补益论对形觉剥夺性弱视大鼠视皮质神经元中c-fos蛋白表达的研究

发布时间：2018-01-06 07:37

本文关键词：补益论对形觉剥夺性弱视大鼠视皮质神经元中c-fos蛋白表达的研究　出处：《长春中医药大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:通过P-VEP检测验证针刺治疗弱视的有效性,免疫荧光染色技术观察单眼形觉剥夺性弱视大鼠和正常发育大鼠视皮质神经元中c-fos蛋白的表达情况,探讨在视觉发育可塑期内,正常大鼠及单眼剥夺性弱视大鼠视皮质17区功能形态的变化规律。从分子水平来探索补益论对弱视治疗的效果。为针刺治疗弱视提供理论基础,在临床预防与治疗弱视提供新的思路。方法:实验选取刚出生14天,健康wistar大鼠18只,雌雄不限(由吉林大学动物中心提供,清洁动物)。将此18只大鼠随机分为正常组(NC)、单眼形觉剥夺模型组(MD1)、单眼形觉剥夺针刺组(MD2),各6只。根据大鼠视觉发育可塑期为出生后14d~45d为依据[22],在大鼠14d时,NC组不给予任何处理,MD1与MD2组行单眼眼睑缝合术,建立单眼形觉剥夺弱视模型。术后每天早晚检查眼睑创口情况,检查缝线是否有脱落,每天滴抗生素眼液、涂红霉素眼膏以预防感染。如缝线抓脱或伤口感染导致漏光或眼睑闭合不严密的动物弃用。形觉剥夺性弱视动物模型的标准为术眼上下睑粘连。各组幼鼠均在同等环境下散放喂养。单眼形觉剥夺针刺治疗组在术后第7天开始给予针刺治疗,单眼形觉剥夺模型组只进行单眼剥夺缝合术,正常组不给予任何处置。针刺治疗组选取“睛明穴”、“攒竹穴”、“肾俞穴”、“脾俞穴”等4个穴位。睛明穴为眼内角直刺0.3cm,攒竹穴斜刺0.5cm,脾俞穴、肾俞穴直刺0.6cm。采用平补平泻手法,1次/1d进行治疗,1次/15min,治疗周期为7天。检测治疗后各组幼鼠的P-VEP的变化。MD组大鼠心脏灌注成功后分别切取MD1、MD2单眼形觉剥夺弱视眼对侧视皮层(VC)的视皮质17区,NC组切取任意一侧视皮质17区。所得视皮质17区标本均浸入4%多聚甲醛磷酸盐缓冲液中,放入4℃冰箱里后固定24h以上。经石蜡切片后行免疫荧光染色技术观察各组视皮质17区组织的c-fos蛋白表达的情况。结果:1.正常组P-VEP检测结果波峰明显、稳定性好、正常变异小。MD1与MD2相比P100潜伏期明显延长,振幅降低。MD1与NC、MD2比较,其P-VEP的振幅明显降低,潜伏期延长,波形不稳定,并且经统计学分析两组差异有统计学意义(P0.05);MD2的P-VEP检测结果与MD1结果比较,其波形较稳定,潜伏期缩短、振幅提高,经统计学分析,两组之间P100潜伏期与P100振幅比较差异有统计学意义(P0.05)。2.免疫荧光染色结果:针刺治疗结束后,在光镜下观察MD2大鼠视皮质17区内可见到c-fos阳性表达神经元分布散在、均匀,着色显强荧光;MD1视皮质17区内c-fos免疫阳性神经元密度及阳性细胞荧光数量都明显低于NC与MD2,经统计学分析,此两组差异有统计学意义(P0.05)。MD1与MD2大鼠视皮质17区内c-fos免疫阳性神经元密度及阳性细胞荧光数量比较,两组差异有统计学意义(P0.05)。结论:1.视觉发育敏感期内补益论对单眼形觉剥夺大鼠的P-VEP波形改变具有明显的改善作用。2.视觉发育关键期内补益论对单眼剥夺弱视大鼠治疗有明显的效果。3.补益论对单眼剥夺大鼠视皮质17区内组织中c-fos蛋白表达变化,提示针刺对弱视视功能改善的机制可能是通过c-fos蛋白表达的改变而实现的,c-fos参与了视觉发育及视觉发育可塑性调节,影响了大鼠视觉系统的可塑性变化。
[Abstract]:Objective: to verify the validity of P-VEP detection by acupuncture treatment of amblyopia, immunofluorescence staining to observe the expression of monocular visual deprivation amblyopia rats and the normal development of rat visual cortical neurons in c-fos protein, investigate in rat visual development period, changes in normal rats and single eye amblyopia rat visual cortex the 17 district function form. From the molecular level to explore the benefit on the effect of treatment of amblyopia. Provide a theoretical basis for acupuncture treatment of amblyopia, and provide a new way of thinking in the clinical prevention and treatment of amblyopia. Methods: this experiment was born just 14 days, 18 healthy Wistar rats, male and female (provided by the animal center of the Jilin University clean. This animal) 18 rats were randomly divided into normal group (NC), monocular deprivation model group (MD1), monocular deprivation acupuncture group (MD2), 6 rats in each rat. According to the visual development period after birth can be plastic 14d~45d According to [22], in 14d rats, NC group were not given any treatment, MD1 and MD2 group underwent monocular lid suture, establish the model of monocular deprivation amblyopia. Postoperative eyelid wound every day sooner or later check, check whether there is a suture off, every drop of antibiotic eye drops, with Erythromycin Eye Ointment such as suture grasping to prevent infection. Removal or wound infection caused leaked or lagophthalmus strict animal abandoned. Form deprivation amblyopia animal model standard for eye surgery on lower eyelid adhesions. The pups were in the same environment scattered