结缔组织生长因子促进半月板无血管区损伤愈合  

Enhancement of meniscal tearing damage repairing in the avascular zone using connective tissue growth factor (CT-GF) in the rabbit model

作　　者：何蔚[1] 刘明[1] 青祖宏[1] 高巍[1] 刘玉杰[2] 黄潇楠[3] 

机构地区：[1]解放军305医院骨科,北京100017 [2]解放军总医院骨科,北京100853 [3]首都师范大学化学系,北京100037

出　　处：《中国骨伤》2013年第8期681-688,共8页China Journal of Orthopaedics and Traumatology

基　　金：全军医学科技"十二五"科研项目(编号:CWS11C290);国家自然科学基金青年基金(编号:00912210010006);国家高技术研究发展计划(863计划)(编号:2006AA02A136)~~

摘　　要：目的:探讨结缔组织生长因子(CTGF)对纤维软骨细胞外基质分泌、VEGF表达及促进半月板无血管区的损伤修复中的作用。方法:将新西兰大白兔半月板白区,经胶原酶消化、离心分离后,提取半月板纤维软骨细胞,培养至第2代。用流式细胞仪鉴定该细胞表面CD31,CD44,CD45和CD105标志物,并用Ⅱ型胶原抗体做免疫细胞化学鉴定,以证明所培养、传代的细胞是纤维软骨细胞。分别将细胞培养在浓度100ng/ml的CTGF培养基中3、14d后,通过实时定量聚合酶链反应,检测Ⅰ型胶原、Ⅱ型胶原和VEGF基因的表达变化情况。造模,在兔半月板中央区,制作长3mm的纵行撕裂。将45只大白兔随机分为3组,处理方式为:半月板单纯缝合术,缝合术加填充PBS-纤维蛋白胶,缝合术加填充1.5μgCTGF-纤维蛋白胶。术后第1、4、10周用荧光免疫组织化学分析法显示Ⅰ型、Ⅱ型胶原和VEGF在损伤处的表达与分布情况,直观撕裂处的愈合情况。结果:体外实验第14天,定量RT-PCR结果显示,100ng/mlCTGF组中的Ⅰ型、Ⅱ型胶原和VEGFmRNA表达比PBS对照组,明显增加。体内实验的荧光免疫组织化学结果显示,在术后第10周,CTGF治疗组中的Ⅰ型、Ⅱ型胶原和VEGF已完全填充撕裂损伤处。PBS-蛋白胶组中,损伤处仍有明显裂隙。结论:CTGF可促进半月板无血管区重要的细胞外基质(Ⅰ型、Ⅱ型胶原)的合成,同时损伤处VEGF的表达活性明显增强,有利于促进无血管区半月板撕裂损伤的愈合。Objective：To investigate effect of connective tissue growth factors （CTGF） on secretion of extracellular matrix synthesis of meniscal fibrochondrocytes,expression of vascular endothelial growth factors （VEGF）,and angiogenesis during the repair of meniscal tearing damage. Methods：Meniscal fibrochondrocytes were isolated from the inner -1 / 2 of rabbits＇ meniscus by collagenase enzymatic digestion,centrifugal separation,and treated with 100 ng / ml CTGF in vitro. Characteriza- tion of fibrochondrocytes was identified by flow cytometry analysising CD31 ,CD44,CD45 and CD105,and was further tested by typeⅡcollagen immunocytochemistry. Changes in gene expression of meniscal fibrochondrocytes were monitored by quantitative real-time polymerase chain reaction. In vitro,the sections of the 3 mm of the longitudinal teared in the middle of the rab- bit＇s meniscus,and then the defects were dealed with simple suture,suture and implanting with PBS-fibrin glue,sutured and implanting with 1.5 μg CTGF respectively. Expression and distribution of typeⅠandⅡcollagen and VEGF,the tearing healing were observed by fluorescence-immunohistochemisty analysis on the 1st week ,the 4th week and the 10th week. Results： Quantitative RT-PCR assays showed that type Ⅰand type Ⅱcollagen,and VEGF mRNA expression in the 100 ng / ml CTGF group had been remarkably enhanced than in the PBS group on the 14th day. Consistent with these effects in vitro ,fluores-cence-immunohistochemical analysis revealed that in the group implanted with CTGF-fibrin glue ,typeⅠcollagen,typeⅡcollagen and capillaries completely filled the defect on the 10th week postoperatively. In contrast ,only soft tissue repair occurred after the PBS-fibrin glue was implanted. Conclusion：CTGF can significantly promote extracellular matrix （Ⅰcollagen,Ⅱcollagen） of the meniscal avascular zone synthesis,and CTGF can greatly heighten the expression of VEGF activity at the same time in vitro,so that it can further enhance the repair of meniscal tear

关 键 词：结缔组织生长因子 纤维软骨细胞 半月板 血管内皮生长因子类 胶原Ⅰ型 胶原Ⅱ型 

分 类 号：R692[医药卫生—泌尿科学]