超声微泡介导白细胞介素-1受体拮抗蛋白基因转染兔软骨细胞的研究
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摘要
背景
骨关节炎(osteoarthritis, OA)是一种慢性、渐进性、退行性关节病变,是临床上最常见的关节疾病。随着我国人口老龄化和创伤发生率增高,OA已成为导致老龄者和创伤病人病废的最主要疾病之一。目前研究显示,OA是力学因素和生物学因素作用下软骨合成和降解偶联失衡的结果,细胞因子学说目前并不能解释OA中软骨破坏的全部原因。但是,不论软骨的哪个部位以何种形式发生退变,都不能脱离IL-1等炎症介质的作用。研究表明,IL-1能刺激软骨细胞、成纤维细胞和滑膜细胞分泌基质金属蛋白酶(matrix metalloproteinases, MMPs)、胶原酶和前列环素2 (prostaglandin E2, PGE2),加速软骨基质中蛋白多糖和Ⅱ型胶原的降解,并抑制蛋白多糖和Ⅱ型胶原的合成,改变和破坏了软骨细胞周围环境,促进软骨细胞的凋亡。
几-1受体分为80kD和68kD两种,前者称为IL-1RⅠ,生物体内分布广泛,体外研究发现IL-1通过与软骨细胞膜上的IL-1RⅠ结合,经丝裂素原激活的蛋白激酶(mitogen-activated protein kinases, MAPKs)途径和NF-κB (nuclear factor-κB)途径进行细胞内信号传导,引起MMPs的表达升高、软骨细胞凋亡等一系列反应。白细胞介素受体拮抗蛋白(interleukin 1 receptor antagonist, IL-1Ra)是一种IL-1的天然拮抗剂,可竞争性结合细胞表面的IL-1RⅠ,从而阻断IL-1的生物学作用。然而,单纯依靠外源性IL-1Ra治疗OA,存在半衰期短,局部浓度低等缺点而限制了其使用。IL-1Ra基因治疗在OA病变处可长期稳定的表达IL-1Ra基因,以往的实验研究初步显示出良好的治疗作用。
然而,成功的基因转染依赖于安全、高效的载体系统,当前基因载体主要有病毒载体和非病毒载体。病毒载体转染效率高,但其潜在致突变性和免疫原性限制了其安全使用；非病毒载体如脂质体和阳离子聚合物等较病毒载体安全,但其转染效率低且靶向性差。因此,寻找和建立无毒副作用又具有靶向性的高效基因载体已成为基因治疗研究亟待解决的关键问题。
近年来,国内外的研究显示,通过超声介导携基因微泡破裂法,能促进目的基因安全而有效的定向转移,增加外源基因转染效率和表达水平,该方法有望为OA的基因治疗提供新的突破点。在以往的研究中,微泡提高基因转染效率从几倍到几百倍不等,除了与超声条件和微泡种类、浓度等因素有关外,也与基因、被转染细胞的种类有关。目前,有关超声介导微泡破裂促进基因转染的实验研究多集中于循环系统的缺血性心肌病、溶栓治疗、肿瘤基因治疗等方面,尚未见有关超声微泡转基因系统在骨相关细胞方面的应用。
因此,本研究拟通过DNA重组技术,构建含增强型绿色荧光蛋白(enhanced green fluorescence protein, EGFP)报告基因的重组人IL-1Ra基因的真核表达质粒pEGFP-N1-hIL-1Ra,采用SonoVue脂质微泡为载体,以体外培养的兔软骨细胞为靶细胞,探讨超声介导微泡破裂促进hIL-1Ra基因转染兔软骨细胞的转染效率和可行性,优化该系统的各方面转染条件,为进一步的体内OA基因治疗奠定基础,进而为OA的治疗探索增效的新途径。
实验一pEGFP-N1-hIL-1Ra真核表达质粒的构建和兔软骨细胞的体外培养
目的构建含绿色荧光蛋白报告基因的真核表达质粒pEGFP-N1-hIL-1Ra,体外培养获得表型稳定的兔软骨细胞。
