同种异体脱细胞神经支架复合脑源性神经营养因子和睫状神经营养因子转染骨髓间充质干细胞桥接受损坐骨神经

Chemical extracted acellular nerve allograft matrix laden with BDNF and CNTF-engineered BMSCs repairs sciatic nerve injury

目的:探讨同种异体脱细胞神经支架(CEANA)复合脑源性神经营养因子(BDNF)和睫状神经营养因子(CNTF)转染骨髓间充质干细胞(BMSCs)桥接修复周围神经缺损的优缺点及可行性。方法:取2只成年雄性犬的骨髓进行BMSCs的分离、培养。取传代培养的第3代细胞,采用电转法将重组表达载体pIRES-BDNF、pIRES-CNTF和pIRES-BDNF-CNTF转染至BMSCs。细胞转染后分成5组,采用反转录半定量PCR(RT-PCR)检测各组BMSCs的BDNF和CNTF的mRNA表达水平;采用免疫印迹检测其蛋白表达水平。取3只雄性实验犬,分离双侧坐骨神经,化学法制备CEANA。取15只雄性实验犬制备坐骨神经缺损模型,随机分为CEANA+BDNF组、CEANA+CNTF组、CEANA+BDNF+CNTF组、CEANA组(采用8cm CEANA端端吻合缺损的坐骨神经)、CEANA+BMSCs组。术后16周观察各组实验犬移植段神经横断面轴突数目、比目鱼肌的动作电位、双侧坐骨神经传导速度的差异。结果:手术后前3组实验犬的切口愈合良好。手术后第8周,前3组的髓鞘厚度、有髓神经纤维总数为CEANA+BDNF+CNTF组>CEANA+CNTF组>CEANA+BDNF组,CEANA+BDNF组和CEANA+CNTF组比较差异有统计学意义。结论:CEANA复合BDNF和CNTF转染BMSCs桥接移植修复犬坐骨神经缺损有良好的作用。

To investigate the effect of chemical extracted acellular nerve allograft(CEANA)with brain-derived neurotrophic factor(BDNF)and ciliary neurotrophic factor(CNTF)-engineered bone marrow derived mesenchymal stem cells(BMSCs)on the peripheral nerve regeneration of the canines.Methods:Two adult male canines were selected.Bone marrow was used to isolate and culture BMSCs.The subculture of the third generation of cells was taken to transfect.The recombinant expression vector pIRES-BDNF,pIRES-CNTF and pIRES-BDNF-CNTF were transfected to BMSCs electrotransfection.Cells after transfection were divided into 5 groups.RT-PCR was used to detect mRNA expression of BDNF and CNTF in each group of BMSCs.Western blotting was used to test BDNF and CNTF protein expression level in the BMSCs of each group.Then sciatic nerves were harvested from the three canines to prepare CEANA.The other fifteen canines were used to establish the models of left sciatic nerve defect with eight cm and divided into five groups:CEANA+BDNF,CEANA+CNTF,CEANA+BDNF-CNTF,CEANA,CEANA+BMSCs.8 cm CEANA was anastomosed at the defect of sciatic nerve.The right sciatic nerves were exposed as the control.The nerve grafts were evaluated by transmission electron microscope observed at eight weeks after operation.Results:After operation,the previous three groups of experimental canine incisions healed well.Eight weeks after the surgery,myelin sheath of the previous three groups thickened,and the total number of myelinated nerve fibers in group were as follows:CEANA+BDNF-CNTF>CEANA+CNTF>CEANA+BDNF.The difference between CEANA+BDNF group and CEANA+CNTF group was statistically significant.Conclusion:CEANA supplemented with BDNF,and CNTF-engineered BMSCs can improve functional recovery of peripheral nerve injury.