人胚嗅鞘细胞与鼠胚胎脊髓组织联合移植对大鼠脊髓损伤的治疗

背景:脊髓损伤高发且后果严重,至今尚无有效措施挽救丧失的神经功能。哺乳动物嗅觉系统的一类特殊胶质细胞的移植引起关注。目的:观察人胚嗅鞘细胞和大鼠胚胎脊髓共同移植在促进大鼠横断脊髓轴索再生方面是否产生协同作用。设计:开放性实验。单位:无锡第三人民医院细胞室,苏州大学附属第一医院神经外科,东南大学附属中大医院神经外科。材料:实验于2002-09/2004-10在无锡市第三人民医院细胞实验室完成。①选取清洁级成年雌性SD大鼠36只,随机数字表法分成4组:人胚嗅鞘细胞组10只、鼠胚胎脊髓组10只、联合移植组10只,模型组6只。②取12周左右的流产新鲜人胚胎(产妇知情同意)用于嗅鞘细胞的培养纯化。③取孕14d的SD大鼠1只,施行剖腹手术将胎鼠连同胎膜一同取出,用于新鲜胚胎脊髓的制备。方法:①4组大鼠均建立脊髓半切洞模型。模型组在损伤洞腔内填塞明胶海绵加全培养基8μL,在损伤上下各1mm处注射相同培养基2μL;人胚嗅鞘细胞组明胶上给予嗅鞘细胞悬液8μL,损伤上下各1mm处注射细胞悬液2μL;鼠胚胎脊髓组将组织碎块直接填塞在洞腔内,外覆明胶;联合移植组将同样大小的胚胎脊髓填塞在洞腔内,然后用微量加样器将8μL的嗅鞘细胞悬液注入洞腔,外覆明胶,在洞腔上下各1mm处注射细胞悬液2μL,按层缝合肌层皮肤。②定期对各组大鼠进行行为学评定,结合病理学观察,并通过辣根过氧化物酶-四甲基联苯胺逆行示踪技术,评价嗅鞘细胞和胚胎脊髓对神经元存活、纤维再生的影响。主要观察指标:①人胚嗅鞘细胞体外培养及纯化。②体外免疫细胞化学分析。③大鼠后肢运动功能BBB评分测定结果。④移植物及受损脊髓修复的免疫组化检测结果。⑤辣根过氧化物酶-四甲基联苯胺示踪对各组被标记的皮质及中脑红核神经元的定量分析情况。结果:①人胚嗅鞘细胞大部分呈双极纺锤型,培养5～7d左右,细胞相互交织成网状,可见大量细胞分裂相。纯化后的细胞纯度为85%。②P75阳性细胞率为(83±7)%,大约(81±6)%的细胞呈胶质纤维酸性蛋白阳性,(91±9)%的细胞呈Vimentin阳性,Nestin阳性率为(77±5)%。③术后3～5d,模型组伤肢开始挛缩,正常侧下肢活动稍受限,其余3组少见明显挛缩症状。从术后2周开始,各组动物行为功能的恢复幅度明显增快,联合移植组BBB评分明显高于人胚嗅鞘细胞组、鼠胚胎脊髓组、模型组(6.2±1.13),(5.0±1.15),(3.9±0.88),(3.3±1.03)分,P<0.05]。④人胚嗅鞘细胞组、联合移植组均在移植部位及移植区2.0～5.0mm范围内见到P75及胶质纤维酸性蛋白阳性双极或多极细胞,同时多极细胞中发现碱性蛋白( )颗粒。鼠胚胎脊髓组、联合移植组脊髓缺损灶内存在大量MAP2阳性反应的细小神经元。人胚嗅鞘细胞组、鼠胚胎脊髓组、联合移植组在脊髓缺损区内都不同程度观察到神经丝阳性纤维存在,尤以联合移植组最为明显,模型组未能找到神经丝阳性纤维存在。⑤模型组损伤侧神经元基本无辣根过氧化物酶标记,而联合移植组标记的皮质、中脑红核神经元的数量均显著高于人胚嗅鞘细胞组、鼠胚胎脊髓组(P<0.05)。结论:嗅鞘细胞和胚胎脊髓联合移植对损伤脊髓具有明显保护作用且促进宿主脊髓轴突再生,在加快大鼠的功能恢复中起到了互补和协同的作用。

Combined transplantation of human fetal olfactory ensheathing cells and rat embryonic spinal cord tissues in the treatment of spinal cord injury in rats

