| Atherosclerosis (AS), a kind of general arterial disease, is a leading dangerous disease over the world with high morbidity and mortality. The etiopathogenisis of this disease, however, need to be further determined, which leads to unsatisfactory curative effect. Therefore, the study of its etiopathogenisis and effective treatment is very significant. Some research showed that gene therapy had extensive perspective and offered a promising new approach for the treatment of AS. In our study, we tried to transduce bone marrow endothelial progenitor cells (EPCs) with recombinant adeno-associated virus encoding human paraoxonase-1 (rAAV-PON1), and compare the effect of transplating EPCs transduced by rAAV-PON1 (PON 1/EPCs), EPCs transduced by rAAV-GFP (GFP/EPCs) and rAAV-PON1 on AS.1. Isolation and culture of bone marrow endothelial progenitor cellsRat bone marrow-derived EPCs were isolated and purified by density gradient centrifugation and direct adherence. The phenotype of endothelial progenitor cells was observed with cell morphology, staining with fluorescein isothiocyanate-labeled vWF, Flk-1 and incorporation of Dil-Ac-LDL. The clonogenic and proliferative potential of EPCs was observed by culture of different cell number and different passages. These results suggested that the cultured cells were positive for vWF and Flk-1, and also could incorporate Dil-Ac-LDL. EPCs have high proliferative potency. The endothelial cell colonies could be formed when the cells were plated in low density. There was a linear relationship between cell number and colonies formation number.2. The establishment of AS rat modelForty male healthy SD rats were randomly divided into 5 groups. The control group (A) rats were fed with basic food, while in other 4 groups were loaded with high fat diet for 3 months. The levels of plasma total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (FIDL-C) and low density Hpoprotein-cholesterol (LDL-C) were detennined with biochemical techniques each month. The pathological and ultrasonic changes in the aorta were determined after 3 months. The results showed that no changes were observed in the aorta of control group with ultrasound and pathology, while the atherosclerotic plaque presented in the aorta of experimental group accompanied with the obvious decrease of serum lipid after 3 months.3. The method of Packing and purify rAAV-PONl-IRES-GFPIn our study, the 1068bp fragment of PON 1 was produced from human fetal liver by RT-PCR. The human P0N1 cDNA fragment was cloned into the multiple cloning site of pAAV-IRES-GFP. The plasmid pAAV-PONl-IRES-GFP (pAAV-PONl) was identified by digesting with restriction enzyme and sequence analysis. The recombinant AAV-PONl-IRES-GFP (rAAV-PONl) was packaged through cotransfecting HEK293 cells with plasmid pAAV-PONl, pAAV-RC and pHelper, and purified with the method single-step column purification (SSCP). The titer of rAAV was 1.77×1010v.g/ml, which was determined by quantitative DNA dot blots. The high purity of rAAV-PONl was achieved, which was determined by SDS-PAGE. We measured the transduction efficiency of EPCs infected with different titers rAAV-PONl particles per cell, and 1×105 rAAV-PONl particles per cell was optimal for transduction experiment by counting green fluorescent cells under the inverted fluorescent microscope. And RT-PCR analysis demonstrated that PON1 mRNA was expressed in PON 1-transduced EPCs.4. Comparison of the effect of PONl/EPCs, GFP/EPCs and rAAV-PONl on AS in vivoIn order to compare the effect of PONl/EPCs, GFP/EPCs and rAAV-PONl on AS, the animal experiment in vivo was performed. In our study, PONl/EPCs, GFP/EPCs and rAAV-PONl were transplanted separately to the AS model of rats via the tail vein. After 2 months, the following tests were performed:the level of serum lipid, HE staining, frozen section for GFP-labeled cells; RT-PCR for eNOS, ICAM-1 and apoE in artery vessel and c-reaction protein in liver; the immunohistochemistry for eNOS, ICAM-1, apoE and ERK on artery vessel; and Western blot method for the levels of human PON1 protein of the aorta. The effects of PONl/EPCs, GFP/EPCs or rAAV-PONl on AS in rats were compared. The results showed that the labeled EPCs were found in the endothelial monolayer of artery vessel and the livers of the PONl/EPCs-treated group and the GFP/EPCs-treated, as well as the livers of the rAAV-PON1-treated group. The level of serum lipid was obviously lower in PON1/EPCs-treated group and rAAV-PON1-treated group than the untreated. The pathological and ultrasonic analysis showed a reduction of atherosclerosis at the aortic arch level in the PON1/EPCs-treated group and rAAV-PON1-treated group compared with the untreated group, in particular in the PON1/EPCs-treated group. The lipid deposits in aortic endothelium in the GFP/EPCs-treated group were less than those in the untreated group. RT-PCR analysis of aortas revealed the eNOS mRNA expression in each treated group increased significantly compared with the untreated group, especially in PON1/EPCs-treated group. ICAM-1, apoE and c-reaction protein mRNA expression levels in the untreated group were significantly higher than those of control group and each treated group. The results of immunohistochemistry were in accordance with the results of RT-PCR. The expression of ERK was decreased in each treated group. And Western-blot analysis detected the human PON1 protein in aortas of PONl/EPCs-treated group and rAAV-PON1-treated group. Taken together, all these results demonstrated that the curative effect of PONl/EPCs-treated group was more obviously than rAAV-PONl-treated group and GFP/EPCs-treated group.Conclusion:EPCs were successfully isolated and cultured in vitro. PON1 were expressed successfully in PON 1-transduced EPCs mediated by rAAV. The AS model of rats was established successfully. The effective therapy for AS was showed in PONl/EPCs-treated group, rAAV-PON1-treated group and GFP/EPCs-treated group, but the effect of EPCs transduced with PON1 gene was more obvious than the other groups.
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