方法通过RT-PCR扩增hIL-1Ra基因,并在两端添加HindⅢ和BamHⅠ酶切位点。将pEGFP-N1载体经BamHⅠ酶切后电泳鉴定,利用定向克隆技术将经HindⅢ和BamHⅠ双酶切后的hIL-1Ra基因片段和pEGFP-N1载体经T4连接酶进行连接,重组的pEGFP-N1-hIL-1Ra在大肠杆菌DH5α感受态细胞内扩增,经过酶切电泳鉴定和DNA测序证明质粒构建是否成功。选用出生28天的新西兰兔一只,取其胫骨和股骨远端的关节软骨,经0.2%Ⅱ型胶原酶消化、分离、培养原代软骨细胞。对原代和传代软骨细胞行Ⅱ型胶原和Ⅰ型胶原免疫组化染色鉴定。
结果通过对重组质粒pEGFP-N1-hIL-1Ra进行酶切鉴定以及DNA序列测定分析,证明真核表达质粒pEGFP-N1-hIL-1Ra构建成功,开放阅读框架正确。体外培养软骨细胞5代以内能维持正常表型,分泌Ⅱ型胶原。
结论利用DNA重组技术能成功将hIL-1Ra基因克隆入pEGFP-N1载体中,构建出真核表达质粒pEGFP-N1-hIL-1Ra,体外培养的软骨细胞在5代内可用于后续实验研究。
实验二超声介导微泡破裂促进pEGFP-N1-hIL-1Ra转染兔软骨细胞的实验研究
目的探讨超声介导下携白介素-1受体拮抗蛋白基因(hIL-1Ra)的SonoVue微泡体外转染兔软骨细胞的效率和表达情况。
方法体外分离培养的兔软骨细胞,分为单纯质粒组(P)每孔加入50μg/ml质粒、微泡+质粒组(M+P)每孔加入30%携质粒微泡、超声+质粒组(U+P)每孔加入50μg/ml质粒,超声+微泡+质粒组(U+M+P)每孔加入30%携质粒微泡,其中(U+P)和(U+M+P)组以辐照强度1.5w/cm2,辐照时间30s进行超声照射。照射后48小时,荧光显微镜和流式细胞术检测转染效率,RT-PCR和Western blot技术检测hIL-1Ra基因的mRNA和蛋白表达,MTT比色法检测细胞生存率。
结果(U+P)和(U+M+P)组能观察到绿色荧光蛋白的表达,U+M+P组转染效率较其它三组明显提高,其转染效率为11.6±1.0%. pEGFP-N1-hIL-1Ra质粒经超声微泡介导转染后可表达hIL-1Ra的mRNA和蛋白,其细胞生存率下降至75.8%。
结论超声微泡可介导hIL-1Ra基因在兔软骨细胞内的转染和表达,可望成为OA基因治疗的新方法。
实验三超声介导微泡破裂促进基因转染兔软骨细胞的参数优化的实验研究
目的观察不同的超声声强、辐照时间、质粒浓度、微泡浓度和有无血清对基因转染效率和细胞生存率的影响,探讨超声介导SonoVue微泡系统转染兔软骨细胞的最佳条件。
方法分别采用不同的超声声强0.5w/cm2、.0w/cm2、1.5w/cm2、2.0w/cm2,不同的辐照时间5s、10s、30s、60s、120s、180s,不同的质粒浓度5μg/ml、10μg/ml、20μg/ml、30μg/ml、50μg/ml、100μg/ml,不同的微泡浓度5%、10%、20%、30%、50%以及加入或不加入血清进行基因转染,以荧光显微镜和流式细胞术检测基因转染的效率,采用MTT比色法观察细胞生存率。
结果在超声声强为1.5w/cm2,暴露时间为30s,微泡浓度为30%,DNA浓度为50μg/ml的条件下能获得理想的基因转染效率和细胞生存率,血清的存在对基因转染效率和细胞生存率无影响。
结论超声介导微泡破裂技术在基因转染时会对细胞产生一定的损伤,合理把握该系统应用时的各项条件是提高转染效率和减小组织细胞损伤的关键。
Background
Osteoarthritis(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 chondrocyte
Objective 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 destruction
Objective 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 chondrocytes
Objective 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.
