针刺和神经干细胞联合治疗实验性脑性瘫痪的机制研究

发布时间：2018-01-13 18:12

本文关键词：针刺和神经干细胞联合治疗实验性脑性瘫痪的机制研究　出处：《广州医学院》2011年硕士论文　论文类型：学位论文

【摘要】：目的: 探讨针刺和神经干细胞(Neural stem cell, NSC)联合治疗对缺血缺氧性脑损伤(Hypoxic-ischemic brain damage,HIBD)幼鼠肢体运动功能、学习和记忆能力、脑组织凋亡基因bax及抗凋亡基因bcl-2的蛋白表达和病理形态学的影响,并对针刺联合NSC疗法治疗实验性脑性瘫痪(Cerebral palsy,CP)的机制进行探索,为临床小儿CP的治疗提供实验依据。内容和方法: 1、实验动物及分组:7日龄SPF级SD幼鼠68只(体重:12g～20g),随机选取12只作为①正常对照组;HIBD成功模型随机分成以下4组:②模型组;③单纯针刺组;④单纯NSC组;⑤针刺+NSC组,每组12只。 2、治疗阶段:包括针刺治疗和NSC治疗,其中NSC采用幼鼠尾静脉注射。 3、行为学检测:包括悬吊试验、斜坡试验和Y迷宫试验。 4、取材:术后21天对正常组和实验组幼鼠断颈取材,用HE染色法观察其海马组织病理形态学改变,并计数海马组织正常神经细胞数目;用TUNEL法检测其脑皮质神经细胞凋亡情况并测出凋亡神经细胞数目;免疫组化法检测其脑组织bcl-2、bax基因的蛋白质表达。 5、数据整理,统计学分析。结果: 1、行为学检测:悬吊试验、斜坡试验、Y迷宫试验中,③单纯针刺组和④单纯NSC组与②模型组相比,各项均数差异均无统计学意义(p 0.05),表明单纯针刺和单纯NSC均不能明显改善幼鼠的肢体运动功能和学习记忆能力;⑤针刺+NSC组与②模型组相比,各项均数差异有统计学意义(p 0.05),表明针刺联合NSC能显著提高幼鼠的肢体运动功能和学习记忆能力(智力水平)。 2、bcl-2基因和bax基因的蛋白质表达:⑤针刺和NSC组与②模型组相比,均数差异均有统计学意义(p 0.05),表明针刺联合NSC能够明显促进抗凋亡基因bcl-2的蛋白质表达、抑制凋亡基因bax的蛋白质表达;③单纯针刺组及④单纯NSC组与②模型组相比差异均无统计学意义(p 0.05)。 3、脑组织肉眼观察:取材可见②模型组左侧脑组织出现大面积坏死,空泡,右侧脑组织组织结构正常;③单纯针刺组、④单纯NSC组、⑤针刺+NSC组左侧脑组织都出现了不同程度的坏死和空泡区域,但程度都比②模型组较轻,其中⑤针刺+NSC组比③单纯针刺组和④单纯NSC组左侧脑组织损伤程度更轻。 4、脑组织HE切片:与②模型组相比,③单纯针刺组、④单纯NSC组、⑤针刺+NSC组幼鼠海马变性神经细胞数量减少,病理形态学明显改善,其中⑤针刺+NSC组海马变性神经细胞数目最少,脑组织病变程度最轻。 5、神经细胞计数:(1)海马正常神经细胞数目:②模型组与①正常组相比,均数更少,差异有统计学意义(p 0.05),表明HIBD后幼鼠海马正常神经细胞数目显著减少;③单纯针刺组和④单纯NSC组与②模型组相比,均数差异均无统计学意义(p 0.05),表明单纯针刺治疗和单纯NSC治疗都不能明显提高HIBD后幼鼠海马正常神经细胞数目;⑤针刺+NSC组与模型组相比,均数更多,差异有统计学意义(p 0.05),表明⑤针刺+NSC组可明显提高HIBD后幼鼠海马正常神经细胞数目。(2)脑皮质组织细胞凋亡:③单纯针刺组和④单纯NSC组与③模型组相比,均数差异均无统计学意义(p 0.05),表明单纯针刺和单纯NSC都不能明显抑制幼鼠脑皮质神经细胞凋亡;⑤针刺+NSC组与②模型组相比,均数差异有统计学意义(p 0.05),表明针刺联合NSC治疗可明显抑制幼鼠脑皮质神经细胞的凋亡。结论: 1、单纯针刺或单纯NSC都不能明显改善缺血缺氧性脑瘫模型幼鼠肌力、随意运动、学习记忆能力(智力)。 2、针刺和NSC联合治疗对缺血缺氧性脑瘫模型幼鼠的肌力增长、随意运动的恢复、学习和记忆能力(智力)的改善有明显效果。 3、针刺和NSC联合治疗对缺血缺氧性脑瘫模型幼鼠脑组织细胞凋亡基因bax的蛋白质表达有明显的抑制作用、对抗凋亡基因bcl-2的蛋白质表达有明显的促进作用。
[Abstract]:Objective:
To investigate the effects of acupuncture on neural stem cells (Neural and stem cell, NSC) injury combined treatment on hypoxic ischemic brain (Hypoxic-ischemic brain damage, HIBD) in limb motor function, learning and memory ability and brain tissue apoptosis gene Bax and anti apoptosis gene Bcl-2 protein expression and morphological changes, and paralysis of the acupuncture combined with NSC therapy treatment of experimental cerebral (Cerebral palsy CP) to explore the mechanism, to provide experimental basis for clinical treatment of children with CP.
Content and methods:
1, experimental animals and groups: 68 SPF rats (7 to 20g) at the age of 7 days (body weight: 12g to 20g), 12 were selected as normal control group, HIBD successful model was randomly divided into the following 4 groups: Group II, model group, 3 simple acupuncture group, 4 simple NSC group, and 12 +NSC group.
2, treatment stage, including acupuncture treatment and NSC treatment, of which NSC was injected into the tail vein of young rats.
3, behavioral test: including suspension test, slope test and Y labyrinth test.
4, material: 21 days after the operation, the normal group and experimental group of rats by cervical specimens, observe pathological changes in hippocampus by HE staining, and count the number of nerve cells in hippocampus of normal cerebral cortex; to detect the apoptosis of neural cells and measured the number of apoptotic nerve cells by TUNEL method; immunohistochemistry the brain tissue Bcl-2 protein expression of Bax gene.
5, data sorting, statistical analysis.
Result:
1, the behavior detection: suspension test, slope test, Y maze test, the acupuncture group compared with the NSC group and the model group, the mean differences were not statistically significant (P, 0.05) showed that acupuncture and NSC alone could not significantly improve rat limb movement function and learning and memory compared with the acupuncture ability; +NSC group and model group, the mean difference was statistically significant (P 0.05), indicating that acupuncture combined with NSC can significantly improve the rat limb motor function and the ability of learning and memory (Intelligence).
