Experimental Research Of Interleukin-1 Receptor Antagonist Gene Transfected Into Rabbit Chondrocyte Cells By Ultrasound-mediated Microbubble Destruction

BackgroundOsteoarthritis(OA) is a chronic and degenerative disease of joint and is one of the most common joint disorders.OA is becoming one of the principal diseases leading to disability with aging of population and high incidence of trauma.Previous research in the field of OA has characterized that OA is the result of disequilibrium between cartilage degeneration and synthesis controlled by the mechanic and biologic factors.The effect of inflammation mediators,such as interleukin-1(IL-1),is necessary in the cartilage degeneration although cytokines is not enough.According to the previous researches,IL-1 stimulates chondrocytes,fibroblasts and synoviocytes to synthesize prostaglandin E2(PGE2),collagenases and matrix metalloproteinases (MMPs),and accelerates the degeneration of main compositions of cartilage,such as proteoglycan and type II collagen,destroys the environment of chondrocytes and promotes their apoptosis.There are two kinds of IL-1 receptors,80kD and 68kD in molecular weight respectively and the former is called IL-1R I expressing extensively in organisms.When coupled with IL-l,serine and threonine residues in IL-1R I are phosphorylated and IL-1R I become activated to signal transduction.It has been certificated that mitogen-activated protein kinases(MAPKs) and nuclear factor-KB(NF-κB) signaling pathways participate in the process IL-1 induced MMPs express and chondrocytes apoptosis.IL-1Ra is a naturally occurring protein that binds to the type I IL-1 cell surface receptor,preventing its ability to interact with IL-1. But relying on the exogenous IL-1Ra only,the effective time and local concentration of IL-1Ra are too limited that can hinder its affect. The transfer of gene encoding IL-1ra to the joint can be considered a new treatment candidate for OA because it can provide gene sustained synthesis at sites of lesions, which the previous experiment studys had showen therapeutic benefits.While, the success of gene therapy is largely dependent on the development of vectors or vehicles that can selectively and efficiently deliver a therapeutic gene to cells or target tissues safely. The main categories of techniques that have been used to deliver genes are viral vectors and nonviral vectors. Viral vectors are really efficient but they have limitations such as wild-type reversion and immunogenicity. Meanwhile, Non-viral delivery systems including Cationic phospholipids and cationic polymers for gene therapy are more safely than methods,while these techniques suffer from lower transfection efficiencies. The viral and non viral delivers all have merits and demerits, and developing a novel and effective method to deliver gene becomes a new aim of the gene therapy research.Recently, some studies demonstrate that ultrasound-mediated microbubble destruction can enhance the transfection efficieney and expression of the exogenous gene to a certain orientation safely and effectively.This method would become a new breakthrough of the gene therapy fo OA. In previous studies, the efficiency of microbubbles to enhance the gene transfer is from a range of times to hundreds of times, in addition to the conditions with ultrasound and microbubble type, concentration and other factors, but also with the genes, cells were transfected with the categories. At present, the ultrasound microbubble-mediated gene transfer focused on the ischemic cardiomyopathy in circulatory system, thrombolytic therapy, cancer gene therapy, et al.It has not yet to see on the ultrasound system in genetically modified bone-related cell applications.Therefore, this study planned to construct recombinant eukaryotic expression plasmid pEGFP-N1-hIL-1Ra containing the enhanced green fluorescence protein (EGFP) and the recombinant human interleukin 1 receptor antagonist (IL-1Ra) by gene recombination technology, investigate the transfection efficieney of IL-1Ra gene in rabbit chondrocytes by ultrasound-mediated microbubble destruction, optimized the parameter and condition of this gene deliver system,and evaluate its feasibility of this method used in OA gene therapy in vivo.Part 1 Construction of recombination eukaryotic expression vector pEGFP-N1-hIL-1Ra and cultivation of rabbit articular chondrocyteObjective To construct a eukaryotic expression vector containing the