NT-6/TGF-β1双基因转染嗅鞘细胞自体移植对骨盆骨折致腰骶神经损伤的修复作用

Pelvic fractures caused by high-energy can damage lumbosacral nerve. The effect of treatment is poor. It is the problem and the hot spots of orthopedics. Due to factors such as anatomy, lumbosacral nerve cannot be treated by conventional methods, such as direct anastomosis, nerve graft to repair. That causes the patient difficult to recover, and high morbidity. Can genetic engineering be applied to solve the problem and seek the method to repair lumbosacral nerve? It is the goal of orthopaedics. As a kind of new technology, Genetic engineering has been successfully applied in many fields. Looking for targeted gene and adapted carrier is the decisive factor for successful gene engineering. The gene transfection can make the expression of cell function better, but multi-gene transfection technique is difficult in genetic engineering. We have preliminary master double gene transfection technique and successful transfected and expressed BMP - 2 and bFGF successfully. We intend to use olfactory ensheathing cells as the best carrier and a retrovirus mediated NT-6/TGF-beta 1 double gene transfection to OECs and autograft to achieve the repair of lumbosacral nerve. Olfactory ensheathing cell itself has the function of spinal cord repair, and combined with nerve growth factor gene transfection, it is more effective to make the olfactory ensheathing cells close to the function of the nerve cells, and achieve the goal of lumbosacral nerve repair.

高能量导致的骨盆骨折易损伤腰骶神经，治疗效果差，是目前骨科治疗的难题、探讨的热点。由于解剖等因素，腰骶神经无法通过常规方法，如直接吻合、神经移植等来修复，导致病人难以康复，致残率高。能否应用基因工程的方法探索寻求治疗特殊部位的腰骶神经损伤，是骨科界期盼的目标。基因工程作为一种新技术，已成功应用多个领域。寻找定向基因与适配载体是基因工程成功的决定因素，且多基因转染可使细胞功能更好的表达，但多基因转染技术是基因工程的一个难点。我们已初步掌握双基因转染技术，并将BMP-2及bFGF两个基因成功转染表达。我们拟应用嗅鞘细胞作为基因工程最佳适配载体，采用逆转录病毒介导NT-6/TGF-β1双基因转染自体OECs并移植，达到腰骶神经修复目的。借助嗅鞘细胞本身具有的脊髓修复功能，附加神经生长因子多基因转染，更加有效表达NT-6和TGF-β1，使嗅鞘细胞具有接近神经细胞的功能，达到修复腰骶神经功能的目的。

我们通过构建了双拷贝逆转录病毒载体PN2A- NT-6/TGF-β1， PA317-NT-6/TGF-β1，感染嗅鞘细胞后，筛选获得OECs- NT-6/TGF-β1基因工程细胞株。建立了骨盆骨折致腰骶神经损伤模型的大鼠模型。并对大鼠移植OECs- NT-6/TGF-β1基因工程细胞株，进行电生理和和后肢功能观察检测。成功构建双拷贝逆转录病毒载体，并建立了OECs- NT-6/TGF-β1基因工程细胞株，对腰骶神经损伤的大鼠模型进行细胞移植后，定期进行电生理及后肢功能观察检测，测定了神经功能恢复情况，并通过神经组织染色验证了OECs- NT-6/TGF-β1细胞移植对腰骶神经损伤的修复作用。NT-6/TGF-β1双基因转染嗅鞘细胞自体移植可以促进腰骶神经损伤的恢复，具体作用机制可能与促进神经纤维再生、抑制神经细胞凋亡相关，在今后骨盆骨折所致的腰骶神经损伤患者中，有潜在的临床应用价值。