牙髓干细胞移植治疗大鼠脊髓损伤的实验研究

Research on dental pulp stem cells in the treatment of spinal cord injury in rat

中文关键词:  脊髓损伤  牙髓干细胞  移植  细胞凋亡

英文关键词:Dental pulp stem cells  Spinal Cord Injury  Apoptosis

中文摘要:

  【摘要】 目的: 探讨牙髓干细胞移植治疗脊髓损伤的神经修复及运动功能恢复情况。方法：培养取材第2代来源于第三磨牙的人牙髓干细胞，鉴定其表面标记物，应用B27、碱性呈纤维细胞生长因子和胰岛素转铁蛋白硒进行神经诱导，并行免疫荧光染色检测。改良Allen′s法制作SD大鼠脊髓损伤模型，3d后随机分为实验组及对照组，每组各20只大鼠。分别于脊髓损伤处注入人牙髓干细胞及生理盐水，于造模后1d、治疗前1d、治疗后3d、治疗后7、14、28d进行动物后肢运动功能检测。28d时脊髓取材进行HE染色，观察脊髓空洞形成，计算空洞面积；Tunnel法检测两组细胞凋亡情况；免疫荧光双标法标记HuNu-NeuN和HuNu-GFAP，观察人牙髓干细胞体内生长分化情况。结果：细胞培养传代后呈长梭形，细胞形态均匀，胞浆丰富、胞核增大，细胞平行排列呈漩涡状或螺旋状。流式细胞仪分析人牙髓干细胞诱导分化后高表达CD44、CD90和CD146，低表达STRO-1，CD34、CD45表达阴性。人牙髓干细胞神经诱导14d，免疫荧光标记GFAP、NeuN呈阳性。细胞移植3d、7d，两组间BBB评分无统计学差异；细胞移植14d及28d实验组BBB评分分别为3.8±0.8、7.2±1.6，对照组BBB评分为2.2±0.8、3.6±1.1，两组间比较差异有显著性（P＜0.05）。28d时HE染色两组均可见脊髓出血、炎性细胞浸润、小血管增生及空洞形成。实验组脊髓空洞面积百分比（26.75±2.50）%，对照组为（49.50±6.25）%，两组差异有统计学意义（P＜0.05）；Tunnel法检测实验组神经细胞凋亡百分比（32.33±1.54）%，对照组为（46.33±1.53）%，实验组显著减少神经细胞的凋亡（P＜0.05）。免疫荧光双标法检测到部分细胞为带有HuNu-NeuN和HuNu-GFAP双抗体细胞。结论：人牙髓干细胞能够在体外特定条件下及移植入脊髓损伤大鼠体内可分化为神经细胞，用于治疗脊髓损伤时可减少神经细胞的凋亡，促进后肢运动功能的恢复。

英文摘要:

  【Abstract】 Objectives: To investigate the neural restoration and recovery of motor function after transplantation of human dental pulp stem cells in the treatment of spinal cord injury. Methods: Human dental pulp stem cells (hDPSCs), which derived from the second passage of third molars, were cultivated, identified and induced by B27, bFGF and insulin transferrin selenium. Immunofluorescence staining was performed after induction. Animal model of acute spinal cord injury was established by improved Allen′s method. The model rats were randomly divided into 2 groups in 3 days, with 20 rats in each group. The experimental group was injected with hDPSCs, and the control group ones were injected with normal saline. The locomotion function of hind limbs was evaluated by using BBB locomotor score before and after either hDPSCs or normal saline injection. 28 days later, hematoxylin and eosin staining was performed to observe the formation of spinal cord cavity and calculate void area. Cell apoptosis in two groups was detected by tunnel experiment. Double antibody with HuNu-NeuN and HuNu-GFAP were detected by immunofluorescence staining. Results: The hDPSCs after subculture were observed to be long fusiform and whirlpool. Cell morphology presented with more uniform. Its cytoplasm was abundant, and the nucleus were larger. Flow cytometry showed that CD44, CD90 and CD146 expressions were positive in the surface antigens of hDPSCs, STRO-1 was low level, and CD45 and CD34 expressions were negative. The hDPSCs after 14-day neuronal differentiation were detected by immunofluorescent labeling with GFAP and NeuN. The majority of cells were positive stained. BBB scores between two groups showed no difference after transplantation in 3 days or 7 days. However, in 14 days and 28 days after transplantation, BBB score in experimental group was 3.8±0.8 and 7.2±1.6 respectively. In control group, BBB score was 2.2±0.8 and 3.6±1.1 respectively, which showed significant difference between two groups(P＜0.05). Spinal cord hemorrhage, inflammatory cells infiltration, vascular proliferation and cavity formation were observed by HE staining in two groups after 28-day transplantation. Area percentage of syringomyelia in experimental group was (26.75±2.50)% and (49.50±6.25)% in control group(P<0.05). Based on Tunnel results, the percentage of neural cell apoptosis in experimental group was (32.33±1.54)%, while in control group, the percentage rose to (46.33±1.53)%. Compared with control group, there was significant difference(P<0.05). Double immunofluorescent staining was used to detect some cells with HuNu-NeuN and HuNu-GFAP expression, in experimental group, some cells were positive with the double antibody. Conclusions: Human dental pulp stem cells can differentiate into neural cells under some special conditions in vitro and in vivo, which can reduce the apoptosis of nerve cells and promote the hind limbs motor function recovery.