切断周围神经后局部嗅鞘细胞移植对脊髓及神经节内神经元的作用

Effects of olfactory ensheathing cells transplantation into local part on neurons in spinal cord and ganglion after transection of peripheral nerve

目的:观察周围神经切断后局部嗅鞘细胞移植对大鼠脊髓及神经节内神经元的作用,为嗅鞘细胞移植治疗周围神经损伤提供实验依据。方法:实验于2005-08/2006-01在西安交通大学医学院动物实验中心完成。选用健康成年SD大鼠55只,随机数字表法分为3组:对照组25只、实验组25只、空白组5只。①对照组与实验组大鼠在坐骨切迹处游离坐骨神经的各分支,在远端切断,在半腱肌内侧肌膜上切口,将坐骨神经近断端的各游离分支包埋进肌肉,神经外膜与肌膜缝合固定,实验组在缝合口两侧2mm处肌肉中各注射1×109L-1的嗅鞘细胞0.1mL,对照组注射细胞培养液DMEM。空白组仅暴露坐骨神经而不进行切断。②术后1,2,3,7和14d,分批处死对照组和实验组大鼠,每次5只,空白组于14d后处死,分别进行病理及TUNEL法观察神经元的改变。结果:实验共选用大鼠55只,全部进入结果分析。①坐骨神经损伤后各组不同时间点苏木精-伊红染色观察伤侧前角运动神经元存活率变化:术后7,14d,实验组大鼠脊髓前角运动神经元的形态改变与对照组相似,但变性数目明显少于对照组,存活神经元数目明显多于对照组(P<0.01)。②坐骨神经切断后各组坐骨神经损伤侧前角运动神经元凋亡指数变化:在术后第3,7,14天,对照组明显多于实验组犤(2.1±1.1)%,(1.2±0.8)%;(3.1±1.1)%,(1.4±0.6)%;(6.1±1.8)%,(4.1±1.3)%,P<0.05犦。③坐骨神经切断后各组坐骨神经损伤侧神经节神经元凋亡指数变化:7,14d时,实验组的神经节内的感觉神经元凋亡阳性细胞要较对照组少犤(2.1±0.32)%,(4.4±0.56)%;(4.3±1.8)%,(6.7±2.5)%,P<0.05犦。结论:在周围神经损伤后脊髓及神经节内神经元有凋亡发生,嗅鞘细胞移植对神经元凋亡有保护作用。

AIM： To observe the effects of local transplantation of olfactory ensheathing cells （OECs） on neurons in spinal cord and ganglion after incision of peripheral nerves, so as to provide experimental basis for treatment of peripheral nerve injury by OECs transplantation. METHODS： The experiment was conducted in the Experimental Center of Animals, Medical College of Xi ＇an Jiao Tong University from August 2005 to January 2006. Fifty-five healthy SD adult rats were selected and randomly divided into 3 groups： control group （n=25）, experiment group （n=25） and blank control group （n=5）. ①Rats in the control group and experiment group were liberated of all branches of sciatic nerve in the ineisura isehiadica and transected at the distal end. A incision was made on the inner lateral tunica muscularis of semitendinosus to embed all free branches of broken end into the muscle. The perilemma and tunica museularis were sutured to fix, and rats in the experiment group were injected with 0.1 mL of OECs （1×10^9 L^-1） at the muscle 2 mm away from the bilateral parts of incision. Rats in the blank control group were injected with cell culture fluid DMEM. Rats in the blank control group were exposed the sciatic nerve without transection. ②Rats in the control group and experiment group were executed in batch respectively at 1, 2, 3, 7 and 14 days after operation with 5 rats each time, while rats in the blank group were executed at 14 days after operation, and changes in neurons were observed with pathological and TUNEL methods. RESULTS： A total of 55 enrolled rats were involved in the analysis of results. ①Changes in the survival rate of motor neurons of anterior horn were studied by HE staining in each group after sciatic nerve injury at different time-point： At 7 and 14 days after operation, the morphous changes of motor neurons of cornu anterius medullae spinalis in rats of the experiment group were similar to those in the control group, while the amount of degeneration were significantly less than those in the control group, and the survival nerve neurons were obviously greater than those in the control group （P 〈 0.01）. ②Changes in apoptotic index of motor nerve in the anterior horn of injured side in each group after transection of sciatic nerve： Those in the control group at 3, 7 and 14 days after operation were remarkably greater than those in the experiment group [（2.1±1.1 ）%, （1.2±0.8） %; （3.1±1.1）%, （1.4±0.6） % ; （6.1±1.8）%, （4.1±1.3）%, P 〈 0.05]. ③ Changes in apoptotic index of neurons in the ganglion of injured side in each group after transection of sciatic nerve： the apoptotic positive cells of sensory neurons in ganglion at 7 and 14 days later were less in the experiment group than those in the control group [（2.1±0.32）%, （4.4±0.56）%; （4.3±1.8）%, （6.7±2.5）% ,P 〈 0.05]. CONCLUSION： There are apoptotic neuron in the spinal cord and ganglion after peripheral nerve injury, while the OECs has protective effect on apoptotic neurons.