针刺对单眼剥夺大鼠视网膜生长蛋白（GAP-43）的影响

发布时间：2018-04-26 03:09

本文选题：针刺 + 单眼视觉剥夺　；参考：《长春中医药大学》2014年硕士论文

【摘要】：目的：通过运用免疫组化方法、激光共聚焦技术观察针刺对可塑期内单眼视觉剥夺大鼠视网膜神经元中相关生长蛋白（GAP-43）结构发育的干预效应，从而进一步讨论弱视发病机制与针刺对视觉系统可塑期内的调节作用。为针刺治疗弱视疾病的机制提供新的思路，为临床治疗弱视的提供参考方法。方法：使用出生后14天清洁级实验大鼠60只，采用随机分组的方法将实验大鼠分为五组，即正常组、模型组、早期针刺组、中期针刺组、晚期针刺组，将模型组和早期针刺组、中期针刺组、晚期针刺组采用缝合单侧上下眼睑的方法复制单眼剥夺弱视模型；正常组不给予任何治疗处理，五组实验大鼠全都放在一样的环境中饲养。早期针刺组在单眼剥夺大鼠模型复制后的第7天开始进行针刺治疗；中期针刺组在单眼剥夺大鼠模型复制后的第14天开始进行针刺治疗；晚期针刺组在单眼剥夺大鼠模型复制后的第21天开始进行针刺治疗。选取“睛明”、“攒竹”、“光明”、“风池”四个穴位，在针刺治疗结束后进行P-VEP检测，观察检测视觉诱发电位（P-VEP）和利用免疫组化方法检测目标蛋白在视网膜中成像；剥夺早期针刺组、剥夺中期针刺组在针刺结束后进行检测。麻醉实验动物取弱视大鼠剥夺眼视网膜，用4%多聚甲醛浸泡视网膜10-20分钟，然后每隔1-2小时分别换10%、20%、30%蔗糖，30%蔗糖后置于4摄氏度过夜将在4摄氏度过夜后的大鼠视网膜进行包埋冷冻切片视网膜，视网膜取厚度14微米切片，贴于硅化防脱载玻片上，，免疫组织化学方法观察GAP-43在组织中的阳性表达，最后使用激光扫描共聚焦显微镜观察目标蛋白（GAP-43）细胞荧光强度变化。结果： (1)在进行单眼视觉剥夺模型复制4周后，模型组P-VEP波形有明显改变，表现为P100的明显延迟和N45-P100幅值降低，其与正常组的波形比较有较为明显的差异（P＜0.01）；针刺治疗后，单眼视觉剥夺针刺组与模型组相比时值与幅值均有显著变化，其P-VEP波形P100出现的时值明显提前（P＜0.05），N45-P100幅值明显升高（P＜0.05） (2)视网膜：正常组和模型组相比，大鼠视网膜GAP-43阳性神经元的表达显著下降，有显著性差异（P＜0.01）；针刺组与模型组比较，大鼠视网膜GAP-43阳性神经元表达水平均明显提高，有显著性差异（P＜0.05）。结论：（1）实验发现针刺能改善单眼视觉剥夺大鼠异常的P-VEP波形；单眼剥夺大鼠的P-VEP波形变化和视觉系统的相关生长蛋白GAP-43的表达降低有关。（2）针刺对视觉发育敏感期内单眼剥夺组大鼠视网膜GAP-43阳性细胞表达有调节作用，对视功能的恢复具治疗的意义。（3）针刺在视觉发育关键期内干预单眼剥夺大鼠其视网膜中GAP-43阳性表达升高，提示针刺对弱视视功能有改善机制,GAP-43与视觉发育及可塑性有密切关系。
[Abstract]:Objective: to observe the effect of acupuncture on the development of GAP-43 in retinal neurons of rats with single eye visual deprivation during the plastic period by using immunohistochemical method and laser confocal technique. The mechanism of amblyopia and the regulating effect of acupuncture on the plastic period of visual system were discussed. It provides a new idea for the treatment of amblyopia and a reference method for clinical treatment of amblyopia. Methods: sixty clean grade experimental rats were randomly divided into five groups: normal group, model group, early acupuncture group, metaphase acupuncture group, late acupuncture group, model group and early acupuncture group. The amblyopia model of unilateral upper and lower eyelids was made by suturing the upper and lower eyelids in the middle period acupuncture group and the late stage acupuncture group, while the normal group was not treated with any treatment, and the five experimental rats were all fed in the same environment. In the early acupuncture group, acupuncture treatment began on the 7th day after the establishment of the monocular deprivation rat model, and the acupuncture treatment began on the 14th day after the establishment of the monocular deprivation model in the metaphase acupuncture group. The late acupuncture group was treated with acupuncture on the 21 th day after the rat model of monocular deprivation was made. Four acupoints of "Jingming", "Zanzhu", "Guangming" and "Fengchi" were selected. After acupuncture treatment, P-VEP was detected, visual evoked potential (VEP) was detected and target protein was detected in retina by immunohistochemical method. The early acupuncture group was deprived and the mid-stage acupuncture group was tested after acupuncture. Rats with amblyopia were taken from anesthetized rats and deprived of their retina. The retina was soaked in 4% paraformaldehyde for 10-20 minutes. Then every 1-2 hours, 10% 30% sucrose 30% sucrose and 30% sucrose were replaced and placed overnight at 4 degrees Celsius. The retina of the rats after the night was embedded in frozen sections, and the retina was cut 14 microns thick, and applied to the silicified anti-desorption slide glass. Immunohistochemical method was used to observe the positive expression of GAP-43 in tissues. The fluorescence intensity of target protein (GAP-43) cells was observed by laser scanning confocal microscopy (LSCM). Results: (1) after 4 weeks of monocular visual deprivation model replication, the P-VEP waveform of the model group was significantly changed, showing that the P100 delayed and the N45-P100 amplitude decreased, which was significantly different from that of the normal group (P < 0.01), and after acupuncture treatment, there was a significant difference between the two groups (P < 0.01), and there was no significant difference between the two groups (P < 0.01). Compared with the model group, the time value and amplitude of the P-VEP wave P100 in the monocular visual deprivation acupuncture group were significantly changed, and the amplitude of the P-VEP waveform P100 was significantly higher than that of the model group (P < 0.05) (P < 0.05). The amplitude of N45-P100 in the monocular visual deprivation group was significantly higher than that in the model group (P < 0.05). (2) retina: compared with the model group, the expression of GAP-43 positive neurons in the normal group was significantly lower than that in the model group (P < 0.01), and the expression level of GAP-43 positive neurons in the retina of the acupuncture group was significantly higher than that in the model group. There was significant difference (P < 0.05). Conclusion: The results showed that acupuncture could improve the abnormal P-VEP waveforms of monocular visual deprivation rats, and the changes of P-VEP waveforms in monocular deprived rats were related to the decreased expression of growth protein GAP-43 in the visual system. (2) Acupuncture can regulate the expression of GAP-43 positive cells in the retina of the monocular deprivation group during the sensitive period of visual development, and has the therapeutic significance for the recovery of visual function. 3) the positive expression of GAP-43 in the retina of monocular deprived rats was increased after acupuncture intervention during the critical period of visual development, suggesting that acupuncture can improve the function of amblyopia. GAP-43 is closely related to visual development and plasticity.
【学位授予单位】：长春中医药大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R245

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