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目的研究骨形成蛋白2(bone m ophogenetic prote in 2,BM P-2)重组腺病毒(adenov irus)转染骨髓基质细胞(m esenchym a l stem ce lls,M SC s)后,与纤维蛋白凝胶构成的复合物(A d-BM P-2+M SC s-纤维蛋白凝胶)对兔关节软骨缺损修复的影响。方法①A d-BM P-2转染原代培养的M SC s,通过RT-PCR、细胞免疫组织化学染色、甲苯胺蓝染色等观察转染后3~9 d的M SC s其BM P-2、Ⅱ型胶原及蛋白多糖转录、表达水平的变化。②转染后的M SC s种植于纤维蛋白凝胶,在体外培养1~9 d,通过上述指标及透射电镜观察三维培养条件下其软骨基质的产生。③42只日本大耳白兔先制成直径4.5 mm全层软骨缺损模型,随机分为3组(n=14):A组为A d-BM P-2+M SC s-纤维蛋白凝胶修复组,B组为M SC s-纤维蛋白凝胶修复组,C组为空白对照组。术后4、8和12周取材行大体观察、HE染色、甲苯胺蓝染色、Ⅱ型胶原免疫组织化学及12周关节软骨弹性常数检测。结果①A d-BM P-2转基因M SC s的BM P-2、Ⅱ型胶原mRNART-PCR检测分别在3、5 d呈阳性,与对照组比较差异有统计学意义(P<0.05)。②转基因M SC s三维培养物免疫组织化学和甲苯胺蓝染色呈阳性,电镜显示细胞生长良好,有基质合成。③A组各时间点大体、组织形态学和组织化学染色均显著优于B组和C组,12周时力学和组织学已接近正常关节软骨。结论A d-BM P-2能通过促进M SC s分泌BM P-2,诱导Ⅱ型胶原和蛋白多糖的表达,在纤维蛋白三维支架中形成软骨基质,移植入兔关节软骨缺损区可修复直径4.5 mm缺损,其修复组织成分接近正常软骨。
Objective To investigate the effect of recombinant adenovirus isolated from bone morphogenetic protein 2 (BM-2) transfected bone marrow stromal cells (M SCs) (A d-BM P-2 + M SC s-fibrin gel) on repair of articular cartilage defects in rabbits. Methods ① Ad-BM P-2 transfected primary cultured SCCs were observed by RT-PCR, immunohistochemical staining and toluidine blue staining for 3 to 9 days. 2, type Ⅱ collagen and proteoglycan transcription, expression level changes. ② After transfection, M SCs were cultured in fibrin gel and cultured in vitro for 1 to 9 days. The production of cartilage matrix under three-dimensional culture conditions was observed by the above indexes and transmission electron microscopy. Forty-two Japanese white rabbits were divided into three groups (n = 14): 4.5 mm in total thickness of cartilage defect model: Group A was A d-BM P-2 + M SC s-fibrin gel repair group, Group B was M SC s-fibrin gel repair group, and group C was a blank control group. At 4, 8 and 12 weeks after operation, the specimens were observed by HE staining, toluidine blue staining, type II collagen immunohistochemistry and elastic modulus of articular cartilage at 12 weeks. Results ① The mRNA and protein expression of BM-P-2 and collagen-Ⅱ of md-BM P-2 transgenic M SCs were positive at 3 and 5 d, respectively, which was significantly different from that of the control group (P <0.05). Transgenic M SC s three-dimensional culture immunohistochemistry and toluidine blue staining was positive, electron microscopy showed that cells grow well with matrix synthesis. ③A group at each time point roughly, histomorphology and histochemical staining were significantly better than the B group and C group, 12 weeks, mechanical and histological close to normal articular cartilage. Conclusions A d-BM P-2 can induce the expression of type Ⅱ collagen and proteoglycan by promoting the secretion of BM P-2 from M SC s, forming the cartilage matrix in the fibrin three-dimensional scaffold and transplanting into rabbit articular cartilage defect area to repair the diameter 4.5 mm defect, the repair of tissue components close to normal cartilage.