【作者】 李艳；

【导师】 李伟；

【作者基本信息】 山东大学 ， 外科学， 2012， 硕士

【摘要】 研究目的骨性关节炎(Osteoarthritis,OA)是老年人群发病率较高的疾病之一,是在全身因素和局部因素的综合作用下,关节软骨发生生化、结构和代谢改变,最终出现关节软骨软化、破溃和局部剥脱以及关节边缘骨与软骨赘生物形成等病理改变,临床上可产生关节疼痛、活动受限和关节畸形等症状,严重影响老年人的身体健康,是致残的重要原因。在骨关节炎的病理改变中,大量炎性介质的产生和释放是造成局部关节改变的重要因素。其中IL-1和TNF-a是OA病理过程中促进软骨基质降解和关节软骨破坏的两种最重要的细胞因子,其他细胞因子对关节软骨的破坏往往是间接通过IL-1和TNF-a发挥作用。目前国内外对OA的发病机制和治疗研究很多,但疗效不尽如人意,近些年来自杀基因治疗为骨关节炎的治疗提供了一种新的方法。本实验中,我们应用基因治疗的方法,利用重组重组腺相关病毒载体(rAAV),将外源性抑炎性因子基因联合自杀基因直接体内转染兔膝关节后,观察对骨关节炎的治疗效果。有可能在骨关节炎的治疗上去的新的进展。方法采用切断并切除前交叉韧带及内侧半月板的方法将32只新西兰白兔的右后膝关节制成OA的动物模型,将兔子平均分成四组,将有rAAV包装的目的基因通过髌韧带注射入关节腔内,A组]AAV2/1-IL-1Ra1ml,B组rAAV2/1-HSV-tk1ml,C组rAAV2/1-TK-IL-1Ra lml,D组为对照组,转染含空质粒的rAAV2/11mL关节腔内注射24小时后,每只动物给予静脉内注射更昔洛韦(GCV)5mg／天,一天两次,持续三天。注射后第7天,用lml生理盐水灌洗关节腔,抽取关节腔灌洗液用ELISA分析外源基因的表达情况,注射后第14天,处死动物,用无菌生理盐水1ml灌洗关节腔,抽取关节灌洗液进行IL-1β、TNF-a及TGF-β1的ELISA分析,取滑膜组织通过real-time PCR方法检测目的基因的表达,并用相对定量的方法—2-ΔΔCT进行目的基因表达的分析,目的基因包括：IL-1β、TNF-α、及TGF-β1。取关节标本进行固定,包埋,切片和HE染色及番红O染色,镜下观察软骨细胞排列情况,并进行软骨组织学评分。结果通过ELISA检测分析接受IL-1Ra基因治疗的A组及IL-1Ra联合HSV-tk基因治疗的C组在外源基因注射后第7天和第14天,关节液内有稳定的外源基因IL-1Ra的表达,而转染HSV-tk基因的B组及未有基因转染的对照组D组未检测到人IL-1Ra的表达。说明外源基因能在关节腔内表达。通过real-time PCR检测目的基因的表达量,IL-1β的表达量三个实验组明显低于对照组,此外C组即IL-1Ra联合HSV-tk基因治疗组明显低于A组IL-1Ra基因治疗组和B组HSV-tk基因治疗组,差别有统计学意义；TNF-α的表达量对照组高于三个实验组,联合基因治疗组低于单基因治疗组,差别有统计学意义；TGF-β1的表达量对照组明显高于三个实验组。通过ELISA检测,IL-1β在A组、B组、C组的水平明显低于对照组D组,TNF-α的表达水平在对照组明显高于三个治疗组,差别有统计学意义；TGF-β1水平对照组明显高于导入外源性基因的A、B、C三组,统计结果以平均值±SEM表示,n=3,P<0.05。HE染色及番红O示染色未转染基因的对照组关节软骨表面粗糙并有软骨剥脱,外源性基因导入后,软骨损害及软骨基质降解明显减轻。关节软骨的Mankin’s评分显示对照组的评分8.264±0.044明显高于三个基因治疗组,而联合基因治疗组的评分明显低于两组单一基因治疗组。结论我们将携带目的基因的重组腺相关病毒载体注射入关节腔内,进行体内转染试验,使其在体内表达相应的目的基因。根据实验结果我们发现,IL-1Ra基因联合自杀基因HSV-tk对于骨关节炎的基因治疗优于单纯的使用IL-1Ra基因或HSV-tk基因治疗,不仅可以有效地减少主要促炎性因子—IL-16的含量,减轻滑膜炎症,同时还可以减轻软骨损害和软骨基质降解,以此来达到治疗骨关节炎的目的。更多还原