feeding. Monocular deprivation acupuncture treatment group in seventh days after surgery was given acupuncture treatment, monocular deprivation the model group only monocular deprivation suture, the normal group was not given any treatment. Acupuncture group selected "Jingming", "zanzhu acupoint", "Shenshu", "4 points Pishu points. Jingming eye angle into 0.3cm, Acupuncture cuanzhu acupoint 0.5cm, spleen Shu point, Shenshu Point into the 0.6cm. the use of reinforcing reducing techniques, 1 /1d for 1 /15min, the treatment period is 7 days. After the treatment in different group P-VEP changes group.MD perfused rat heart were harvested after successful MD1 deprivation amblyopia on the side MD2 monocular cortex (VC) of the visual cortex area 17, NC group cut from any side of the visual cortex area 17. The visual cortex area 17 specimens were immersed in 4% paraformaldehyde phosphate buffer, into the refrigerator after 4 degrees Celsius above 24h. The fixed paraffin sections after immunofluorescence staining to observe the expression of each group the visual cortex area 17 tissue c-fos protein. Results: 1. normal results of P-VEP wave obviously, good stability, small variation in.MD1 compared with MD2 normal P100 latency was prolonged and the amplitude decreased.MD1 and NC, MD2, P-VEP significantly decreased the amplitude, latency prolonged, waveform Stable, and statistically significant differences between the two groups (P0.05); the results of P-VEP MD2 compared with MD1 results, the waveform is stable, the latency and amplitude increase, by statistical analysis, the comparison between the two groups P100 latency and amplitude of P100 were statistically significant (P0.05).2. immunofluorescence staining results: Acupuncture Treatment after MD2 rats was observed under light microscope in the visual cortex area 17 can be seen in the positive expression of c-fos neurons in the distribution, uniform coloring was strong fluorescence MD1; visual cortex area 17 the number of fluorescent c-fos immunoreactive neurons and positive cell density were significantly lower than those of NC and MD2, statistical analysis, statistical the significance of the difference between the two groups (P0.05) in visual cortex area 17 fluorescence quantitative comparison of c-fos immunoreactive neurons and density of.MD1 positive cells and MD2 rats, there was significant difference between two groups (P0.05). Conclusion: 1. visual development sensitive During the period of tonic on monocular visual deprivation P-VEP waveform changes of rats can significantly improve the effect of.2. on the critical period of visual development of tonic monocular deprivation amblyopia rats treatment has obvious effect on.3. and on the expression of c-fos protein in monocularly deprived rats visual cortex area 17 in the organization change, as the mechanism of acupuncture to improve the function of amblyopia may be through changes in the expression of c-fos was achieved, c-fos is involved in the visual development and visual plasticity regulation effect of plasticity of the visual system of rats.

【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】：2015
【分类号】：R777.44

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