骨髓源神经样细胞移植对兔SCSG受损修复作用的研究
发布时间：2018-11-09 19:05
【摘要】：目的 1、人工体外分离培养兔骨髓间充质干细胞（Bone marrow mesenchymal stem cellsBMSCs）并诱导其分化为骨髓源神经样细胞。 2、兔颈上交感神经节（superior cervical sympathetic ganglion SCSG）损毁模型的建立。 3、将骨髓源神经样细胞植入到SCSG损伤模型体内，观察模型兔瞳孔的变化，并通过测定损伤局部及海马组织中脑源性神经因子（brain-derived neurotrophic factor，BDNF）、睫状神经生长因子（Ciliary Neurotrophic Factor，CNTF）的表达情况，探讨骨髓源神经样细胞移植对兔颈上交感神经节受损修复作用及可能的作用机制。 方法 利用持针钳压榨兔SCSG，制备单侧（右侧）SCSG损伤模型。将体外经密度离心联合贴壁方法分离培养得到BMSCs，用碱性成纤维细胞生长因子（Basic FibroblastGrowth Facto，，bFGF）预诱导，再经CNTF诱导分化为骨髓源神经样细胞。随机地将模型兔分为三组，每组15只。A组为对照组，B组培养液组，C组为细胞移植组。模型制作成功第7天时，A组无处理，B组经耳缘静脉注入细胞培养液1ml，C组注入含骨髓源神经样细胞1×106个的细胞培养液1ml。移植后1天、7天、10天、14天、21天观察兔瞳孔变化，移植后第21天处死兔，分离神经节，进行HE染色，并采用免疫组化分别检测兔SCSG之BDNF和CNTF的表达水平。取兔海马区标本进行HE染色并检测BDNF的表达水平。 结果 1、兔瞳孔直径(mm)的比较：颈上交感神经节损伤后1、7、10、14、21天时，A组兔瞳孔直径分别为：5.81+0.42，5.84+0.34，5.68+0.29，5.91+0.31，5.80+0.36；B组兔瞳孔直径分别为：5.84+0.32，5.74+0.32，5.63+0.37，5.83+0.27，5.58+0.23；C组兔瞳孔直径分别为：5.64+0.33，5.71+0.35，6.78+0.33，7.97+0.28，8.05+0.21。C组在10天、14天时间点，瞳孔直径均明显大于A组（P0.05）。A组、B组不同时间点比较无明显差异（P0.05）。C组不同时间点比较，差异明显（P0.05），14天、21天瞳孔直径明显大于1天、7天、10天（P0.05），14天与21天比较，瞳孔直径差异无统计学意义（P0.05）。 2、BDNF免疫组化染色：A组、B组、C组兔海马区BDNF阳性细胞数分别为：17.13+2.19，18.53+1.35，20.20+4.29，C组比A组、B组BDNF阳性细胞数明显要多，差异有显著性（（P0.05）），A组与B组比较无统计学意义（P0.05）。三组兔损伤侧SCSG之BDNF阳性细胞数分别为：6.67+1.11，6.80+1.26，12.18+7.93，C组比A组、B组BDNF阳性细胞数明显要多，差异有显著性（（P0.05）），A组与B组比较无统计学意义（P0.05）。 3、CNTF免疫组化染色：A组、B组、C组CNTF阳性细胞数分别为：15.6+1.83，15.6+1.54，37.73+7.26，C组明显高于A组、B组（P0.05），而A组与B组比较无统计学意义（P0.05）。 结论 1、骨髓源神经样细胞移植治疗兔SCSG损伤，可促进其受损神经功能的修复。 2、骨髓源神经样细胞移植修复兔SCSG损伤的机制可能与上调BDNF及CNTF有关。
[Abstract]:Objective 1. Rabbit bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cellsBMSCs) were cultured in vitro and induced to differentiate into bone marrow derived neural cells (BMSCs). 2. The (superior cervical sympathetic ganglion SCSG) lesion model of superior cervical sympathetic ganglion in rabbits was established. 3. Bone marrow-derived neuronal cells were implanted into SCSG injury model to observe the changes of pupillary, and to measure the brain derived neurofactor (brain-derived neurotrophic factor,BDNF) in local and hippocampal tissue. The expression of ciliary nerve growth factor (Ciliary Neurotrophic Factor,CNTF (ciliary nerve growth factor) and its possible mechanism in repairing the injury of superior cervical sympathetic ganglion (SCG) in rabbits were investigated by bone marrow-derived nerve-like cell transplantation. Methods unilateral (right) SCSG injury model was established by pressing rabbit SCSG, with needle holding forceps. BMSCs, was preinduced by basic fibroblast growth factor (Basic FibroblastGrowth Facto,bFGF) by density centrifugation and adherent method in vitro, and then differentiated into bone marrow-derived nerve-like cells induced by CNTF. The model rabbits were randomly divided into three groups: control group (group A), culture medium group (group B) and cell transplantation group (group C). On the 7th day after the model was made, no treatment was found in group A, and 1ml of cell culture medium (1 ml) containing 1 脳 106 myeloid nerve cells was injected into group B via auricular vein. Pupillary changes were observed on day 1, day 7, day 10, day 14 and day 21 after transplantation. The ganglion was isolated and stained with HE on the 21st day after transplantation. The expression of BDNF and CNTF in rabbit SCSG were detected by