【Abstract】 ObjectiveOsteoarthritis(OA) is one of the high incidence of disease in older population, in the combination with whole body factors and with local factors, articular cartilage biochemical, structure and metabolic happened changes, eventually appear articular cartilage softening, burst and local peeling and bone and cartilage edge joint formation vegetations pathological changes. It can produce clinical joint pain and restricted movement and joint deformities and other symptoms and seriously influence the health of old people, is the important reason for the disabled. In OA, excessive release of proinflammatory cytokines attributes greatly to the pathological changes in local joint. Among these cytokines, IL-1and TNF-a are the most important of cytokines in the pathogenic process, which promote cartilage matrix destruction and articular cartilage degradation. Other cell factors on the destruction of articular cartilage is often indirectly by IL-1and TNF-a. In present so many research about the pathogenesis and treatment of OA, but the effect is an unsatisfying, in recent years suicide gene therapy offers a radical different approach to the treatment of osteoarthritis. This study demonstrates that direct in vivo transfer of the exogenous anti-inflammatory cytokine gene combined with suicide gene by rAAV into osteoarthritic rabbit knee joints using intra-articular injections to observe the therapeutic effect of osteoarthritis.This avenue may therefore represent a promising future treatment for osteoarthritis. MethodsOA was induced in right knees of32New Zealand white rabbits by the excision of medial meniscectomy plus the anterior cruciate ligament transaction. Approximately1×1010v.p./ml of rAAV were injected into the joint space of the knee through the patellar tendon. A total of32operated rabbits were divided into four groups. Four rabbit groups received rAAV encoding IL-1Ra gene as Group A, HSV-tk gene as Group B as well as combined-gene transferred Group C and the control group D, being transfected by the rAAV carrying no target gene. Twenty-four hours following intra-articular injection the animals received intravenous infusions of ganciclovir (GCV) at a close of5mg/kg. The ganciclovir was administered twice daily for a total of3days. One week after the rAAV injection, keen joints were lavaged with1ml of0.9%(w/v) physiological saline solution through the patellar tendon. Concentration of exogenous IL-1Ra was determined by ELISA.Two weeks after the rAAV injection, the rabbits were sacrificed. Keen joints were lavaged with1ml of0.9%(w/v) physiological saline solution through the patellar tendon. Concentration of IL-1Ra、 IL-1β、TNF-a and TGF-β1expression in recovered lavage fluids were measured using a cytokine ELISA kit.. Levels of IL-1Ra,IL-1β,TNF-a, TGF-β1and HSV-tk expression in synovium were measured using rear-time PCR. Gene expressions in all samples were analyzed by applying the2-AACt relative quantification method. Cartilage from the lesion areas of medial femoral condyle were fixed, embedded, sectioned and stained with hematoxylin and eosin and Safranin O. The samples were examined by light microscopy. Histologic quanitative assessment was performed by Mankin’s score.ResultsConcentration of exogenous hIL-1Ra was determined by ELISA assays. At seven days and14days after gene transfection, the exogenous hIL-1Ra was expressing stability in the synovial fluid, while the transfection of the B group of the HSV-TK gene and group D no gene transfection control group does not detect the expression of human IL-1Ra. The exogenous gene can express in the intra-articular.According to the measurement of real-time PCR and the analysis of relative quantification-comparative CT method, expression levels of IL-lβwere significantly decrease in group A,B and C than in group D. The expression levels of TNF-a were significantly increase in group D than in in group A,B and C. The expression of TGF-β1,all of the gene transfection groups showed significant decreases compared with the non-transfected group.Concentration of exogenous hIL-1Ra was determined by ELISA assays. The expression of hIL-1Ra was high following hIL-1Ra gene transfection in synovial fluids. Such trends were also notable at protein level for IL-1β、TNF-a and TGF-(31by ELISA assays. The level of IL-1β was markedly higher in group D than group A,B and C. The level of TNF-a was lower in gene transfection groups than group D. The level of TGF-β1was lower in gene transfection groups than group D. The results of ELISA was shown as mean±SEM, n=3and P<0.05. In the control group, the articular cartilage surface was rough and had some superficial leakage and ulcers. After exogenous genes were transfected, the cartilage lesions and matrix degradation were less evident.Conclusions:The target gene were successfully transferred to the joint space of the knee in vivo carried by rAAV. The transfected genes were expressed in synovial fluid of rabbits. This study suggests that combination-gene transfection show better results than transfection gene alone, gene transfection is effective in treating experimental OA. Combination-gene transfection has better biologic effects on OA.更多还原