腺病毒介导TGF-βGF-β_1基因转染腘绳肌腱对兔前交叉韧带重建术后腱-骨愈合的组织形态学影响

EFFECT OF HAMSTRING TENDON TRANSFECTED WITH ADENOVIRUSMEDIATED TRANSFORMING GROWTH FACTOR β_1 GENE ON HISTOMORPHOLOGY OF TENDON-BONE INTERFACE HEALING AFTER ANTERIOR CRUCIATE LIGAMENT RECONSTRUCTION IN RABBITS

目的 探讨腺病毒介导TGF-β_1基因转染腘绳肌腱对兔前交叉韧带（anterior cruciate ligament,ACL）重建术后腱-骨愈合组织形态学的影响。方法 取重组腺病毒载体Ad TGF-β_1与腺病毒空载体Ad GFP,以DMEM培养液稀释其滴度至5×10~8 PFU/m L。取新西兰大白兔48只,体质量1.6~2.5 kg,随机分为A、B、C 3组（n=16）,以兔右后肢膝关节制备自体同侧腘绳肌腱重建ACL模型;ACL重建前A、B组腘绳肌腱分别以Ad TGF-β1与Ad GFP转染培养12 h,C组以DMEM培养作为对照。转染后12 h,荧光显微镜观察A、B组腘绳肌腱绿色荧光表达,ELISA检测A组腘绳肌腱中TGF-β1蛋白含量。重建术后观察各组动物一般情况,并于2、4、8、12周取材行HE及Masson染色,计数腱-骨界面成纤维细胞以及参照Buark评价分级标准对腱-骨愈合进行评价。结果A、B组腘绳肌腱转染培养后,均可见绿色荧光表达;A组转染12 h时腘绳肌腱组织中TGF-β1蛋白表达量为（221.0±12.2）ng/m L。组织学观察示：A组术后腱-骨界面见成纤维细胞生成及胶原纤维逐渐增多,Sharpey纤维逐步形成;至12周时界面区见到规整Sharpey纤维,部分区域组织有直接愈合倾向。而B、C组移植肌腱与骨隧道间成纤维细胞较A组少且腱-骨连接疏松;至12周时界面区组织未见直接愈合倾向。各时间点A组腱-骨界面成纤维细胞计数以及腱-骨愈合分级均显著优于B、C组,比较差异有统计学意义（P〈0.05）;B、C组间比较差异均无统计学意义（P〉0.05）。结论 腺病毒介导TGF-β1基因转染方法可促进自体腘绳肌腱重建兔ACL术后腱-骨愈合。

Objective To investigate the effect of hamstring tendon transfected with adenovirus-mediated transforming growth factor β1 （AdTGF-β1） genes on the histomorphology of tendon-bone interface healing after anterior cruciate ligament （ACL） reconstruction in rabbits. Methods AdTGF-β1 and AdGFP were diluted to 5× 10^8 PFU/mL with DMEM. Forty-eight New Zealand white rabbits were divided into 3 groups randomly （n=16）, weighing 1.6-2.5 kg for ACL reconstruction with hamstring tendon autograft. Hamstring tendon was cultured and transfected with AdTGF-β1 （group A） and AdGFP （group B） for 12 hours before ACL reconstruction, and was cultured with DMEM in group C. After 12 hours of transfection, the expression of green fluorescence was observed in groups A and B under fluorescence microscopy; TGF-β1 protein level was detected by ELISA in group A. At 2, 4, 8, and 12 weeks after operation, the specimens were harvested for HE and Masson staining; the number of fibroblasts was counted, and the Buark grading was used to evaluate tendon-bone interface healing. Results Green fluorescence was observed after 12 hours of transfection in groups A and B. TGF-β1 protein level reached （221.0±12.2） ng/mL at 12 hours in group A. The histological observation showed that few fibroblasts and collagen fibers were found, and Sharpey fibers appeared in group A; regular Sharpey fibers were seen in the interface, and integrity interface in some areas at 12 weeks. But fibroblasts of groups B and C were less than those of group A, with loose tendon-bone interface; no integrity interface was observed at 12 weeks. The number of fibroblasts and Buark grading of group A were significantly higher than those of groups B and C （P〈0.05）, but no significant difference was found between groups B and C （P〉0.05）. Conclusion Hamstring tendon transfected with AdTGF-β1 gene can promote the healing of tendon-bone interface after ACL reconstruction.