Caspase-3在形觉剥夺性弱视大鼠视皮层及外侧膝状体的表达

发布时间：2018-05-16 02:26

本文选题：半胱氨酸天冬氨酸蛋白酶3 + 形觉剥夺性弱视　；参考：《南华大学》2013年硕士论文

【摘要】：目的研究细胞凋亡因子半胱氨酸天冬氨酸蛋白酶3（Cysteine protease protein32KD，Caspase-3）在形觉剥夺性弱视大鼠视皮层及外侧膝状体的表达，探讨其与弱视发病机理的关系，期望能对弱视的治疗产生积极的影响。方法 1.刚出生的健康SD（Sprague Dawley）大鼠40只，饲养14天后随机选择20只行单眼眼睑缝合术为单眼形觉剥夺组（Monocular Deprivation，MD），正常组（Normal Postnatal， NP）不做特殊处理。根据饲养天数分别在14天、21天、45天、120天处死动物。MD组即分为MD14、MD21、MD45、MD120；NP组分为NP14、NP21、NP45、NP120，每组各5只。 2.将NP21、MD21组的大鼠称重麻醉后行P-VEP（Pattern Visual EvokedPotential）检测，记录P100波的潜伏期和波幅值。 3.采用HE（Hematoxylin Eosin）染色和免疫组化法以及图像分析系统对正常大鼠和单眼形觉剥夺弱视大鼠视皮层、外侧膝状体中Caspase-3免疫阳性神经元进行定位观察，，并根据测定的平均灰度值定量研究其变化。结果 1.P-VEP检测：MD21组大鼠弱视眼的P-VEP P100波峰潜伏期检测值较NP21组同侧眼显著延长(P0.01)，波幅明显降低(P0.01)，提示MD21组弱视眼的视觉电生理已改变，表明可以通过单眼睑缝合术成功构建形觉剥夺弱视模型。 2.（1）HE染色：NP45大鼠大脑视皮层神经元的结构正常，细胞轮廓清晰，可清晰地显示神经元的形态结构及染色，MD45剥夺组神经元细胞排列不整齐，胞膜结构不清，神经元变性，部分神经元细胞核固缩，呈强嗜碱性，胞浆深染，结构不清。（2）Caspase-3免疫阳性神经元在视皮层中的表达：Caspase-3经染色后呈棕黄色，阴性细胞为蓝色。出生后14天（P14）Caspase-3的平均灰度值在NP14和MD14两组无显著性差异（P=0.074），而出生后21天、45天、120天（P21、P45、P120），Caspase-3免疫反应产物在MD组大鼠视皮层中表达密集，NP组表达产物较前基本无改变。P21、P45、P120三个时段MD组平均灰度值均较同时期NP组显著性升高（P＜0.05）。在MD组MD21和MD45组间无显著性差异（P0.05），MD21和MD45与MD14、MD120之间均有差异显著性（P＜0.05）。（3）Caspase-3免疫阳性神经元在外侧膝状体的表达：在MD组大鼠LGN中颗粒状物分布密集，但不同时期其密度也不同。NP组大鼠LGN的Caspase-3表达产物分布稀疏，P14：MD组和NP组两组无显著性差异(P=0.168)，P21、P45、P120三个时段MD组均较同时期NP组平均灰度值显著性升高(P＜0.05)。在MD组MD14、MD21、MD45和MD120组间两两比较均有差异显著性（P＜0.05）。（4）MD组Caspase-3在大鼠视皮层及外侧膝状体的表达量随剥夺时间变化，其在视觉发育关键期形觉剥夺开始时增加，到早中期持续高表达，视觉发育关键期结束后稍降低，但仍高于形觉剥夺开始时。结论 1.在大鼠视觉发育关键期的早期，应用单侧眼睑缝合术即使剥夺是短暂的（7天）可以成功建立形觉剥夺性弱视模型。 2.大鼠单眼形觉剥夺会引起视皮层神经细胞发生形态学改变，引起细胞类凋亡。 3.大鼠单眼形觉剥夺后，Caspase-3在视皮层和外侧膝状体中的表达：14天开始增加，21天明显增高，45天达到峰值，120天时较前稍下降，但仍高于14天开始剥夺时。 4.单眼形觉剥夺通过引起Caspase-3的表达增加，进而导致神经元细胞凋亡损伤，可能是弱视的发病机理之一。
[Abstract]:objective
The expression of cysteine aspartic proteinase 3 (Cysteine protease protein32KD, Caspase-3) in the visual cortex and lateral geniculate body of rats with form deprivation amblyopia is studied to explore the relationship with the pathogenesis of amblyopia, hoping to have a positive effect on the treatment of amblyopia.
Method
1. newly born healthy SD (Sprague Dawley) rats, and 14 days after feeding, 20 single eye eyelid sutures were randomly selected as monocular form deprivation group (Monocular Deprivation, MD). The normal group (Normal Postnatal, NP) did not do special treatment. The animals were killed in 14 days, 21 days, 45 days and 120 days respectively. The.MD groups were divided into MD14, MD21, and 120 days. MD120; NP were NP14, NP21, NP45 and NP120, 5 in each group.
2. the rats in group NP21 and group MD21 were anesthetized and P-VEP (Pattern Visual EvokedPotential) was detected. The latency and amplitude of P100 wave were recorded.
3. the HE (Hematoxylin Eosin) staining, immunohistochemical method and image analysis system were used to locate the Caspase-3 immunoreactive neurons in the visual cortex and the lateral geniculate body of normal rats and monocular deprivation amblyopia rats, and the changes were quantified according to the average gray value measured.
Result
1.P-VEP detection: the detection value of P-VEP P100 wave peak latency in the amblyopia of group MD21 rats was significantly longer than that in the NP21 group (P0.01), and the amplitude of the wave decreased significantly (P0.01), suggesting that the visual electrophysiology of the amblyopia was changed in MD21 group, indicating that the form deprivation amblyopia model could be constructed successfully by monocular eyelid suture.
