小干扰RNA沉默TNF-α表达并抑制骨溶解的实验研究  

EXPERIMENTAL STUDY ON SMALL INTERFERING RNA SILENCING EXPRESSION OF TUMOR NECROSIS FACTOR α AND INHIBITING OSTEOLYSIS

作　　者：余博[1] 郝绍文[1] 孙首选[1] 郭浩辉[1] 杨小春[1] 马晓军[1] 金群华[1] 

机构地区：[1]宁夏医科大学总医院骨三科,银川750004

出　　处：《中国修复重建外科杂志》2013年第8期994-999,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(81060146)~~

摘　　要：目的通过构建沉默TNF-α的重组腺病毒并观察其对小鼠假体骨溶解模型的影响,探讨基因治疗人工关节假体周围骨溶解的可能性。方法设计沉默TNF-α的小干扰RNA(small interfering RNA,siRNA)编码序列的引物,扩增后利用RAPAD腺病毒包装系统将该序列载入腺病毒,并构建出E1、E3区双缺失的重组腺病毒Ad5-TNF-α-siRNA-CMVeGFP。取8～10周龄SPF级BABL/C雌性小鼠64只,体重20～25 g,随机分为4组(n=16)。空白对照组(A组)制备假体模型,颗粒对照组(B组)、空病毒组(C组)、治疗组(D组)制备假体松动骨溶解模型;造模第2周开始向关节腔内分别注入以下溶液(两次各注射40μL):A组注入PBS溶液,24 h后再次注入;B、C、D组注入钛颗粒悬浊液,24 h后分别注入PBS溶液、腺病毒Ad5 E1-CMVeGFP溶液(1×109PFU/mL)、重组腺病毒Ad5-TNF-α-siRNA-CMVeGFP溶液(1×109PFU/mL);每隔2周重复1次至第10周。术后观察小鼠一般情况,术后12周取材进行组织学观察及Westernblot检测TNF-α表达。结果目的基因载入腺病毒载体后经双切酶鉴定及DNA测序确定获得阳性克隆,重组腺病毒Ad5-TNF-α-siRNA-CMVeGFP成功转染HEK293细胞。术后各组小鼠均存活至实验完成。组织学观察示,A组炎性细胞及破骨细胞少,骨质形成良好;B、C组见大量炎性细胞浸润,破骨细胞多,骨质破坏明显;D组少量炎性细胞浸润,破骨细胞较B、C组少,未出现明显骨质破坏。各组界膜厚度及破骨细胞计数均为A组<D组<B组<C组,组间比较差异均有统计学意义(P<0.05)。Western blot检测显示各组均有TNF-α表达,A、B、C、D组TNF-α表达量分别为0.235±0.022、0.561±0.031、0.731±0.037、0.329±0.025,组间比较差异均有统计学意义(P<0.05)。结论成功构建沉默TNF-α的重组腺病毒,并可有效沉默小鼠假体周围组织中TNF-α的表达,从而抑制骨溶解。Objective To investigate the possibility of gene therapy of osteolysis around artificial joint prosthesis by constructing the recombinant adenovirus which can silence tumor necrosis factor α（TNF-α）. Methods The primer of small interfering RNA（siRNA） coding sequence of silent TNF-α was designed and amplified,and then RAPAD adenovirus packaging system was used to load the sequence to adenovirus,and the recombinant adenovirus Ad5-TNF-α-siRNA-CMVeGFP which lacked both E1 and E3 regions was constructed.Then 64 female BABL/C mice（weighing,20-25 g） were randomly divided into 4 groups（n=16）： blank control（group A）,positive control（group B）,simple adenovirus（group C）,and treatment group（group D）.The prosthetic-model was established in group A,and the prosthetic-loosening-model in groups B,C,and D.At 2 weeks after modeling,PBS solution was injected first,and then the same solution was injected 24 hours later in group A;titanium particle solution was injected,and then PBS solution,Ad5 E1-CMVeGFP（1 × 109 PFU/mL）,and Ad5-TNF-α-siRNA-CMVeGFP（1 × 109 PFU/mL） were injected,respectively in groups B,C,and D 24 hours later,every 2 weeks over a 10-week period.The general condition of mice was observed after operation.The tissues were harvested for histological observation,and the expression of TNF-α was detected by Western blot at 12 weeks after operation. Results The positive clones were achieved by enzyme digestion and confirmed by DNA sequencing after loading the target genes into adenovirus vector,and then HEK293 cells were successfully transfected by recombinant adenovirus Ad5-TNF-α-siRNA-CMVeGFP.All mice survived to the completion of the experiment.Histological observation showed that there were few inflammatory cells and osteoclasts in group A,with a good bone formation;there were a large number of inflammatory cells and osteoclasts in groups B and C,with obvious bone destruction;inflammatory cells and osteoclasts in group D was less than those in groups B and C,with no obvio

关 键 词：关节置换 骨溶解 基因治疗 TNF-Α 重组腺病毒 小干扰RNA 小鼠 

分 类 号：R681[医药卫生—骨科学]