2, the protein expression of bcl-2 gene and Bax gene: the acupuncture and NSC group and the model group, the mean difference was statistically significant (P, 0.05) showed that the protein expression of acupuncture combined with NSC can significantly promote the anti apoptosis gene Bcl-2 and apoptosis inhibiting gene Bax protein expression; the acupuncture group and the simple the NSC group and the model group, the differences were not statistically significant (P 0.05).
3, to observe the brain tissue in model group were visible to naked eye: the left brain tissue showed a large area of necrosis, cavitation, right brain tissue structure was normal; the acupuncture group, the simple NSC group, the acupuncture group +NSC left brain tissue have suffered different degrees of necrosis and cavitation region, but the degree than in model group the lighter, which the +NSC acupuncture group than in the acupuncture group and the NSC group on the left side of brain injury degree lighter.
4, brain tissue HE staining: compared with the model group, the acupuncture group, the simple NSC group, the number of the acupuncture group +NSC hippocampus nerve cell degeneration reduced, pathological morphology significantly improved, in which the number of the acupuncture group +NSC hippocampal nerve cell degeneration at least, brain lesions lightest.
5, nerve cell count: (1) number of normal nerve cells in hippocampus: compared with the normal group, model group, the number was less, the difference was statistically significant (P, 0.05) showed that the number of normal nerve cells in hippocampus in rats after HIBD decreased significantly; the acupuncture group compared with the simple NSC group and model group. The mean differences were not statistically significant (P, 0.05) showed that acupuncture treatment and NSC treatment alone can significantly improve HIBD after normal nerve cells in hippocampus in rats of the acupuncture group +NSC number; compared with the model group, the number was more, the difference was statistically significant (P 0.05), the +NSC group showed that acupuncture can significantly improve HIBD after the normal nerve cells in hippocampus in rats (2). The number of apoptotic cells in the cerebral cortex tissue: the simple acupuncture group compared with the NSC group and the model group, the mean differences were not statistically significant (P, 0.05) showed that the simple acupuncture and simple NSC can inhibit Neuronal apoptosis in the cerebral cortex of young rats. 5. There was a significant difference between the acupuncture group +NSC and the model group (P 0.05), indicating that acupuncture combined with NSC treatment could significantly inhibit the apoptosis of neurons in the cerebral cortex of young rats.
Conclusion:
1, pure acupuncture or simple NSC can not significantly improve the muscle strength, random movement and learning and memory ability (Intelligence) of hypoxic cerebral palsy model young rats.
2, acupuncture combined with NSC has a significant effect on the increase of muscle strength, the recovery of voluntary movement, and the improvement of learning and memory ability in rats with hypoxic ischemic cerebral palsy.
3, acupuncture combined with NSC has a significant inhibitory effect on the protein expression of apoptosis gene Bax in the hypoxic cerebral palsy model rats, and significantly promotes the protein expression of the anti apoptotic gene bcl-2.

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R246.6;R742.3

【参考文献】