2. (1) HE staining: the structure of the neurons in the visual cortex of the brain of NP45 rats is normal and the cell outline is clear. The morphological structure and dyeing of the neurons can be clearly displayed. The neuron cells in the MD45 deprivation group are in irregular arrangement, the membrane structure is not clear, the neurons are denatured, and the nuclei of some neurons are strongly basophilic, the cytoplasm is deeply dyed and the structure is not clear.
(2) the expression of Caspase-3 immunoreactive neurons in the visual cortex: Caspase-3 was brown in brown, and the negative cells were blue. The average gray value of 14 days after birth (P14) Caspase-3 had no significant difference in NP14 and MD14 two groups (P=0.074), while 21 days after birth, 45 days, 120 days (P21, P45, P120), Caspase-3 immunoreaction products in MD group rats. The expression in the cortex was dense, and the expression products of NP group had no changes in.P21, P45, and P120 were significantly higher in MD group than that in the same period NP group (P < 0.05). There was no significant difference between the MD21 and MD45 groups in MD group (P0.05), and there was a significant difference between MD21 and MD.
(3) expression of Caspase-3 immunoreactive neurons in the lateral geniculate body: the distribution of granules in LGN of group MD rats was dense, but the density of the Caspase-3 expression products of LGN in group.NP was sparse in different period, and there was no significant difference between group P14:MD and NP group (P=0.168), P21, P45, and P120 groups were all flat compared with the same period. The mean gray value increased significantly (P < 0.05). There was a significant difference between 22 groups in group MD, MD14, MD21, MD45 and MD120 (P < 0.05).
(4) the expression of Caspase-3 in the visual cortex and lateral geniculate body of the MD group changes with the deprivation time. It increases at the beginning of visual development at the critical stage of form deprivation, and continues to express high in the early and middle stages. The critical stage of visual development decreases slightly after the end of visual development, but still higher than the beginning of form deprivation.
conclusion
1. in the early stage of the critical period of visual development in rats, unilateral eyelid suture was applied, even if deprivation was transient (7 days), and the form deprivation amblyopia model could be successfully established.
2. rats with monocular deprivation can cause morphological changes of neurons in the visual cortex and induce cell apoptosis.
The expression of Caspase-3 in the visual cortex and lateral geniculate body after the monocular deprivation of the 3. rats: the increase in the visual cortex and the lateral geniculate body: the increase in the 14 day, the 21 day increase, the peak of the 45 day, and a slight decrease at 120 days, but still higher than the 14 days of deprivation.
4. monocular deprivation can induce neuronal apoptosis by increasing the expression of Caspase-3, which may be one of the pathogenesis of amblyopia.

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R777.44

【共引文献】