不同频率电针对急性坐骨神经损伤大鼠L4-L5脊髓中Bcl-2、Eax及P53的表达影响

发布时间：2018-05-04 10:51

本文选题：坐骨神经损伤 + 不同频率　；参考：《辽宁中医药大学》2016年硕士论文

【摘要】：目的:通过比较不同频率(2Hz,100Hz)电针对坐骨神经钳夹损伤大鼠的实验观察,以“Bcl-2”“P53”“Bax”作为检测指标,揭示电针治疗坐骨神经损伤抗凋亡作用的机制,探求周围神经损伤后抑制神经元凋亡的最佳治疗频率。材料与方法:参考钳夹法制作模型。选用SPF级SD大鼠48只,体重250g-350g,7-9周,雌雄各半。采用随机数字表分配法分为四组:空白组、模型组、治疗组1(2Hz)、治疗组2(100Hz),每组各12只。空白组:正常饲养,将动物固定在自制的塑料筒内,鼠尾和两后肢露在筒外固定,持续15min,不做任何治疗。模型组:造模成功后,将动物固定在自制的塑料筒内,鼠尾和两后肢露在筒外固定,持续15min,不做任何治疗。治疗组1:造模成功后,于造模第8天深刺“环跳”穴。连接电针仪进行治疗,另一端制成无关电极,选用频率为2Hz的连续波,电针治疗强度以针刺部位局部轻微抽动为准,每次治疗15min,1次/天,连续治疗14天。治疗组2频率为100Hz,除频率与治疗组1不同外,其余予相同处置。各组在造模第8天、22天进行足底印迹检查。取损伤处坐骨神经钳夹处约上下2mm的坐骨神经及同侧L4-L5节段脊髓,用来制作成切片,通过HE染色,Western blot免疫组化等方法测定神经细胞凋亡相关蛋白Bcl-2、P53及Bax的表达。结果:1.一般状态观察摄食饮水及大小便正常,造模后无感染;除空白组其余各组呈现拖趾状或蹦跳状步态,各组间差异不明显。治疗组1和治疗组2在造模第15天步态开始恢复。2.坐骨神经功能指数(SFI)测定治疗组与模型组相比SFI值升高(P0.01),且治疗组1优于治疗组2(P0.01)。3.HE染色模型组神经纤维排列呈不规则、紊乱的形式,且神经纤维周围的雪旺细胞增加,轴突和髓鞘发生裂解,几乎完全消失。治疗组神经纤维病理变化较轻,神经纤维出现新的生长,两者对比治疗组1较轻。4.Western blot定性、定量检测4.1 Western blot Bcl-2检测结果:四组实验中bcl-2蛋白均有明显条带,测定灰度值,(1)与空白组比较,模型组、治疗组1、治疗组2中bcl-2蛋白表达明显升高(P0.01);(2)与模型组相比,治疗组1和治疗组2中bcl-2蛋白表达明显升高(P0.01);(3)与治疗组1相比,治疗组2中bcl-2蛋白表达降低(P0.05)。4.2 Western blot Bax检测结果:四组实验中Bax蛋白均有明显条带,测定的灰度值,(1)与空白组相比,模型组和治疗组2中Bax蛋白表达明显升高(P0.01),治疗组1中Bax蛋白表达升高(P0.05);(2)与模型组相比,治疗组1和治疗组2中Bax表达明显降低(P0.01);(3)与治疗组1相比,治疗组2中Bax表达升高(P0.05)。4.3 Western blot P53检测结果:四组实验中P53均有明显条带,测定的灰度值,(1)与空白组相比,模型组和治疗组2中P53蛋白表达明显升高(P0.01),治疗组1中P53蛋白表达升高(P0.05);;(2)与模型组相比,治疗组1和治疗组2中P53蛋白表达明显降低(P0.01);(3)与治疗组1相比,治疗组2中P53蛋白表达升高(P0.05)。5.免疫组织化检测结果5.1 Bcl-2免疫组织化检测结果:四组实验中bcl-2蛋白均有表达,(1)与空白组相比,模型组中bcl-2蛋白表达升高(P0.05),治疗组1与治疗组2中bcl-2蛋白表达明显升高(P0.01);(2)与模型组相比,治疗组1中bcl-2蛋白表达明显升高(P0.01),治疗组2中bcl-2蛋白表达升高(P0.05);(3)与治疗组1相比,治疗组2中bcl-2蛋白表达升高(P0.05)。5.2 Bax免疫组织化检测结果:四组实验中Bax蛋白均有表达,(1)与空白相比,模型组和治疗组2中Bax蛋白表达明显升高(P0.01),治疗组1中Bax表达均升高(P0.05);(2)与模型组相比,治疗组1和治疗组2中Bax蛋白表达明显降低(P0.01);(3)与治疗组1相比,治疗组2中Bax蛋白表达升高(P0.05)。5.3 P53免疫组织化检测结果:四组实验中P53均有表达,(1)与空白组相比,模型组和治疗组2中P53表达明显升高(P0.01),治疗组1中P53表达升高(P0.05);(2)与模型组相比,治疗组1和治疗组2中P53表达明显降低(P0.01);(3)与治疗组1相比,治疗组2中P53表达升高(P0.05)。结论:1.电针可以改善急性坐骨神经损伤大鼠坐骨神经运动功能的恢复,且2Hz组优于100Hz组。2.电针可调节急性坐骨神经钳夹损伤后脊髓前角细胞运动神经元凋亡及相关蛋白Bcl-2、bax及p53的表达,机制可能与增强抗凋亡作用因子Bcl-2,降低促凋亡因子Bax、P53的表达有关。3.电针可调节急性坐骨神经钳夹损伤后脊髓前角细胞运动神经元凋亡及相关蛋白Bcl-2、bax及p53的表达,频率2Hz优于100Hz。
[Abstract]:Objective: To compare the experimental observation of sciatic nerve clamp injury in rats with different frequency (2Hz, 100Hz) electroacupuncture, with "Bcl-2" "P53" "Bax" as the detection index, to reveal the mechanism of anti apoptosis by electroacupuncture in the treatment of sciatic nerve injury, and to explore the best treatment frequency for the inhibition of neuron apoptosis after peripheral nerve injury. The model was made by clamp method. 48 SPF class SD rats were selected, weight 250g-350g, 7-9 weeks, and male and female. The random digital table distribution method was divided into four groups: blank group, model group, treatment group 1 (2Hz), treatment group 2 (100Hz), each group was 12. The blank group was kept in the homemade plastic tube, the rat tail and two hind limbs were fixed outside the tube. Continuous 15min, without any treatment. Model group: after the model was successful, the animal was fixed in the homemade plastic tube, the rat tail and