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

English Keywords:Dental pulp stem cells  Spinal Cord Injury  Apoptosis

English Abstract:

  【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.