enhanced green fluorescence protein and recombinant human interleukin 1 receptor antagonist and culture rabbit articular chondrocyte which have stable phenotype.Method The HindⅢand BamH I primers specific for amplifying the DNA fragment encoding hIL-1Ra were designed and synthesized.The targeted DNA fragment was obtained from human totally RNA by RT-PCR. The pEGFP-N1 vector had been enzyme digestion by BamH I.Cutting pEGFP-N1 vector and hIL-1Ra with HindⅢand BamH,and using T4 ligase to connect the pEGFP-N1 vector and rhIL-1Ra.The recombinant plasmid pEGFP-N1-hIL-IRa was first propagated in E.coli 5α,and then was confirmed to contain hIL-1RacDNA sequence by agarose gel electrophoresis and DNA sequence analysis.Articular cartilage were removed from the distal end of tibia and femurs of the 28 days fatal New Zealand white rabbit,primary chondrocytes were digested with 0.2% typeⅡcollagenase. TypeⅠcollagen and typeⅡcollagen immunohistochemistry had been taked to the primary chondrocytes and passaged chondrocytes to make sure that whether the cell still has its phenotype.Result The construction of the recombinate eukaryotic expression plasmid pEGFP-N1-hIL-1Ra and the correction of the open reading frame were confirmed through restriction enzyme maping analusis and DNA sequencing.The chondrocytes can retain its phenotype and excrete typeⅡcollagen before five generations.Conclusion By gene recombinant technology,the hIL-1Ra gene can be cloned into pEGFP-N1 vector to construct the recombinant eukaryotic expression plasmid pEGFP-N1-hIL-1Ra The chondrocytes within five generations can be used in the later experiment.Part 2 Experimental Research of interleukin-1 receptor antagonist gene transfected into rabbit chondrocytes by ultrasound-mediated microbubble destructionObjective To explore the efficiency and expression of transfering of interleukin-1 receptor antagonist gene by ultrasound-mediated microbubbles destruction.Method Cultured rabbit chondrocytes in vitro were grouped to the followings. P group:plasmid DNA alone; M+P group:mirobubbles and plasmid DNA; U+P group: ultrasound irradiation and plasmid; U+M+P group:ultrasound irradiation, mirobubbles and plamid DNA. The ultrasound intensity was 1.5w/cm2, ultrasound exposure time was 60s. Forty-eight hours later, the transfering efficiency was observed under fluorescence microscopy and flow cytometry. RT-PCR and Western blot analysis was used to examined the expression of IL-1ra mRNA and protein. Cell viability was assayed by MTT.Result Green fluorescence was observed in U+P and U+M+P gruop by fluorescence microscopy.The transferring efficiency was highest in the U+M+P group, the trasfection expression rate is 11.6±1.0%. The IL-1ra expression increased after the ultrasound-mediated microbubbles irradiation.The survival rate of the chondrocytes reduced to 75.8%.Conclusion Ultrasound-mediated microbubbles destruction can increase the transfection of hIL-1ra gene in chondrocyte cells. It may become a novel method of gene therapy for cartilage lesions.Part 3 Optimization of parameters in ultrasound-mediated microbubble destruction enhance gene delivery in rabbit chondrocytesObjective To observe the the trasfection expression rate and survival rate of the chondrocytes in different parameters of intensity of ultrasound,times of irradiation, concentration of plasmid, concentration of microbubble and the presence of the serum or not.Method The gene transfection were act in the conditions of different intensity of ultrasound as 0.5w/cm2. 1.0w/cm2、1.5w/cm2、2.0w/cm2, different times of irradiation as 5s、10s、30s、60s、120s、180s, different concentration of plasmid as 5μg/ml、10μg/ml、20μg/ml、30μg/ml、50μg/ml、100μg/ml, different concentration of microbubble 5%、10%、20%、30%、50% respectively.The rate of gene transfection in rabbit chondrocytes was detected by fluorescence microscopy and flow cytometry.The survival rate of rabbit chondrocytes were evaluated by MTT.Result We can obtain a fine transfection efficiency and survival rate when intensity was 1.5w/cm2,duration was 30s,dose of plasmid and microbubble was 50μg/ml and 30%. The trasfection expression rate and survival rate of the chondrocytes has no significant difference whether the presence of the serum or not.Conclusion Ultrasound-mediated microbubble destruction may injured the chondrocytes.To chose a optimal parameters is the key of raise the trasfection expression rate and decrease the harmful of the cells.