Trypan blue assays. And theoptimal exposure protocols were determined finally. Results The group of P+U+Mexpressed the most fluorescence protein at48hours. Then this group was set differentparameters. Fix exposure time and plasmid concentration, EGFP transfectionefficiency would be significantly increased if the intensity of ultrasound andmicrobubble concentration were enhanced. However, if intensity of ultrasound is over1.5W／cm2and microbubbles concentration over30%, gene expression would notincrease significantly or could even lower down, and coupled with obviouslydecreased cells viability(P<0.01). Fix intensity(1.5W／cm2), microbubblesconcentration(30%)、Plasmid concentration（8μg／ml）, change exposure time（15s、 30s、45s、60s），we find that when exposure time achieve30s, gene expression wouldbe higher compared with other exposure time. When fix microbubblesconcentration(30%)，US intensity (1.5W／cm2) and exposure time（30s）, EGFPtransfection efficiency would be significantly increased if the Plasmid concentrationwere increased. However, if plasmid concentration is over16μg/ml, gene expressionwould not increase significantly. Increasing plasmid concentration cannot affect thecells viability. Conclusion Ultrasound-targeted microbubbles destruction and PEI canenhance the efficiency of EGFP gene transfection in H9C2cells and US intensity (1.5W／cm2), microbubbles concentration(30%), exposure time（30s）and plasmidconcentration（16μg／ml）are relative optimal transfection parameters. Part2. The vitro study of PHD2-shRNA gene transfection in H9C2cells mediated by ultrasound-targeted microbubble destruction andPEI silencing the expression of PHD2and inducing angiogenesisfactor expressionObjective After constructing the targeting PHD2-shRNA eukaryotic expressionvector, the PHD2-shRNA gene transfection in H9C2cells mediated byultrasound-targeted microbubbles destruction and PEI study the effect ofPHD2-shRNA on silencing PHD2and the expression of HIF-1α and its downstreamangiogenesis factor(VEGF、TGF-β and bFGF). Methods We construct the targetingPHD2-shRNA eukaryotic expression plasmid and the control plasmid （EGFPplasmid）. Culture normoxia and anoxic H9C2cells, and all H9C2cells are dividedinto four groups:(1) control plasmid normoxia group;（2）PHD2-shRNA plasmidnormoxia group;(3) control plasmid anoxic group;(4) PHD2-shRNA plasmid anoxicgroup. After48hours of the PHD2-shRNA and the control plasmid gene transfectionin H9C2cells mediated by ultrasound-targeted microbubbles destruction and PEI，observe enhanced green fluorescent protein with inverted flurescence microscopy.The protein expression of PHD2、HIF-1α were tested by Western-blot and the mRNAexpression of VEGF、bFGF and TGF-β were measured by RT-PCR. Results Theresults of sequence analysis confirm that the recombinant plasmid is constructedsuccessfully. The enhanced green fluorescent proteins are visible in four groups afterH9C2cells cultured for48hours. The intensity of enhanced green fluorescent proteinin four groups has no difference. Western-blot shows that the PHD2protein bands ofthe PHD2-shRNA plasmid normoxia and anoxic groups are dimmer than thecorresponding control plasmid normoxia and anoxic groups. However, the HIF-1αprotein bands of the PHD2-shRNA plasmid normoxia and anoxic groups are brighterthan the corresponding control plasmid normoxia and anoxic groups. The difference of PHD2／GAPDH and HIF-1α／GAPDH absorbance ratio is significant betweenthe groups. RT-PCR show that the VEGF、TGF-βand bFGF mRNA bands of thePHD2-shRNA plasmid normoxia and anoxic groups are brighter than the correspondcontrol plasmid normoxia and anoxic groups. The expression of VEGF、TGF-β andbFGF is significant between the groups. Conclusion The PHD2-shRNA genetransfection in H9C2cells mediated by ultrasound-targeted microbubbles destructionand PEI can significantly regulate the gene expression of PHD2down and increasethe expression of HIF-1α、VEGF、TGF-β and bFGF. It is prepared for the study ofthe gene therapy for ischemic heart disease in vivo. |
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