| Part ?Background:Expression of foreign gene would be enhanced and prolonged by sustained releasing plasmid DNA (pDNA) complexes from biodegradable materials. In our previous study, a biodegradable thermoresponsive dextran-poly(e-caprolactone)-2-hydroxylethylmethacrylate-poly(N-isopropylacrylamide)(Dex-PCL-HEMA/PNIPAAm) hydrogel was synthesized. Moreover, it has been demonstrated that expression of foreign gene was enhanced and prolonged by sustained releasing a target gene to cells from the same hydrogel, but the pDNA-loaded hydrogel did not direct contact with the target cells in the previous study. The interaction between hydogel and cells is important for the gene transfer. However, it was still unclear whether gene transfer could be achieved by directly seeding cells on the top of DNA-loaded Dex-PCL-HEMA/PNIPAAm hydrogel.Objective:This study was designed to investigate whether gene transfer could be achieved by seeding cells on the top of pDNA-loaded Dex-PCL-HEMA/PNIPAAm hydrogel and whether the concentration of hydrogel would influence gene delivery.Methods:Hydrodel solutions were prepared by mixing the hydrogel in phosphate buffer solution (PBS, pH7.4and1.0wt%,1.5wt%or3.0wt%). The morphology of hydrogel was investigated by scanning electron microscopy. Then the cumulative release of pDNA from the hydrogel was calculated. H9C2cells were seeded onto the pDNA-loaded gels with hydrogel contents of1.0wt%,1.5wt%or3.0wt%, respectively. Cell viability was determined by the cell counting kit (CCK)-8assay after transfection for48h. Flow cytometry analysis was employed to quantify the percentage of EGFP-expressing cells following pDNA/EGFP transfection. H9C2cells were harvested for EGFP assay at7d and14d post-transfection.Results:The pore size of lower concentration gel is significant larger than that of higher concentration gel (P<0.05). The rate of pDNA release from1.5wt%gel was lower than that from1.0wt%gel (P<0.05), but it was increased as compared to3.0wt%gel (P<0.05). The cytotoxcity induced by lipoplexes was significantly decreased by encapsulating the complexes in hydrogel (P<0.05). Plasmid-loaded1.5wt%gel mediated a higher expression of EGFP in comparison with the other two concentration of the hydrogels after H9C2cells were transfected at37?for7days and14days (P<0.05).Conclusion:Gene transfer could be achieved by directly seeding H9C2cells on the top of pDNA-loaded Dex-PCL-HEMA/PNIPAAm gel. Moreover, the expression of transgene is regulated via changing the concentration of Dex-PCL-HEMA/PNIPAAm hydrogel which affects the morphological structure of the gel and the pDNA release. So the appropriate concentration of gel may be important for gene transfer.Part IIBackground:Although the ventricular remodeling and dysfunction after AMI are considerably improved by the restoration of blood flow, reperfusion itself results in additional myocardial ischemia-reperfusion (I/R) injury. Angiotensin converting enzyme inhibitors (ACEI) attenuated apoptotic cardiomyocytes induced by I/R. However, it seems that the effect of angiotensin converting enzyme (ACE) can not be completely blocked because ACEI may up-regulate ACE level in myocardium and plasma through a feedback mechanism. In order to avoid the feedback elevation of ACE during using of ACEI and more completely block the effects of ACE, silencing of ACE gene by RNA interference (RNAi) may be a better selection. plasmids containing short-hairpin RNA (shRNA) could induce the silencing of target gene by RNAi. Moreover, it is difficult to evaluate the effects of inhibiting intracellular ACE in vivo study.Objective:The objective of this study was to demonstrate whether the apoptosis in H9C2cardiomyocytes following anoxia/reoxygenation (A/R) would be improved via silencing of intracellular ACE by RNAi.Methods:The cultured H9C2cardiomyocytes were randomly divided into different groups. In the control (Con) group, the H9C2cardiomyocytes were cultured under normal conditions for12h. The A/R group was operated as described in the preceding section. In the negative control (NC) group and the ACE-shRNA plasmid-treated group (shRNA group), the H9C2cardiomyocytes subjected to A/R48h after being transfected with negative control ACE-shRNA plasmid or ACE-shRNA plasmid respectively. Cell viability, caspase-3activity, and apoptosis were analyzed by the CCK-8assay, colorimetric method and flow cytometry, respectively. The mRNA and protein of ACE were detected by quantitative real-time RT-PCR and western blot analysis. ACE2, Bcl-2and Bax protein