骨关节炎(osteoarthritis, OA)是一种慢性、渐进性、退行性关节病变,是临床上最常见的关节疾病。随着我国人口老龄化和创伤发生率增高,OA已成为导致老龄者和创伤病人病废的最主要疾病之一。目前研究显示,OA是力学因素和生物学因素作用下软骨合成和降解偶联失衡的结果,细胞因子学说目前并不能解释OA中软骨破坏的全部原因。但是,不论软骨的哪个部位以何种形式发生退变,都不能脱离IL-1等炎症介质的作用。研究表明,IL-1能刺激软骨细胞、成纤维细胞和滑膜细胞分泌基质金属蛋白酶(matrix metalloproteinases, MMPs)、胶原酶和前列环素2 (prostaglandin E2, PGE2),加速软骨基质中蛋白多糖和Ⅱ型胶原的降解,并抑制蛋白多糖和Ⅱ型胶原的合成,改变和破坏了软骨细胞周围环境,促进软骨细胞的凋亡。
几-1受体分为80kD和68kD两种,前者称为IL-1RⅠ,生物体内分布广泛,体外研究发现IL-1通过与软骨细胞膜上的IL-1RⅠ结合,经丝裂素原激活的蛋白激酶(mitogen-activated protein kinases, MAPKs)途径和NF-κB (nuclear factor-κB)途径进行细胞内信号传导,引起MMPs的表达升高、软骨细胞凋亡等一系列反应。白细胞介素受体拮抗蛋白(interleukin 1 receptor antagonist, IL-1Ra)是一种IL-1的天然拮抗剂,可竞争性结合细胞表面的IL-1RⅠ,从而阻断IL-1的生物学作用。然而,单纯依靠外源性IL-1Ra治疗OA,存在半衰期短,局部浓度低等缺点而限制了其使用。IL-1Ra基因治疗在OA病变处可长期稳定的表达IL-1Ra基因,以往的实验研究初步显示出良好的治疗作用。
然而,成功的基因转染依赖于安全、高效的载体系统,当前基因载体主要有病毒载体和非病毒载体。病毒载体转染效率高,但其潜在致突变性和免疫原性限制了其安全使用；非病毒载体如脂质体和阳离子聚合物等较病毒载体安全,但其转染效率低且靶向性差。因此,寻找和建立无毒副作用又具有靶向性的高效基因载体已成为基因治疗研究亟待解决的关键问题。
近年来,国内外的研究显示,通过超声介导携基因微泡破裂法,能促进目的基因安全而有效的定向转移,增加外源基因转染效率和表达水平,该方法有望为OA的基因治疗提供新的突破点。在以往的研究中,微泡提高基因转染效率从几倍到几百倍不等,除了与超声条件和微泡种类、浓度等因素有关外,也与基因、被转染细胞的种类有关。目前,有关超声介导微泡破裂促进基因转染的实验研究多集中于循环系统的缺血性心肌病、溶栓治疗、肿瘤基因治疗等方面,尚未见有关超声微泡转基因系统在骨相关细胞方面的应用。
因此,本研究拟通过DNA重组技术,构建含增强型绿色荧光蛋白(enhanced green fluorescence protein, EGFP)报告基因的重组人IL-1Ra基因的真核表达质粒pEGFP-N1-hIL-1Ra,采用SonoVue脂质微泡为载体,以体外培养的兔软骨细胞为靶细胞,探讨超声介导微泡破裂促进hIL-1Ra基因转染兔软骨细胞的转染效率和可行性,优化该系统的各方面转染条件,为进一步的体内OA基因治疗奠定基础,进而为OA的治疗探索增效的新途径。
实验一pEGFP-N1-hIL-1Ra真核表达质粒的构建和兔软骨细胞的体外培养
目的构建含绿色荧光蛋白报告基因的真核表达质粒pEGFP-N1-hIL-1Ra,体外培养获得表型稳定的兔软骨细胞。
方法通过RT-PCR扩增hIL-1Ra基因,并在两端添加HindⅢ和BamHⅠ酶切位点。将pEGFP-N1载体经BamHⅠ酶切后电泳鉴定,利用定向克隆技术将经HindⅢ和BamHⅠ双酶切后的hIL-1Ra基因片段和pEGFP-N1载体经T4连接酶进行连接,重组的pEGFP-N1-hIL-1Ra在大肠杆菌DH5α感受态细胞内扩增,经过酶切电泳鉴定和DNA测序证明质粒构建是否成功。选用出生28天的新西兰兔一只,取其胫骨和股骨远端的关节软骨,经0.2%Ⅱ型胶原酶消化、分离、培养原代软骨细胞。对原代和传代软骨细胞行Ⅱ型胶原和Ⅰ型胶原免疫组化染色鉴定。
