微囊化转染神经生长因子基因细胞移植在周围神经再生中的作用

目的探讨应用免疫隔离技术进行转染神经生长因子（NGF）基因的3T3细胞移植促进周围神经损伤后再生的可行性.方法采用海藻酸钠-聚赖氨酸-海藻酸钠（APA）微胶囊包埋NGF-3T3细胞并进行培养;制备坐骨神经横断损伤SD大鼠模型,96只SD大鼠随机分A组（微囊化NGF-3T3细胞组）、B组（空胶囊组）、C组（转染NGF的3T3细胞组）和D组（阴性对照组）.分别采用神经干动作电位（NAP）、神经传导速度（NCV）和坐骨神经功能指数（SFI）检测坐骨神经功能恢复情况.结果微囊化NGF-3T3细胞培养后保持活性和增殖能力,将具有分化潜能的大鼠嗜铬细胞瘤细胞与其共培养,7 d时细胞胞体分化成多边形或锥形,形成突起;培养10 d左右NGF分泌量最高,达269 pg/ml;培育50 d仍然保持在208 pg/ml.移植术后4、8及12周时,A组大鼠的NAP及NCV均大于B、C、D组（P＜0.05）,而B、C、D三组之间无显著性差异（P＞0.05）;A组大鼠的SFI恢复情况优于B、C、D组（P＜0.05）,而B、C、D三组之间无显著性差异（P＞0.05）.结论微囊化NGF-3T3细胞在体外培养一定时间后,仍保持增殖能力和生物活性,移植到大鼠周围神经损伤局部后可长时间存活,并通过持续分泌NGF起到促进周围神经修复的作用.

Effect of Transplanting Encapsulated Cells Transfected with Nerve Growth Factor Gene on Peripheral Nerve Regeneration Following Nerve Injury

ObjectiveTo investigate the effect of microencapsules cells transfected with nerve growth factor (NGF) gene to the sciatic nerve regeneration following sciatic nerve injury in rats.MethodsMicroencapsules containing cells transfected with NGF gene were prepared using drop generative technique and cells were cultured in vitro. Animal model of sciatic nerve cut and sutured was established with Sprague-Dawely rats, and ninety-six animals were randomly divided into group A (in vivo implantation of microencapsules cells transfected with NGF gene), group B (in vivo implantation of microencapsule), group C (in vivo implantation of cells transfected with NGF gene), and group D (negative control group). The nerve conductive velocity (NCV), nerve action potential (NAP), sciatic nerve function index (SFI) were detected in the 4th, 8th and 12th week postimplantation.ResultsThe microencapsules cells transfected with NGF gene in microencapsules retained reliable cell viability and function. The expanded cells formed cell aggregates, with confocal laser scanning microscopy (CLSM), exhibited green fluorescence material in the cell. The NGF concentration in supernatant were arriving at 269 pg/ml when cultured for 10 days. The results of NCV, NAP and SFI tests in group A were higher than those in the other groups ( P<0.05).ConclusionAfter implantation, microencapsules cells transfected with NGF gene may secrete NGF continuously in vivo, and has significant improvement effect on nerve regeneration following sciatic nerve injury.