were also examined by western blot analysis. The levels of Ang? in the culture medium were assessed by enzyme-linked immunosorbent assay.Results:Forty-eight hours after ACE-shRNA plasmids transfection,76.8%±5.1%H9C2cardiomyocytes were positive for green fluorescence. There were no significant differences in viability between experimental groups before A/R (P>0.05). Compared with control group, in A/R group cell viability was significantly decreased, the expression of ACE and ACE2?the level of Ang ??apoptotic H9C2cardiomyocytes and caspase-3activity were significantly increased,and the Bax?Bcl-2and Bax/Bcl-2ratio were also significantly increased after A/R (P<0.05). Compared with A/R group, in shRNA group cell viability was significantly increased, the expression of ACE and Bax?the level of Ang ??apoptotic H9C2cardiomyocytes?caspase-3activity and Bax/Bcl-2ratio were significantly decreased, and the Bcl-2protein and ACE2protein were significantly increased after A/R (P<0.05). However, these results had not obvious difference between BPS group and NC group (P>0.05).Conclusion:Gene silencing of intracellular ACE holds great potential in treatment of cardiomyocyte apoptosis after I/R injury by regulating the intracellular RAS, consequently regulating the intrinsic pathway of apoptosis.Part IIIBackground:Although non-viral gene therapy using pDNA encoding for target genes is safer and cheaper than viral gene therapy, its transfection efficiency is lower and duration of gene expression is shorter. Thus, sustained delivery of pDNA from biodegradable materials is an attractive approach to extend the duration of foreign gene expression. The expression of foreign gene was enhanced and prolonged by sustained releasing a target gene to cells from Dex-PCL-HEMA/PNIPAAm hydrogel in vitro. Moreover, we have demonstrated that injection of the same hydrogel improved post-infarct ventricular remodeling.Objective:To explore whether intramyocardial injection of plasmid containing the ACE-shRNA with the same hydrogel enhances the cardioprotective effects superior to either alone after rat myocardial infarction.Methods:In this study, equal volume PBS, lOug ACE-shRNA plasmids, hydrogel containing lOug negative control ACE-shRNA plasmids and hydrogel containing lOug ACE-shRNA plasmids were shortly injected into the infarct area of rats after myocardial infarction, respectively. At30days after treatment, Fluorescence microscopy was used to detect enhanced green fluorescent protein (EGFP) expression in cells around the injection sites, left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD) and left ventricular fractional shortening (FS) were evaluated by transthoracic echocardiography. The left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP), as well as its first derivative (+dp/dtmax) were measured with a pressure-volume catheter. Infarct size and apoptosis were analyzed by Masson staining and histological TUNEL staining, respectively. Collagen deposition and neovascularization following myocardial infarction were evaluated by histopathological examinations. The mRNA and protein expression of ACE were detected by real-time RT-PCR and western blot analysis. The levels of Ang? in heart were assessed by enzyme-linked immunosorbent assay.Results:Two days after infarction, initial echocardiography showed no significant difference of LVEDD, LVESD and LVFS between the four groups (P>0.05). EGFP was detected in the pDNA+Gel group, while not in the pDNA group30days after myocardial infarction. Direct intramyocardial injection of negative control ACE-shRNA plasmid-loaded hydrogel significantly decreased LVESD and LVEDD, inhibited the expression of local ACE expression and Ang? level, reduced cell apoptosis, infarct size and collagen content, and increased FS and+dp/dtmax compared with the injection of PBS30d after myocardial infarction in rats (P<0.05), and the effects were significantly enhanced by injection of ACE-shRNA plasmid-loaded hydrogel. However, these results had not obvious difference between BPS group and pDNA group (P>0.05). The arterial density in the peri-infarct area is not significant difference between the four groups (P>0.05).Conclusion:Dex-PCL-HEMA/PNIPAAm hydrogel-mediated ACE-shRNA plasmid transfer significantly extended the duration of gene expression in heart. Moreover, the combination of ACE-shRNA plasmids and the hydrogel may have synergistic benefits superior to either alone in rats with myocardial infarction. |
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