摘要目的：研究和探讨敏感期内、末不同时限单眼斜视和剥夺动物模型视觉系统凋亡调节抑制基因Bcl-2塑性变化的规律。方法：应用免疫细胞化学SABC技术染色和计算机图像分析了Bcl-2蛋白在N、MS₁、MD₁、MS₂和MD₂猫同侧视皮质17区Ⅳ层及外侧膝状体相应层的表达。结果：在正常发育幼猫的视皮质和外侧膝状体主要是在神经元核浆，呈深棕黑色免疫阳性反应，胞浆淡染。在MS₁和MD₁眼输入的同侧视皮质17区Ⅳ层及外侧膝状体相应层(A1)表现为Bcl-2蛋白表达的免疫阳性神经元数目减少，核浆染色淡，表明Bcl-2 mRNA表达水平降低。在同侧视皮质17区Ⅳ层表达浓度和数密度：N组与MS₁和MD₁组比较差异具有非常显著性(P＜0.001)；N组与MS₂和MD₂组比较差异无显著性(P＞0.05)。结论：MS₁和MD₁组幼猫实验眼同侧视皮质17区Ⅳ层及同侧外侧膝状体Al层神经元在视觉发育敏感期内，由于长期的斜视及形觉剥夺效应以致功能低下，对Bcl-2调节的敏感性降低，以及对NTs的营养保护及上调Bcl-2 mRNA表达水平应答能力降低，在双眼竞争的神经元发育及突触连接中处于劣势，使对侧眼形成优势循环。这也可能解释在敏感期内当对侧眼遮盖时暴露弱视眼所出现反转弱视的戏剧效应。Bcl-2与NTs有交互作用，它们协同促进神经元的分化，发育和连接重塑。在正常的视环境中，双眼视平衡发育，凋亡抑制基因Bcl-2起着删除细胞死亡程序并保护调节NGF家族的生物效能。

Expression of the apoptosis inhibitory gene Bcl-2 in the neurons of visual system of MS and MD kittens during visual development

Shao Ligong,Guo Jingqiu.Department of pediatric Ophthalmology,First Teaching Hospital of Beijing Medical University,General Hospital of the People's Liberation Army,Beijing 100853

AbstractObjective:To investigate plasticity changes in the regulation of the expression of apoptosis inhibitory gene bcl-2 in the visual systems of normal (N),monocularly strabismic(MS) and monocularly deprived(MD) kittens during the critical period of development.The product (bcl-2 protein) of bcl-2 gene expression is an important bioactivator.It mediates neurtrophine and protective action by the NGF family.The studies provide a basis for understanding the mechanisms and for finding effective methods to prevent and treat amblyopia due to strabismus and deprivation in children.Methods:Surgery was performed on kittens during the critical period (age 2～4 wks) to induce esotropic MS by sectioning the lateral rectus muscle of one eye and MD by lid suture.Before surgery,the animals were randomly divided into 3 groups:N,MS and MD.After the animals were reared for sixteen weeks under the same environmental conditions,they were examined with 6-Channel Pattern Visual Evoked Potentials (6CPVEP).Then,visual cortex area 17 was investigated with polyclonal antibodies for bcl-2,which was used to label the neurons of visual cortex area 17 using the immunocytochemical Streptavidin Biotin Peroxidase Complex(SABC) method.Data were analyzed from photographs from optical microscopy and Computer Image Analysis.Results:Gene expression level of bcl-2mRNA (numeric density and concentration of bcl-2 immunopositive neurons) in layer Ⅲ of the ipsilateral visual cortex area 17 and the laminas receiving ipsilateral geniculate input from the strabismic and deprived eyes are significantly lower than the input from the untreated eyes.Conclusion:Results indicate that the functional status of neurons in layer Ⅲ of visual cortex area 17 and the laminas receiving lateral geniculate input from the treated eyes during the critical period of visual development are greatly weakened in the long term in response to convergence strabismus and form deprivation.That is,the treated eyes are hyposensitive to bcl-2 regulation.Their ability to respond to neurotrophine and the protective action of NTs (neurotrophic factors) is also reduced,with causes the synaptic connections of neurons in layer Ⅲ of visual cortex area 17 and layer Al of ipsilateral geniculate input from the treated eyes to be underdeveloped because the susceptibility of neurons to bcl-2 determines whether neurons in the visual pathways are affected by the dominant during binocular viewing so that critical synaptic links develop normally.

