Experimental Research On Treatment Of Liver Fibrosis Induced By Hepatocyte Growth Factor Mediated By Ultrasound-Targeted Microbubble Destruction

Liver fibrosis is the final common pathway of most chronic liver diseases of various etiologies. In normal liver this space contains a non electron-dense basement membrane-like matrix which is essential for maintaining the differentiated function of all resident liver cells. As the liver becomes fibrotic, the total content of collagens and non-collagenous components increases and it is accompanied by a shift in the type of extracellular matrix in the subendothelial space from the normal low density basement membrane-like matrix to interstitial type matrix containing fibril-forming collagens (mostly collagens I and III).In recent years it became clear that the fibrotic process is reversible. Gene therapy may provide novel treatment strategies for a wide range of liver fibrosis. However, the lack of a safe and effective method for gene delivery to a specific organ or cell remains an obstacle to clinical application. Recent studies showed that mechanical and cavitation effects caused by ultrasound-targeted microbubble destruction (UTMD) were able to increase membrane permeability and enhance exogenous genetic materials into targeted cells. UTMD is a new noninvasive gene transfer technology, which can provide a new method of gene therapy on liver fibrosis.This study had prepared a ultrasound contrast agent which can load with gene efficiently. And the UTMD was applied in treatment of liver fibrosis induced by HGF in vitro and in vivo, and observed the effect of gene transfection and therapeutic effect of the technique mediated HGF gene treating liver fibrosis. The study would provide reliable experimental evidences for studying and evaluating gene transfer mediated by UTMD, which could be a novel method in gene therapy of liver fibrosis. The study included the following three parts:PARTâ… : EXPERIMENTAL STUDY ON PREPARATION OF LIPID ULTRASOUND MICROBUBBLES LOADED GENEObjective To study the self-made lipid ultrasound contrast agents ability of gene-loaded, and to assess its physical characteristics and its enhancement effects in contrast imaging on normal rabbit liver. Methods Apply the layer-by-layer (L-b-L) assembly technique to attach multiple layers of gene and polyllysine onto preformed lipid-coated microbubbles to increase the gene loading capacity. and its appearance, distribution, concentration, mean diameter, zeta potential and the loaded-gene ability of the microbubbles were detected. Five rabbits were examined to observe the enhancement time and intensity in liver. Results The mean diameter and concentration of the self-made lipid microbubbles loaded polylysine, polylysine and gene, polylysine and gene and polylysine was no significant statistical difference (p<0.05 ). With the increasing of polylysine and gene, It's surface potential was reversed to positive or negative. The amount of adsorbed gene, increased linearly with the fraction of cationic lipid in the shell. Contrast imaging showed that the loaded gene and polyllysine ultrasound microbubbles could significantly enhance the echo intensity of liver. Conclusions The lipid ultrasound microbubbles loaded gene and polyllysine are consistent to the standard of ideal ultrasound contrast agent, and the efficiency of loaded-gene are high. It may become a new vehicle carrying genes or drugs.PARTâ…¡: CONTRAST AGENTS ENHANCED HGF GENE TRANSFECTION IN VITROObjective To study the transfection effects of contrast agents enhanced HGF gene in hepatic stellate cell (HSC-T6). Methods HSC-T6 was seeded in the 24-well plates, and divided into 5 groups. The first group was as contrast group and the second group was given proper dose of plasmid ( pIRES2-EGFP/ HGF). Add loaded-gene contrast agents in the third group. Pulsed-ultrasound exposed liposome was the fourth group. Add loaded-gene contrast agents to the fifth group and exposed to pulsed-ultrasound,too. Expression of the report gene EGFP was observed on fluorescent microscopy and quantified by FACS analysis. The viability of HUVEC was measured using MTT assay. Western Blotting detected the expression of HGF peptide. Results Green fluorescence intensity and transfection efficiency in the fifth group were higher than that of other groups, and cell viability had no significant difference. HGF protein in the fifth group was higher than that of other groups. Conclnsions Ultrasound microbubbles can enhance the efficiency of gene transfection without obvious damage to cell viability in HSC-T6. This method offers a new strategy to increase the transfection efficiency of non-viral genetic vector.PART III: THE TREATMENT OF LIVER FIBROSIS INDUCED BY HEPATOCYTE GROWTH FACTOR DIRECTED ULTRASOUND-TARGETED MICROBUBBLE DESTRUCTION INRATSObjective The purpose of this study was to explore the feasibility of using ultrasound-targeted microbubble destruction to treat liver fibrosis induced by hepatocyte growth factor (HGF). Methods Forty Wistar rats were divided into 5 groups after the models of liver fibrosis were prepared: (1) HGF, ultrasound, and microbubbles (HGF+US/MB), (2) HGF and ultrasound (HGF+US), (3) HGF and microbubbles (HGF+MB), (4) HGF (HGF) and (5) model lone(MA). All rats were killed after being transfected for 14 days. Recovery of the liver was detect by DWI and pathological methods .Collagen I expression was detected by immunohistochemistry. Hepatocyte growth factor expression in the liver was detected by western blotting. Results The results of DWI and Pathological examination showed the recovery of liver in HGF+US/MB group were better than those of other groups. In HGF+US/MB group, collagen I expression was less, and HGF protein was the highest among all the groups. Conclusions Ultrasound-targeted microbubble destruction could deliver HGF into the fibrotic liver and produce an anti-fibrosis effect, which could provide a novel strategy for gene therapy of liver fibrosis.