| Liver fibrosis is the wound-healing response to various liver damage，in the form of a progressive accumulation of fibrosis．It alters the tissuestructure and function of liver，leading to cirrhosis and liver failure, Genetherapy may provide novel treatment strategies for a wide range of liverfibrosis. HGF was originally characterized as a potent mitogen for maturehepatocytes, has emerged as a potent anti-fibrotic cytokine that preventstissue fibrosis in various organs, including the liver.[，However, the lack of asafe and effective method for gene delivery to a specific organ or cellremains an obstacle to clinical application. Microbubbles are contrast agentsfor medical ultrasound imaging, improve transfection efficiency afterultrasound-induced cavitation, However, microbubbles are generallyunstable and their mean diameter of around1–6μm is too large forintravascular applications. Moreover, functional particles such as targetingmolecules are difficult to modify on the surface of microbubbles., Theirsurface should be easily modified with functional molecules for targeting after injection into the blood and ultimately， Liposomes have someadvantages as drug, antigen and gene delivery carriers, Their size can beeasily controlled and they can be modified to add a targeting function.but thegene expression was very low Therefore, we developed novelBio-MB+Bio-CNLP, Here, we assessed the feasibility ofBio-MB+Bio-CNLP for gene delivery after cavitation induced byultrasound.Chronic diffuse liver disease is a worldwide problem.Whereashistology remains the gold standard by which to diagnose hepatic fibrosis,Several clinical limitations are associated with the use of liver biopsy.It isan invasive and costly procedure prone to complications, sampling error isinherent in the technique, Alternative techniques for assessingthe severityof diffuse liver disease are urgently needed. and such a tool would reducebiopsy related risks and costs, could be useful for guiding anti-fibrotictreatments, monitoring treatment efficacy, and helpful in the clinicalevaluation of new anti-fibrotic drugs. Furthermore, the identification ofoccult advanced fibrosis, may direct further management, and has essentialprognostic implications.We established a bile duct ligation (BDL) rat model of hepatic fibrosis.Bio-MB+Bio-CNLP-pCDH-HGF carrier was administered through tail-veininjections. The gene therapy efficacy was evaluated in vivo. Before and aftertreatment, the rats were examined with diffusion-weighted imaging （DWI）、 Perfusion imaging (PWI) and magnetic aesonancespectroscopy(MRS) respectively. Magnetic resonance functional imagingcould potentially provide a more accurate representation of the diseaseprocess. Recently, in vivo Magnetic resonance functional imaging hasbeen suggested for the evaluation of chronic liver disease and discuss thevalue of MR functional imaging in early diagnosis、quantization analysisand staging of hepatic fibrosis by analyzing the changes of perfusionparameters of MR functional imaging of different degree of hepaticfibrosisPart I Enhanced effect of HGF on liver cell growth with specialemphasis on hepatic stellate cells by a novel modified ultrasoundtargeted microbubble gene delivery systemPurpose: To evaluate the efficacy and safetyof human hepatocytegrowth factor (HGF) delivery by a novel ultrasound targeted microbubblesystemusing avidin to crosslink biotinylated ultrasound microbubbles(Bio-MB) and biotinylated cationic nano-liposomes (Bio-CNLP), which isreferred to as Bio-MB+Bio-CNLP.Methods: HGF was linked to Bio-MB+Bio-CNLP, and transfected tohepatic stellate cells (HSC-T6) and normal liver cell line L02according tothe following four groups:(1) Bio-MB+Bio-CNLP+HGF, no ultrasonicirradiation group;(2) Bio-MB+Bio-CNLP+HGF+ultrasonic irradiation group;(3) liposomes+HGF;(4) Bio-MB+Bio-CNLP+ultrasonicirradiation group. Protein expression, L02cell viability, and HSC-T6cellapoptosis rate were determined respectively.Results: Compared with liposome mediated transfection, Bio-MB+Bio-CNLP mediated transfection rate increased markedly, with the highestprotein expression in HGF transfected HSC-T6cells at72hours aftertransfection. Compared with mock transfection control group, the apoptosisrate was32.8%and7.5%respectively. Cell viability significantly increasedin HGF transfected L02compared with normal L02.Conclusion: Introducing HGF using an ultrasound-mediated Bio-MB+Bio-CNLP system improved gene transfection rate and promoted normalliver cell growth. This appears to be a promising approach for targetedtherapy.Part Ⅱ Efficacy of HGF for treating hepatic fibrosis and itsrelationship with MRI functional imaging parametersObject: The aims of the present study were: i) to explore the interest oftreating liver fibrosisusing human hepatocyte growth factor (HGF)expression vector carried by a novel ultrasound targeted microbubbledelivery system.; b) to explore the diagnostic interest of MR functionalimaging parameters in evaluating liver fibrosisMethod: We established a rat model of hepatic fibrosis by bile duct ligation. The rats were administered HGF linked to biotynilated ultrasoundmicrobubbles and biotynilated cationic nano-liposomes, and compared tosham-operated animals. The impact on hepatic fibrosis was evaluated byhisto-pathology, hydroxyproline content, with special focus on MRfunctional imaging parameters.Result: Our targeted gene therapy produced a significant anti-fibrosiseffect, as shown by liver histology and decreased hepatic collagen content.Moreover, proved of interest as indicated by the respective evolution of MRfunctional imaging parameters.Conclusion: This is the first in vivo report of using ultrasoundmicrobubble-cationic nanoliposomes complex for gene delivery. The dataindicate that, in our experimental model, this approach is efficient tocounteract the fibrosis process. MRI functional imaging parameters appearsa promising imaging technique for reflecting liver fibrosis.. |
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