结果通过对重组质粒pEGFP-N1-hIL-1Ra进行酶切鉴定以及DNA序列测定分析,证明真核表达质粒pEGFP-N1-hIL-1Ra构建成功,开放阅读框架正确。体外培养软骨细胞5代以内能维持正常表型,分泌Ⅱ型胶原。
结论利用DNA重组技术能成功将hIL-1Ra基因克隆入pEGFP-N1载体中,构建出真核表达质粒pEGFP-N1-hIL-1Ra,体外培养的软骨细胞在5代内可用于后续实验研究。
实验二超声介导微泡破裂促进pEGFP-N1-hIL-1Ra转染兔软骨细胞的实验研究
目的探讨超声介导下携白介素-1受体拮抗蛋白基因(hIL-1Ra)的SonoVue微泡体外转染兔软骨细胞的效率和表达情况。
方法体外分离培养的兔软骨细胞,分为单纯质粒组(P)每孔加入50μg/ml质粒、微泡+质粒组(M+P)每孔加入30%携质粒微泡、超声+质粒组(U+P)每孔加入50μg/ml质粒,超声+微泡+质粒组(U+M+P)每孔加入30%携质粒微泡,其中(U+P)和(U+M+P)组以辐照强度1.5w/cm2,辐照时间30s进行超声照射。照射后48小时,荧光显微镜和流式细胞术检测转染效率,RT-PCR和Western blot技术检测hIL-1Ra基因的mRNA和蛋白表达,MTT比色法检测细胞生存率。
结果(U+P)和(U+M+P)组能观察到绿色荧光蛋白的表达,U+M+P组转染效率较其它三组明显提高,其转染效率为11.6±1.0%. pEGFP-N1-hIL-1Ra质粒经超声微泡介导转染后可表达hIL-1Ra的mRNA和蛋白,其细胞生存率下降至75.8%。
结论超声微泡可介导hIL-1Ra基因在兔软骨细胞内的转染和表达,可望成为OA基因治疗的新方法。
实验三超声介导微泡破裂促进基因转染兔软骨细胞的参数优化的实验研究
目的观察不同的超声声强、辐照时间、质粒浓度、微泡浓度和有无血清对基因转染效率和细胞生存率的影响,探讨超声介导SonoVue微泡系统转染兔软骨细胞的最佳条件。
方法分别采用不同的超声声强0.5w/cm2、.0w/cm2、1.5w/cm2、2.0w/cm2,不同的辐照时间5s、10s、30s、60s、120s、180s,不同的质粒浓度5μg/ml、10μg/ml、20μg/ml、30μg/ml、50μg/ml、100μg/ml,不同的微泡浓度5%、10%、20%、30%、50%以及加入或不加入血清进行基因转染,以荧光显微镜和流式细胞术检测基因转染的效率,采用MTT比色法观察细胞生存率。
结果在超声声强为1.5w/cm2,暴露时间为30s,微泡浓度为30%,DNA浓度为50μg/ml的条件下能获得理想的基因转染效率和细胞生存率,血清的存在对基因转染效率和细胞生存率无影响。
结论超声介导微泡破裂技术在基因转染时会对细胞产生一定的损伤,合理把握该系统应用时的各项条件是提高转染效率和减小组织细胞损伤的关键。
Background
Osteoarthritis(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 chondrocyte
Objective 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 destruction
Objective 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 chondrocytes
Objective 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.
引文
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