BACKGROUND: Spinal cord injury occurs frequently and its consequence is very severe. There is no effective method to rebuild the function of demylinated nerves. Transplantation of a kind of special glial cells in olfactory system of mammal attracts more attention.OBJECTIVE: To observe whether combined transplantation of human fetal olfactory ensheathing cells (human OECs) and rat embryonic spinal cord tissues (rat ECS) possesses synergistic effect in promoting axonal regeneration in the rats following spinal cord transection.transection.DESIGN: Open experiment.SETTING: Cell Room, Third People's Hospital of Wuxi City; Department of Neurology, First Hospital Affiliated to Soochow University; Department of Neurosurgery, Zhongda Hospital Affiliated to Central South University MATERIALS: This experiment was carried out in the cell laboratory of the Third People's Hospital of Wuxi from September 2002 to October 2004. ① Totally 36 adult female SD rats, of clean grade, were selected and randomly divided into 4 groups: human OECs group (n=10), rat ECS group (n=10), combined transplantation group (n=10) and sham-operation group (n=6). ② Fresh 12-week aborted human embryo was used for culture and purification of human OECs (Informed consent was obtained from the parturient). ③One SD rat at embryonic 14 days underwent caesarean operation, and fetal rat and fetal membrane were taken out together and used for preparing new embryonic spinal cord.METHODS: ①Rats of 4 groups were all created into hemisection cavity models. Gelatin sponge and complete culture medium of 8 μL were packed into the injured cavity of rats in the model group, and the same culture medium of 2 μL was injected at 1 mm above or below injure; Human OECs suspension of 8 μL was added to gelatin sponge in human fetal Human OECs group, and human OECs suspension of 2 μL was injected at 1 mm above and below injure; rat embryonic spinal cord tissue of rat ECS group was chipped into pieces, which were packed into the cavity,and gelatin sponge was spread on the injury part. Embryonic spinal cord with the same size was packed into the cavity of combined transplantation group, then 8 μL human OECs suspension was injected into cavity with micro sample injector, and gelatin sponge was spread on the injury part, and then cellular suspension of 2 μL was injected at 1 mm above and below the cavity, and muscular layer skin was sutured layer by layer. ②The rats of each group were performed ethological evaluation periodically. Combined with pathological observation, effect of human OECS and rat ECS on neuronal survival and regeneration was evaluated by performing horseradish peroxidase-tetramethyl benzidine tracer technique.MAIN OUTCOME MEASURES: ①In vitro culture and purification of human fetal human OECs. ② In vitro immunocytochemistrical analysis. ③BBB scoring of motor function of hindlimb of rats. ④ Immunohistochemical detection of implants and injured spinal cord repair⑤ Quantitative analysis on labeled neurons at the cortex and mesencephalic red nucleus ineach group with horseradish peroxidase-tetramethyl benzidine tracer technique.RESULTS: ① Most of human fetal OSCs presented double-polar spindle.Five to seven days after culture, OSCs weaved into net and a lot of mitosis phases were found. The cellular purity was 85%. ② The rate of P75 positive cells was (83±7)%. Glial fibrillary acidic protein was found in about (81±6)% of cells and Vimentin in (91±9)% of cells and the rate of Nestin positive was (77±5)%. ③Three to five days after operation, affected limb of rats of sham-operation group began to contract, the activity of hindlimb of intact side was limited a little. Fewer obvious contraction symptoms were found in the other 3 groups. From 2 weeks after operation,behavioral function recovered significantly fast in each group. BBB scores of combined transplanted group were significantly high than those of human OECs group, rat ECS group and sham-operation group (6.2±1.13) vs.(5.0±1.15)vs.(3.9±0.88)vs.(3.3±1.03)scores,P ＜ 0.05]. ④In bipolar or multipolar cells, in which basic protein(+)granules were found, P75 and glial fibrillary acidic protein positive were found at the implanted part in the range of 2.0 to 5.0 mm of transplanted region in the human OECs group and combined transplantation group. A great many of small MAP2 positive neurons were found in the spinal defected focus in the rat ECS group and combined transplantation group. Nerve plexus positive fibers were observed in spinal defected region of human OECs group, rat ECS group and combined transplantation group to different extents, especially significantly in the combined transplantation group, but they were not found in the model group. ⑤ Horse radish peroxidase labeling was hardly found in neurons at the injured side of sham-operation group, while the number of labeled neurons at the cortex and nesencephalic red nucleus was significantly higher in the combined transplantation group than in the human OECs group and rat ECS group (P ＜ 0.05).CONCLUSION: Combined transplantation of OECs and ESC can obviously protect injured spinal cord, promote host spinal axonal regeneration and play s a complementary and synergetic effect in speeding up the functional recovery of rats.