the two hind limbs were exposed to the tube and fixed outside the tube. 15min was sustained without any treatment. After the successful model of 1: in the treatment group, the "ring jump" point was deeply prickled for eighth days. The electroacupuncture instrument was connected to the treatment, the other end was made into the independent electrode and selection frequency. For the continuous wave of 2Hz, the strength of the electroacupuncture treatment was based on the local mild aspiration of the acupuncture site. Each time was treated with 15min, 1 times / day and continuous treatment for 14 days. The 2 frequency of the treatment group was 100Hz, except for the frequency of 1 different from the treatment group. The same disposal was given in the treatment group for eighth days and 22 days. The sciatic nerve clamp at the injury site was about 2mm The sciatic nerve and the L4-L5 segmental spinal cord of the same side were used to make slices. The expression of Bcl-2, P53 and Bax of neuronal apoptosis related proteins was measured by HE staining and Western blot immunohistochemical method. Results: 1. the normal state of drinking water and urine and stool were observed in general state, and no infection after the model was made; the other groups in the blank group showed toes or jumping. In the treatment group 1 and the treatment group 2, the SFI value of the.2. sciatic nerve function index (SFI) in the treatment group was increased (P0.01) compared with the model group (P0.01), and the treatment group was better than the treatment group 2 (P0.01).3.HE staining model group with irregular, irregular form, and the nerve fiber week. The Schwann cells in the circumference were increased, the axon and myelin sheath were cracked and almost completely disappeared. The pathological changes of the nerve fibers in the treatment group were lighter, and the nerve fibers appeared new growth. The comparison between the two groups was 1 light.4.Western blot, and the quantitative detection of the results of 4.1 Western blot Bcl-2 detection: the bcl-2 protein in the four groups had obvious bands, and the gray level was measured. Value, (1) compared with the blank group, the model group, the treatment group 1, the bcl-2 protein expression in the treatment group was significantly increased (P0.01); (2) the expression of Bcl-2 protein in the treatment group 1 and the treatment group was significantly higher than that in the model group (P0.01); (3) compared with the treatment group 1, the bcl-2 protein expression decreased (P0.05).4.2 Western blot Bax detection results in the treatment group 2: four group experiments Ba. Compared with the blank group, the expression of Bax protein in the model group and the treatment group increased significantly (P0.01), and the expression of Bax protein in the treatment group increased (P0.05). (2) the expression of Bax in the treatment group and the treatment group was significantly lower than that in the model group (2). (3) the expression of Bax in the treatment group was higher than that in the treatment group (3) (P (P) (P) (P). (3) the expression of Bax in the treatment group was increased (P) (P). 