bFGF/BMP-2基因修饰骨髓间充质干细胞促进前交叉韧带重建术后腱骨愈合的机制研究

The integration of tendon grafts anchored within bony tunnels is still sometimes unsatisfactory after anterior cruciate ligament (ACL) reconstruction. The application of bone mesenchymal stem cells (BMSCs) has shown some improvements in the healing of tendon grafts in bone tunnels. However, BMSCs alone are insufficient to augment tendon-bone healing where the repair site may lack the cellular and/or molecular signals necessary to induce appropriate differentiation of the transplanted cells. We found that basic fibroblast growth factor (bFGF) could promote the proliferation of cells and cartilage matrix repair in our previous work. There has few reports about BMSCs genetically modified with bFGF gene to augment tendon-bone healing at home and abroad. It was also confirmed that the bone morphogenetic protein 2 (BMP-2) was conducive to tendon-bone healing. Adenoviral vectors encoding human bFGF, BMP-2 were constructed in the study. Then, BMSCs genetically modified with bFGF or/and BMP-2 gene were implanted in the tendon-bone interface in the rabbit model of ACL reconstruction. The quality of the tendon-bone healing was evaluated. At the same time, the genetically modified rabbit BMSCs were seeded in alginate microspheres and co-cultured with tendon fibroblasts in vitro. The mRNA and protein expression of relative genes were evaluated by real time PCR and western blotting. The aim of the study was to investigate BMSCs genetically modified with bFGF or/and BMP-2 gene on the effect of tendon-bone healing and expect to reach a similar physiological typical 4-layer structure: the tendon, fibrocartilage, mineralized fibrocartilage, and bone. The results will provide new ideas for the solution of tendon-bone healing after ACL reconstruction.

前交叉韧带(ACL)重建术后存在腱骨不愈合或愈合不良等问题。骨髓间充质干细胞(BMSCs)可促进腱骨愈合，但植入腱骨界面后，因缺乏足量诱导分化的细胞因子使其促愈合效应降低。本项目组前期研究发现碱性成纤维细胞生长因子(bFGF)可促进细胞增殖及软骨基质修复。bFGF基因修饰BMSCs促进腱骨愈合的研究国内外鲜见报道。已知骨形态发生蛋白2(BMP-2)有利于腱骨愈合。本研究拟构建bFGF及BMP-2腺病毒载体，将bFGF或/和BMP-2基因修饰的BMSCs植入兔ACL重建模型腱骨界面，评估腱骨愈合质量；将基因修饰的兔BMSCs复合海藻酸钠微球，与肌腱成纤维细胞体外共培养，定量PCR及免疫印迹法检测相关基因mRNA及蛋白的表达，探讨bFGF单独及联合BMP-2基因修饰BMSCs在促进腱骨愈合中的作用，以期达到类似生理状态时ACL止点的典型四层结构，为ACL重建术后腱骨愈合的解决提供新的思路。

背景：腱骨愈合对于前交叉韧带重建术后恢复至关重要。由于腱骨界面愈合能力有限，许多策略包括生长因子和基因转染被用来促进前交叉韧带（ACL）重建术后腱骨愈合。.研究内容：旨在初步探讨bFGF/ BMP2基因转染大鼠BMSCs与韧带成纤维细胞共培养后的生物学效应，并进一步观察家兔ACL重建术后采用bFGF/ BMP2基因转染的BMSCs是否可有效提高腱骨界面的愈合，从而增强其生物力学强度。 .材料与方法：构建编码bFGF和BMP2基因的重组腺病毒载体。细胞水平，培养大鼠BMSCs和韧带成纤维细胞，检测腺病毒对BMSCs的转染效率及转染后目的基因表达。将BMSCs和韧带成纤维细胞三维共培养6天后，检测两种细胞细胞增殖和相关基因mRNA表达改变。动物水平，培养兔BMSCs，将腺病毒转染的BMSCs植入ACL重建术后腱骨界面处。术后4周和12周取材，观察兔膝关节大体形态学，组织学、micro-CT和生物力学分析腱骨界面愈合情况。.结果及结论：细胞水平，流式鉴定和特殊染色表明第3代大鼠BMSCs已经较为纯化且具有多向分化潜能；重组腺病毒载体转染大鼠BMSCs最佳MOI 值为70； AdbFGF和AdBMP2转染大鼠BMSCs后，相应基因mRNA和蛋白表达升高；共培养后，AdbFGF组和AdbFGF plus AdBMP2组BMSCs和韧带成纤维细胞细胞增殖明显增强，但AdBMP2组细胞增殖无明显改变。对于BMSCs，单独的bFGF可促其成纤维细胞分化，单独的BMP2可促其向成骨方向分化，但联合基因促其成骨分化且较单独的BMP2具有更强的促成骨分化能力，对于韧带成纤维细胞，单独的bFGF和联合基因均可降低其基质合成，BMP2对其无明显影响。动物水平，兔BMSCs转染后，相应基因和蛋白表达升高；HE染色表明12周时腱骨界面bFGF组、BMP2组和bFGF plus BMP2组均出现类软骨细胞，且bFGF plus BMP2组软骨区域最宽；micro--CT表明12周时各组胫骨骨隧道面积明显减小，且bFGF plus BMP2组骨隧道面积最小；生物力学结果显示各组最大载荷和刚度在4周和12周时均明显增强，且bFGF plus BMP2组最强。bFGF/BMP2基因修饰的BMSCs 移植能加速腱骨愈合过程，且联合基因较单基因更有效。