immunohistochemistry. The hippocampal specimens of rabbits were stained with HE and the expression of BDNF was detected. Results 1 comparison of pupillary diameter (mm) in rabbits: the pupil diameter of group A was 5.81 0.42n 5.84 0.45.68 0.295.91 0.31 卤5.80 0.36 on day 1421 after injury of superior cervical sympathetic ganglion. The diameter of pupil in group A was 5.81 0.42n 5.84 0.45.68 0.295.91 0.31 and 5.80 0.36 respectively. The pupil diameter of group B was 5.84 0.32 ~ 5.74 0.32 ~ 5.63 0.37 ~ 5.83 0.27 ~ 5.58 and 0.23 respectively. The pupillary diameter of group C was 5.64 0.33 / 5.71 0.35 / 5.71 0.335.78 0.337.97 0.337.97 0.280.280.05 / 0.21.C respectively. The pupil diameter of group C was significantly larger than that of group A at 10 days and 14 days (P0.05). A group). There was no significant difference at different time points in group B (P0.05). The pupil diameter of group B was significantly larger than that of 1 day, 7 days, 10 days (P0.05), 14 days and 21 days, respectively (P0.05). There was no significant difference in pupil diameter (P0.05). 2the number of BDNF positive cells in hippocampus of group A, group B and group C were 17.13 2.19, 18.53 1.35 and 20.20 4.29, respectively, and the number of positive cells of BDNF in group B was significantly higher than that in group A. There was significant difference (P0.05), A group and B group compared with no statistical significance (P0.05). The number of BDNF positive cells in the injured side of SCSG in the three groups was 6.67 1.116.80 1.26 1.26 12.18 7.93 渭 g / L respectively compared with group A, and the number of BDNF positive cells in group B was significantly higher than that in group A (P0.05),). There was no significant difference between group A and group B (P0.05). 3the number of CNTF positive cells in group A, group B and group C were 15.6 1.83n 15.6 15.6 1.54 + 37.73 7.26C, respectively, higher than those in group A (P0.05), group B (P 0.05), group B (P 0.05). There was no significant difference between group A and group B (P0.05). Conclusion 1. Bone marrow-derived neuron-like cell transplantation can promote the repair of injured nerve function in rabbits with SCSG injury. 2. The mechanism of bone marrow derived neuroid cell transplantation in repairing SCSG damage in rabbits may be related to up-regulation of BDNF and CNTF.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741
本文编号：2321300
[Abstract]:Objective 1. Rabbit bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cellsBMSCs) were cultured in vitro and induced to differentiate into bone marrow derived neural cells (BMSCs). 2. The (superior cervical sympathetic ganglion SCSG) lesion model of superior cervical sympathetic ganglion in rabbits was established. 3. Bone marrow-derived neuronal cells were implanted into SCSG injury model to observe the changes of pupillary, and to measure the brain derived neurofactor (brain-derived neurotrophic factor,BDNF) in local and hippocampal tissue. The expression of ciliary nerve growth factor (Ciliary Neurotrophic Factor,CNTF (ciliary nerve growth factor) and its possible mechanism in repairing the injury of superior cervical sympathetic ganglion (SCG) in rabbits were investigated by bone marrow-derived nerve-like cell transplantation. Methods unilateral (right) SCSG injury model was established by