BMP-2/TGF-β1基因修饰韧带干细胞定向分化促进ACL重建术后生物学愈合的实验研究

How to promote tendon-to-bone healing and ligamentation of the transplanted tendon is of great significance for rehabilitation of patients after ACL reconstruction surgery. In our previous study, we confirmed that the healing process of tendon-to-bone could be accelerated by local application of bone marrow-derived mesenchymal stem cells (BMSCs). Recently, we isolated stem cells from human and rat ligament tissues, which represented superior proliferation and chondrogenic differentiation potential when compared to BMSCs. Based on our previous findings, this study intends to transfect BMP-2 and TGF-β1 gene by adenovirus (Ad) to ligament-derived stem cells (LDSCs), which can separately induce the cells to differentiate towards osteogenic and ligamentogenic lineages. Then, TGF-β1 transfected LDSCs (Ad- TGF-β1-LDSCs) were utilized to construct cell sheets to wrap the tendon during the process of ACL reconstruction for accelerating ligamentation process. Meanwhile, cell sheets of BMP-2 transfected LDSCs (Ad- BMP-2-LDSCs) were implanted to the bone tunnel side in the same approach for inducing new bone formation. This study will evaluate the efficacy of the constructed tissue engineered complex on the tendon-to-bone healing and tendon ligamentation process by animal models. Also, the proliferation, differentiation, migration and gene transfection efficacy of the transplanted LDSCs were analyzed by in vivo and in vitro tests. This study tries to render an optimal effect on promoting biological healing after ACL reconstruction by optimum combination of different tissue engineering techniques, including new seed stem cells, genetic modification and cell sheet technology. In summary, this study will provide theoretical basis and new clues for exploring innovating biological treatment strategies for ACL reconstruction.

如何促进ACL重建术后腱骨愈合及移植肌腱韧带化对尽快恢复膝关节功能至关重要，课题组前期发现局部使用骨髓间充质干细胞(BMSCs)能够促进腱骨愈合强度。近期，我们从人和大鼠的韧带组织中分离出韧带来源干细胞（LDSCs），发现其具有更强的增殖和成软骨分化潜能。因此，本课题拟选用LDSCs作为种子细胞，体外转染BMP-2和TGF-β1基因靶向诱导LDSCs分别向骨和韧带细胞分化，并构建细胞膜片定向植入到移植肌腱表面（Ad- TGF-β1-LDSCs）和骨隧道侧(Ad- BMP-2- LDSCs)。通过ACL重建动物模型，综合评价该组织工程技术对移植物生物学愈合的促进作用，观察种子细胞在ACL生物愈合过程中增殖、分化、迁移等生物学行为以及基因修饰对其影响。本课题将新型种子干细胞、基因转染、细胞膜片等组织工程技术进行优化组合，为探索ACL重建术后生物学治疗策略提供理论依据和新的思路。

前交叉韧带（anterior cruciate ligment，ACL）撕裂是最常见的运动损伤之一。其相关疾病是人类劳动能力丧失和生活质量下降最常见的原因，给家庭和社会带来沉重的经济负担。ACL 重建能否成功很大程度上取决于移植肌腱和骨隧道的生物学整合，而早期腱骨结合界面是最薄弱的环节，因此，如何促进腱骨早期愈合具有具有十分重要的理论价值和临床意义。根据我们的前期研究发现，局部植入骨髓间充质干细胞（BMSCs）能够有效促进腱骨早期愈合。另外，我们从人前交叉韧带中成功分离出CD34+细胞（CD34+LDSCs），发现其具有更优的增殖和成韧带分化潜能，动物研究也显示其促进腱骨愈合的治疗作用强于BMSCs。因此，我们推测，韧带来源的CD34+细胞，在腱骨界面微环境可能具有更强增殖分化潜能和修复特性，从而更适合作为促进腱骨愈合的种子细胞。通过本项目的实施，课题组在前期研究基础上采用腺病毒携载 TGF-β1和BMP-2 联合转染LDSCs并植入腱骨界面，以期在局部高浓度生长因子微环境下LDSCs 能够向成骨和成腱双向分化，从而促进腱骨界面的早期愈合。我们成功构建BMP2、 TGFβ1、 BMP2/TGFβ1重组腺病毒，其转染种子细胞效率可，并得到免疫荧光、定量PCR及western blot结果证实；BMP2和 TGFβ1（单一或联合）重组腺病毒显著影响种子细胞增殖与凋亡；其基因转染种子细胞后，可显著影响成骨及成韧带基因的表达模式；成功构建了小鼠前交叉韧带重建模型及AdBMP2/TGFβ1-CD34+LDSCs 细胞膜片，并植入其骨隧道；组织学观察，结果显示与BMP2、TGFβ1转染组比较，BMP2/TGFβ1共转染组腱骨界面愈合较好，并得到生物力学实验结果进一步证实；micro-CT评估新生骨的形成情况，得到类似的结果。本项目系统揭示了促进腱骨界面愈合的分子演变模式，为后续针对腱骨界面愈合的分子机制进一步深入研究奠定了重要的理论基础，更为今后新的腱骨愈合生物学治疗策略提供理论依据。