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Objective Analysis of the effect and the mechanism of adenovirus with down regulation of matrix metalloproteinase inhibitor 1 (TIMP-1) achieved targeting by ultrasound microbubbles combined with ultrasound irradiation for liver fibrosis in rats. Methods Recombinant adenovirus-mediated with down regulation of TIMP-1 gene was constructed and a mixture of recombinant adenovirus and ultrasound contrast agent was prepared.The rat liver fibrosis model was established and divided into 5 groups : model group; adenovirus group (recombinant adenovirus); adenovirus microbubble group (mixture of recombinant adenovirus and ultrasound contrast agent); experimental group (ultrasound irradiation + mixture of recombinant adenovirus and ultrasound contrast agent); ultrasound adenovirus group (ultrasound irradiation + recombinant adenovirus). The rats were sacrificed after 24 hours and liver sections were prepared. The expression of EGFP in each group was observed and the transfection rate was analyzed. The liver slices were stained by Masson to judge the stage of liver fibrosis. ANOVA analysis was used to compare the levels of alanine aminotransferase (ALT) , aspartate aminotransferase (AST) , bydroxyproline (Hyp) , hyaluronic acid (HA) , type IV collagen (CIV) and laminin (LN) in each group. The relative expression levels of TIMP-1 and matrix metalloproteinase 13 (MMP-13) in each group were detected by Western blot. Results The transfection rate of the experimental group was higher than that of the adenovirus group (q = 3.418) , the adenovirus microbubble group (q = 3.756) and the ultrasonic adenovirus group (q = 5.502) , and the difference was statistically significant (P < 0.05); Pathological examination showed that the degree of fibrosis in the experimental group and the grade of liver fibrosis were lower than the other groups (P < 0.01). The activities of ALT, AST, HA, LN, CIV and Hyp in the experimental group were lower than those in the other 4 groups.Western blot showed that the level of TIMP-1 protein expression was highest while the level of MMP-13 protein expression was lowest in the experimental group than those in the other groups. Conclusion Adenovirus with down regulation of TIMP-1 achieved targeting by ultrasound microbubbles combined with ultrasound irradiation can inhibit the activity of TIMP-1 and improve the degree of liver fibrosis. Gene therapy is an potential therapeutic method in the application of treating liver fibrosis.
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Objective To prepare a innovative VP3 gene‐loaded ultrasound microbubble decorated with TATp and SDF‐1 , having the extracellular accumulation and intracellular permeation function ,and characterize their property .Methods VP3 gene‐loaded ultrasound microbubbles were prepared with the method of W/O/W double emulsion .SDF‐1 and TATp were covalently con‐gjugated to the surface of poly‐lactic/acid‐glycolic acid(PLGA) microbubble though thioether bonds to prepare gene‐loaded targeted ultrasound mirobubbles .Their particle size ,distribution and surface potential were determined by Malvern measuring instrument . The conjugation status of TATp and SDF‐1 were evaluated by flow cytometry and confocal microscopy .Their DNA protection were identified by digestion reaction test .The vitro targeting capacity was preliminarily assessed by light microscopy and flow cytometry , and the vitro ultrasound imaging was investigated under high frequency imaging condition .Results The gene‐loaded targeted ultra‐sound mirobubbles showed regularly sphericity .The diameter was (536 .00 ± 16 .55)nm ,and showed a narrow distribution .The zeta potential was(-0 .08 ± 0 .08)mV .The average gene loading was 0 .62% ,with the average rate of 36 .13% gene encapsulation effi‐ciency .The DNA protective effect sustained 60 min without damage .Connection rate of TATp and SDF‐1 coupled with PLGA mi‐crobubbles surface was 69 .84% .The vitro targeting study showed that more targeted microbubbles stably clustered together in the tongue SCC‐15 cell membranes with the connection rate of 91 .44% ,while non‐targeted microbubbles combination rate was 12 .96% .Moreover ,the vitro ultrasound imaging was tiny dot ,even high echo .Conclusion TATp‐SDF‐1‐VP3‐PEG‐PLGA micro‐bubbles were prepared successfully ,which can efficiently target to tongue SCC‐15 cells ,and pass through the cell membranes at a short time in company with outstanding ultrasound imagings in vitro .
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Objective To construct eukaryotic expression vector encoding rat IMD gene and deliver it into rat renal tissue via ultrasound-mircobubbles. Methods IMD gene was inserted into pCDNA3.1 ( + )between Hind Ⅲ and EcoRI enzyme sites. The recombinant plasmid designated as IMD-pCDNA 3.1 wasconfirmed by restrictive enzyme digestion and sequencing. Eighteen male Wistar rats were randomized into 3groups, which were treated with no transfection, empty vector transfection and IMD transfection, respectively, in renal tissue via ultrasound-microbubbles. RT-PCR and Western blotting were applied to detect the expression level of IMD. Results Enzyme- digestion and sequencing data showed that IMD-pCDNA 3.1 was correctly constructed. The differences in ALT, AST, BUN and SCr were not significant; No obvious damage in the glomerular, tubular and interstitial was observed in all the treated groups;Compared with non-transfection group and empty vector-transfection group, IMD mRNA and protein expression in IMD transgenic renal tissue were significantly increased. Conclusion IMD-pCDNA 3.1 expression vector was successfully constructed and well expressed in rat kidney.
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Background The key premise of genetic research and treatment is to select the desired gene vector,ultrasound microbubbles as a new type of gene vector can safe,fast and effectively enhance the gene transfection and expression by reversibility increasing the permeability of cells. Objective This study was to observe the effects of ciliary neurotrophic factor(CNTF)gene mediated by ultrasound microbubbles intraocular transfer on visual function and retinal ganglion cell(BGCs)after optic nerve injury. Methods Fifty-five adult Sprague-Dawley(SD)rats were divided into 6 groups randomly,including normal control group(n=5),sham injury group(n=10),simple injury group(n=10),naked plasmid group(n=10),plasmid+ultrasonic irradiation group(n=10)and ultrasound microbubbles group(n=10).The model of optic nerve injury Was made by forceps clip on the fight optic nerve.and the corresponding intervene was performed in different groups.Flash visual evoked potential(F
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Objective:To investigate the effect of inhibition of vascular endothelial growth factor(VEGF) antisense oligodeoxynucleotides(ASODN) on human bladder carcinoma in nude mice mediated by ultrasound microbubble. Methods:The mice were randomly divided into 3 groups when transplantation tumor was established: For UM+ASODN group, mixture of microbubble and VEGF-ASODN was inject into every mice from caudal vein plus ultrasound tumor local irradiation. For UM group, microbubble was inject into very mice from caudal vein plus local irradiation with the same condition. For control group, physiological saline was inject into every mice from caudal vein.The treatment was performed every 3 days with total 5 times.The volume change of mouse subcutane tumors was observed.Evaluate the expression of VEGF and CD34 and Ki67 with immunohistochemistry dyeing and calculate the microvessel density (MVD) and cell proliferation index;TUNEL assay was used to analyse the apoptosis of tumor cells. Results:The growth speed of nude mice tumor in UM+ASODN group was obviously slower than control group; The expression of VEGF and The MVD in UM+ ASODN group was lower than control group(P