pressing rabbit SCSG, with needle holding forceps. BMSCs, was preinduced by basic fibroblast growth factor (Basic FibroblastGrowth Facto,bFGF) by density centrifugation and adherent method in vitro, and then differentiated into bone marrow-derived nerve-like cells induced by CNTF. The model rabbits were randomly divided into three groups: control group (group A), culture medium group (group B) and cell transplantation group (group C). On the 7th day after the model was made, no treatment was found in group A, and 1ml of cell culture medium (1 ml) containing 1 脳 106 myeloid nerve cells was injected into group B via auricular vein. Pupillary changes were observed on day 1, day 7, day 10, day 14 and day 21 after transplantation. The ganglion was isolated and stained with HE on the 21st day after transplantation. The expression of BDNF and CNTF in rabbit SCSG were detected by immunohistochemistry. The hippocampal specimens of rabbits were stained with HE and the expression of BDNF was detected. Results 1 comparison of pupillary diameter (mm) in rabbits: the pupil diameter of group A was 5.81 0.42n 5.84 0.45.68 0.295.91 0.31 卤5.80 0.36 on day 1421 after injury of superior cervical sympathetic ganglion. The diameter of pupil in group A was 5.81 0.42n 5.84 0.45.68 0.295.91 0.31 and 5.80 0.36 respectively. The pupil diameter of group B was 5.84 0.32 ~ 5.74 0.32 ~ 5.63 0.37 ~ 5.83 0.27 ~ 5.58 and 0.23 respectively. The pupillary diameter of group C was 5.64 0.33 / 5.71 0.35 / 5.71 0.335.78 0.337.97 0.337.97 0.280.280.05 / 0.21.C respectively. The pupil diameter of group C was significantly larger than that of group A at 10 days and 14 days (P0.05). A group). There was no significant difference at different time points in group B (P0.05). The pupil diameter of group B was significantly larger than that of 1 day, 7 days, 10 days (P0.05), 14 days and 21 days, respectively (P0.05). There was no significant difference in pupil diameter (P0.05). 2the number of BDNF positive cells in hippocampus of group A, group B and group C were 17.13 2.19, 18.53 1.35 and 20.20 4.29, respectively, and the number of positive cells of BDNF in group B was significantly higher than that in group A. There was significant difference (P0.05), A group and B group compared with no statistical significance (P0.05). The number of BDNF positive cells in the injured side of SCSG in the three groups was 6.67 1.116.80 1.26 1.26 12.18 7.93 渭 g / L respectively compared with group A, and the number of BDNF positive cells in group B was significantly higher than that in group A (P0.05),). There was no significant difference between group A and group B (P0.05). 3the number of CNTF positive cells in group A, group B and group C were 15.6 1.83n 15.6 15.6 1.54 + 37.73 7.26C, respectively, higher than those in group A (P0.05), group B (P 0.05), group B (P 0.05). There was no significant difference between group A and group B (P0.05). Conclusion 1. Bone marrow-derived neuron-like cell transplantation can promote the repair of injured nerve function in rabbits with SCSG injury. 2. The mechanism of bone marrow derived neuroid cell transplantation in repairing SCSG damage in rabbits may be related to up-regulation of BDNF and CNTF.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741
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