携带LacZ的腺病毒载体在牵引骨痂局部转染成纤维细胞和成骨细胞的表达

Expression of adenovirus-LacZ after injected into the callus to transfect fibroblasts and osteoblasts during distraction osteogenesis

目的:利用小鼠胫骨牵引成骨动物模型,在牵引成骨过程中局部给予携带了LacZ的腺病毒载体后观察其表达情况,以探讨基因治疗促进骨折愈合的可行性。方法:实验于2004-10/2006-01在中南大学湘雅三医院完成。①实验分组:取雄性8周龄CD-1小鼠20只,随机数字表法分为实验组和对照组,每组各10只。②实验方法:所有小鼠接受左胫骨中上段骨干横行截骨安置特制延长外固定架,胫骨牵引过程包括5d静止期,10d牵引期,牵引速率为0.1mm/次,2次/d,共0.2mm/d。牵引期第7天实验组牵引骨痂局部注射5μL携带了LacZ的腺病毒载体,对照组局部注入等量未含LacZ腺病毒液。③实验评估:注射后第3天麻醉后杀取动物,采集左胫骨标本,分别作组织学检查和组织化学分析。结果:纳入小鼠20只,均进入结果分析。在牵引第10天,牵引骨痂中纤维间区形成,两端的新生骨由骨折两端中心生长延伸。骨髓腔内外可见大量新生骨形成。X-Gal底物染色显示,在实验组新生骨组织内可见大量细胞呈阳性染色;而对照组未发现阳性染色细胞。结论:在牵引成骨过程中,骨痂局部注射携带LacZ的腺病毒,能成功转染局部的成纤维细胞及成骨细胞,并表达LacZ基因,为临床应用基因治疗促进骨牵引延长或骨折愈合提供可行性。

AIM： To explore the feasibility of gene therapy for fracture healing by locally injecting the adenovirus-LacZ into the distracted callus of mouse tibia and observing the expression of LacZ. METHODS： The experiment was conducted in Xiangya Third Hospital of Central South University from October 2004 to January 2006.①Twenty male 8-week-old CD-1 mice were selected and randomly divided into experimental group （n =10） and control group （n =10）.②AII the mice received osteotomies on the left tibia and placement of special external distractive fixators. This procedure involved 5 days latency and 10 days distraction with distraction velocity of 0.1 mm, twice daily, totally 0.2 mm a day. On the 7^th day of distraction, 5μL adenovirus-LacZ was injected into the distracted callus of the tibia in the experimental group and 5 μL adenovirus alone was injected into the control group. ③Animals were sacrificed under anesthesia on the third day after injection, and the lengthened left tibia were harvested and analyzed by histological and histochemical techniques. RESULTS： All the 20 mice were involved in the result analysis. On the 10^th day of distraction, fibrous interzone formed and the new bone extended from the both ends of fracture to the centre of the distracted callus. A great deal of new bone was observed inside and outside of the bone marrow cavities. The X-Gal substrate dyeing suggested that many cells presented positive dyeing in the experimental group but no such cells were found in the control group. CONCLUSION： During distraction osteogenesis, the injection of adenovirus-LacZ into the callus could successfully result in the transfection to the fibroblasts and osteoblasts, and expression of Lac-Z in these cells, which offers the feasibility of utilizing the gene therapy for distraction osteogenesis and fracture healing.