Screening For Ultrasound-responsive Elements And The Application In Liver Fibrosis Treatment

With the development of molecular biology,gene therapy has become an important method for treating diseases.The scope of application has become wider and the technical feasibility has improved.By introducing foreign genes,gene therapy enables the cells to express specific proteins,thus performing therapeutic effects.Of all the factors related with gene therapy,gene expression control is a critical one.By controlling the expression level of gene products,we can reduce potential side effects while assuring therapeutic effects.In this way,accident consequences may be prevented.Currently,the major methods used in gene expression control include light-induced gene expression system,tetracycline-induced gene expression system,electromagnetism-induced gene expression system and so forth.Although these methods may work in some extent,they have their own limitations respectively.Take the light-induced gene expression system,which is at the front of research for example,it is able to rapidly induce gene expression,but unable to reach deep tissues and induce gene expression in organs like livers,thus failing to play an effective role.As a kind of noninvasive mechanical wave,ultrasound can penetrate skins and reach tissues deep inside the body.Besides,ultrasound can perform precise orientation.Therefore,to investigate ultrasound-responsive elements and use it as a method of gene expression control is of vital importance in gene therapy for diseases in liver and other deep tissues.Ultrasound is widely used in medicine and main biological effects of ultrasound can be divided as thermal effects and nonthermal effects.By taking advantage of the heat produced by ultrasound to activate promoter HSP70 and induce downstream gene expression is a widely-studied field in spatiotemporal gene expression.However,the temperature required for HSP70 activation is rather high.Normally,obvious elevation of gene expression can be observed with temperatures above 43°C,which often bring damages to surrounding tissues.In recent years,nonthermal effects of ultrasound have drawn more and more attention.It has been reported that low dose of ultrasound can influence gene expression and signaling pathways.Since ultrasound can activate signaling pathways,producing various biological effects,it's reasonable to deduce that there exist some sequences that can respond to low dose ultrasound.By synthesizing enormous random promoter sequences,we were able to screen ultrasound-responsive ones from them.As an important part of reticuloendothelial system,liver is capable of phagocytose large quantity of nanoparticles and microparticles,serving as the main tissue of gene delivery and an ideal organ for gene therapy.Liver fibrosis is a chronic disease induced by liver injury and consequent excessive repair.When liver confronts stimulation like inflammation and injury,the hepatic stellate cell will be activated,collagen secretion will increase,and large quantity of extracellular matrix will deposit in liver.Without timely intervention,sustainable fibrosis would lead to irreversible liver cirrhosis and may transform into liver carcinoma eventually,posing a serious threat to public health.The evolvement of liver fibrosis involves various types of cells,cytokines,and signaling pathways,during which macrophages play important roles.Distinct subtypes of macrophages function differently during liver fibrosis.Therefore,we assumed to set up liver fibrosis as our disease model,introduce foreign genes to manipulate macrophage differentiation preference and control liver fibrosis progress by regulating functions of macrophages.As has been reported,in liver fibrosis models induced by CCL4,mi R155 knock-out mice showed milder fibrosis compared to wildtype ones and this alleviation of fibrosis was closely related with macrophage phenotype and corresponding function.In this research,we intended to utilize ultrasound exposure in combination with ultrasound-responsive element to precisely control the release of miR155 inhibitor,thus regulating the level of miR155 and consequently the differentiation of macrophages and the progress of liver fibrosis.The main experiments and results were as follows.1 Construction of random library and screening for ultrasound-responsive elementWe searched for ultrasound-responsive element by constructing library containing large amounts of vectors with random promoter sequences and screening from the library.Techniques in molecular biology such as restriction enzyme digestion,ligation,cloning were used to construct libraries consisting of pWPI-20NminiCMV-PAC,which can induce expression of resistant gene,the PAC here means puromycin N-acetyl-transferase gene,namely the resistant gene of puromycin.HeLa cells were cultured in medium containing puromycin and observed under microscope to determine the optimal selection concentration of puromycin.HeLa cells were transfected with random vectors of pWPI-20NminiCMV-Puror and exposed to ultrasound prior to the addition of puromycin in culture medium.Cells that survived puromycin were sampled and their genetic information was sequenced for screening of ultrasound-responsive element,which would pave the way for subsequent gene expression control in gene therapy.2 Analysis of gene expression change of bone marrow-derived mesenchymal stem cells(BM-MSCs)upon low intensity ultrasound.The promotion of low intensity ultrasound in bone healing has been widely reported and has become a consensus.In consider of the promotion of ultrasound in bone healing,and multiple differentiation potential and diverse roles of bone marrow mesenchymal stem cells,we did some research on the impact of ultrasound on mice BM-MSCs were extracted from bone marrow of C57BL6 mice using the whole bone marrow adherent method.Ultrasound irradiation(100 mW/cm2,15 min)was induced to MSCs and the technology of RNA sequencing was used to explore the impact of ultrasound on MSCs gene expression profiles.The results of RNA sequencing were analyzed for screening of possible ultrasound-regulated pathways.3 Induction of liver fibrosis and delivery of microRNA(miRNA)to the liverMale C57BL6 mice aged six weeks to eight weeks were used for induction of liver fibrosis.Mice were weighed and injected 25 ml/kg CCL4(diluted 3:7 in olive oil)intraperitoneally while control group mice were injected with live oil alone,all injections were practiced twice a week for 8 weeks.Liver tissues were harvested and examined for fibrosis every two weeks.To make sure that therapeutic miRNA can successfully reach liver macrophage to function,we also explore the optimal method for miRNA in vivo delivery.We tested three different approaches for in vivo miRNA delivery,including chitosan coated PLGA nanoparticles,commercially available microbubble Targesphere and special in vivo RNA delivery reagent Engreen.Dye conjugated miRNA were used to evaluate the efficacy of different drug delivery systems.The results showed that chitosan coated PLGA nanoparticles can barely mediate cellular nucleic acid transfection,let alone in vivo delivery.Targesphere in combination with ultrasound destruction can carry the nucleic acid to the liver,but in a not very obvious level.The in vivo transfection reagent Engreen turned out to be the most effective mediator for liver gene delivery.4 The application of elaborate control of miR155 based on ultrasound-responsive elements in alleviating liver fibrosis.The selected sequences of ultrasound-responsive elements were ligated with miR155 inhibitor,thus the level of miR155 can be tightly controlled.The constructed vectors were transfected into mice with liver fibrosis and were used in combination with ultrasound irradiation to control the expression of miR155 inhibitor and explore the efficacy of ultrasound induced mi R155 inhibitor release and its therapeutic effect on liver fibrosis amelioration.The specific methods would be that to deliver miR155 inhibitor to C57BL6 mice treated with CCL4,and divided mice into groups of CCL4 injection alone,CCL4 injection+miRNA inhibitor NC,CCL4 injection+miR155 inhibitor,CCL4+miR155 inhibitor+LIPUS.The extent of fibrosis be observed and quantified to analyze the impact of miR155 inhibitor on liver fibrosis progression.Currently,this part of experiment is still underway.