Key wordsStrabismusDeprivationVisual systemBcl-2

儿童弱视主要是在小儿视觉发育敏感期内，由于各种影响视觉发育的眼病和(或)视环境的不良，使双眼视长期紊乱、视觉系统神经元功能、形态和神经生化机制异常，临床表现有外眼及眼底检查无特殊变化，又不能完全矫正其低视力(≤0.8)、失立体视和形觉障碍等体征^［1］。关于发病机理，近年来各国学者结合现代高技术对弱视形成的心理物理、神经病理生理和生化过程及变化特征进行了多方位的探讨和研究，为小儿眼科临床弱视防治工作提供了参考依据和新的途径，弱视动物模型能较客观地反映人类弱视形成的特点，其研究成果具有指导意义^［2～4］。为了确定凋亡抑制基因bcl-2在敏感期内、末斜视和剥夺猫视觉系统中的作用和地位，我们应用组织学和免疫细胞化学技术对敏感期内、末斜视和剥夺猫模型视皮质和外侧膝状体神经元的bcl-2基因表达产物变化进行了观察和研究，从形态学、分子神经生物学角度探讨敏感期内和敏感期末单眼斜视(monocular strabismus,MS)和单眼剥夺(monocular deprivation,MD)模型猫视皮质神经元及其突触的可塑性变化机理。

1材料和方法

1.1实验动物和试剂

1.1.1动物采用实验用纯种虎皮猫共计32只。其中4～6wk龄幼猫24只，10～12wk龄幼猫8只。动物随机分为5组：①正常组(normal group,N);②单眼斜视一组(monocular strabismus₁，MS₁)；③单眼剥夺一组(monocular deprivation₁，MD₁)。上述三组每组8只为4～6wk龄，共计24只。④单眼斜视二组(MS₂)；⑤单眼剥夺二组(MD₂)。上述两组每组4只为10～12wk龄，共计8只。

1.1.2试剂及仪器常用试剂为市售纯化学分析试剂，由中国军事医学科学院基础医学研究所提供。主要试剂：SABC(Streptavidin Biotinperoxidae Complex)、正常羊血清和Bcl-2抗体(中山公司)。主要仪器：振动切片机、CQ-970型计算机图像分析仪、多导程图形视觉电生理仪(MPVEP)、Nikon双目自动照像显微镜和脑立体定位仪。

1.2实验方法

1.2.1单眼斜视和剥夺性弱视动物模型手术制作和饲养：单眼斜视组(MS)动物模型手术制作：在微量噻胺酮(Compound Ketamine)0.1ml/kg肌注麻醉下，结膜下注射少量利多卡因(0.5%)，沿角膜缘2mm处剪开球结膜并分离找出一眼外直肌群切除，然后再彻底分离周围结缔组织及外侧韧带，术后实验眼形成内斜视。手术眼共12只(左右眼各6只)其中8只眼为4～6wk幼猫(MS₁)，4只眼为10～12wk猫(MS₂)。

单眼剥夺组(MD)幼猫为剪除眼睑缘周围毛发后，自内眦到外眦剪除上下睑缘各1～1.5mm，皮下及皮肤分层缝合封闭实验眼形成单眼剥夺共12只(左眼、右眼各6只)。其中敏感期内(4～6wk)8只眼(MD₁)，敏感期末4只眼(10～12wk，MD₂)。动物的饲养环境是在宽敞饲养室内。N和MS₁，MD₁，MS₂及MD₂组实验猫共同在自然光线环境中散放喂养。

1.2.2实验动物的灌注固定和取材：实验动物在饲养到第16周时进行MPVEPs检测，视觉电生理实验证实猫是否产生弱视后分批灌注固定动物并快速取材。(1)动物麻醉后行开胸术，剪开心包膜，右心室切开放出心血管系统血液，快速切开左心室并经左心室主动脉插管灌注生理盐水500ml冲洗心血管系统，接快灌注4℃冷固定液(4%多聚甲醛磷酸缓冲液，pH7.2)，再慢灌注100ml含4%多聚甲醛，0.5%戊二醛和0.2%苦味酸的4℃磷酸缓冲溶液(pH7.2)2小时。在4℃冰箱放置过夜后，标本块转至15%蔗糖溶液(PBS pH7.2)中浸泡12小时，待标本块下沉后行冰冻切片或振动切片。(2)标本取材顺序为：①开颅取脑，垂直脑矢状轴切取视皮质组块，部分切片为平行视皮质17区切取，免疫组织化学染色后选取Ⅳ层组织切片照相。②冠状切取同侧和对侧外侧膝状体组织。(3)行免疫细胞化学染色标本应用振动切片，组织片厚35～40μm。(4)免疫细胞化学SABC技术处理切片：①切片入PBS缓冲溶液(0.1mol/L,pH7.2)漂洗；②用甲醇加0.3%H₂O₂处理切片15～30分钟(室温)以封闭内源性过氧化酶的活性；③0.2%Tritonx-100液中10分钟(室温)；④以正常羊血清孵育15～25分钟(室温)，以封闭组织中的非特异性抗原；⑤滴加兔抗人第一抗体(1