0.05).4.3 Western blot P53 detection results: in the four groups, P53 had obvious bands, the measured gray value, (1) the expression of P53 protein in the model group and the treatment group increased significantly (P0.01), and the expression of P53 protein in the treatment group increased (P0.05), compared with the blank group (P0.05); (2) the expression of P53 protein in the treatment group 1 and the treatment group was significantly lower than that in the model group (P0.) 01) (3) compared with the treatment group 1, the expression of P53 protein in the treatment group was increased (P0.05).5. immunohistochemical detection results 5.1 Bcl-2 immunohistochemical detection results: four groups of experimental Bcl-2 protein expression, (1) compared with the blank group, the expression of Bcl-2 protein in the model group increased (P0.05), and the expression of Bcl-2 protein in the treatment group 1 and the treatment group was significantly increased (P0.0). 1) (1) (2) the expression of Bcl-2 protein in the treatment group was significantly higher than that in the model group (P0.01), and the expression of Bcl-2 protein in the treatment group increased (P0.05). (3) the expression of Bcl-2 protein expression increased (P0.05).5.2 Bax in the treatment group (1) compared with the treatment group (1): the Bax protein in the four groups was expressed, (1) compared with the blank, the model group and the treatment group 2 were compared. The expression of Bax protein in the treatment group increased significantly (P0.01), and the expression of Bax in the treatment group increased (P0.05). (2) the expression of Bax protein in the treatment group 1 and the treatment group was significantly lower (P0.01). (3) the expression of Bax protein expression in the treatment group (1) was increased (P0.05) in the treatment group (P0.05).5.3 P53 immunohistochemistry: (1) and (1) Compared with the blank group, the expression of P53 in the model group and the treatment group increased significantly (P0.01), and the expression of P53 in the treatment group increased (P0.05). (2) the expression of P53 in the treatment group 1 and the treatment group was significantly lower than that in the model group (P0.01). (3) the expression of P53 in the treatment group was higher than that in the treatment group (1) (P0.05). Conclusion: 1. electroacupuncture can improve the acute sciatic nerve injury in rats. The recovery of the motor function of the sciatic nerve, and the 2Hz group is better than the 100Hz group.2. electroacupuncture can regulate the apoptosis of the motor neuron in the anterior horn cell of the spinal cord after the acute sciatic nerve clamp injury and the expression of the related protein Bcl-2, Bax and p53. The mechanism may be related to the enhancement of the anti apoptotic factor Bcl-2, the decrease of the apoptosis stimulating factor Bax, and the expression of P53 in.3. electroacupuncture. Apoptosis of motor neurons in anterior horn of spinal cord and expression of related proteins Bcl-2, Bax and p53 after 2Hz were better than 100Hz.

【学位授予单位】：辽宁中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R245

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