Cacna1f基因突变对大鼠神经肌肉功能影响的研究

发布时间：2018-03-15 09:35

本文选题：先天性静止性夜盲　切入点：遗传性疾病　出处：《第四军医大学》2011年博士论文　论文类型：学位论文

【摘要】：先天性静止性夜盲（congenital stationary night blindness, CSNB）是一种遗传性视网膜疾病，表现为视网膜杆体细胞的功能异常，视网膜电图（electroretinogram, ERG）b波降低或消失，暗适应功能丧失或较差，但是通常不伴有进行性的眼底改变，以及病人不会感觉到自己的运动能力下降。而有些运动系统疾病却伴有CSNB疾病表型，如肌营养不良症可以伴有视觉系统异常。其中Duchenne肌营养不良症（Duchenne muscular dystrophy，DMD）具有与CSNB病相似的ERG波形改变，但是没有检索到CSNB疾病与DMD疾病有关联的报道[1，2]。我们发现并正在建立近交系的一种不完全型CSNB（CSNB2）模式大鼠[3，4]，致病基因为Cacna1f，遗传模式为伴性遗传[5]；该品系大鼠除视觉症状外，前期实验中还发现该品系大鼠运动能力可能也存在异常，如爬杆实验、旋转棒实验中均表现为耐力不良（易疲劳）。Cacna1f基因突变使传递神经递质的活动区（active zone，CAZ）的ribbon小体等结构发生改变（前期实验），还能使突触相关蛋白表达下降，从而阻滞视觉信号从光感受细胞向二级神经元传递，这可能是导致视觉表型的重要机制之一。骨骼肌神经肌接头处存在传递神经递质的CAZ，并且也有功能类似于ribbon小体的结构--致密体以及突触相关蛋白。最初认为Cacna1f基因只在视网膜内表达，目前认为Cacna1f所编码的钙通道蛋白也存在于骨骼肌等其它组织中[6，7]。鉴于CSNB大鼠存在骨骼肌运动耐力不良表型，因此我们推测：Cacna1f基因突变也会影响到骨骼肌-神经肌接头处的神经递质释放活性区，使神经递质释放受阻信号传导阻滞，故导致神经肌肉功能的改变。为验证该推测，本文从以下四个方面进行研究：1、Cacna1f突变大鼠视网膜结构及功能发育变化的特点；2、CSNB大鼠肌耐力不良遗传特征的验证；3、Cacna1f突变对大鼠骨骼肌功能变化特征的影响；4、Cacna1f突变大鼠骨骼肌中突触相关蛋白表达的特征。材料与方法利用本实验室发现并培育的CSNB近交系大鼠及同龄野生型SD大鼠，采用视觉电生理检查技术，观察CSNB大鼠视网膜发育各时期的视觉功能变化；通过CSNB大鼠与对照组大鼠杂交，建立F1代杂交鼠，，验证肌肉耐力不良的遗传特征；运用离体比目鱼肌肌条灌流技术，检测CSNB大鼠骨骼肌的收缩功能是否发生改变；利用免疫组化技术，了解CSNB大鼠突触蛋白及视细胞的发育情况，以及骨骼肌、脊髓、大脑上钙通道蛋白的表达特征，并通过激光共聚焦技术对蛋白表达量进行观察；通过免疫印迹技术，半定量CSNB大鼠突触相关蛋白在骨骼肌的表达特征。结果 1、不同时期CSNB大鼠视网膜结构及功能的发育特点在睁眼前1天、睁眼当天，野生型大鼠与CSNB大鼠ERG明适、暗适反应较相似。野生型大鼠睁眼后2天，视杆反应（rod ERG）出现,最大混合反应（Max ERG）出现b波，出现明适反应（Photopic ERG），OPs子波潜时较前期缩短、幅值增大；2天后波形变化不大，仅各项波形幅值增大、潜时延长。CSNB大鼠ERG波形与睁眼前波形一直较相似；出生后第26天（Postnatal26，P26）CSNB大鼠Photopic ERG明显。两组大鼠的Flicker反应在睁眼前似存在。大鼠睁眼前后视觉功能发育曲线显示：从睁眼前一天到P26，野生型大鼠与CSNB大鼠Max ERG的a波幅值无明显差异，但CSNB大鼠潜伏期均延长（P0.05）。野生型大鼠Photopic ERG较CSNB大鼠幅值高、潜时短（P 0.01）均有统计学意义。视觉功能衰退曲线表明：野生型大鼠Max ERG的a波以及Photopic ERG的b波3个月幅值达最大，17个月仍能记录。CSNB大鼠Max ERG的a波21天至1月幅值最大，到13个月a波消失；Photopic ERG的b波P21出现，1月幅值最大，10个月波形消失。 HE染色显示，两种大鼠视网膜形态结构的发育较相似。在睁眼时两种大鼠的视网膜CAZ内的突触相关蛋白SYN与CtBP2，未见明确点状表达，睁眼后各时期CSNB大鼠的两种蛋白表达均较野生型大鼠减少；SYN与CtBP2两者相互伴行，且表达量随视网膜早期发育而增加。两种大鼠的视杆双极细胞数量相似；睁眼时两种大鼠视杆双极细胞突触数量相似；睁眼后2天，野生型大鼠视杆双极细胞突触开始较CSNB大鼠增多。 2、 CSNB大鼠肌耐力不良的遗传特征经行为学及视觉电生理技术的检测，在杂交F1代子鼠中，所有雌鼠ERG表现均为正常，而雄鼠ERG表现均为CSNB表型；F1代雄鼠均表现为肌肉耐力不良，爬杆持续时间较雌鼠显著缩短（P 0.01）。提示CSNB大鼠肌肉耐力不良伴随着CSNB疾病视觉表型，为性连锁隐性遗传特征。 3、 Cacna1f突变大鼠肌肉收缩功能的变化特征野生型大鼠与CSNB大鼠比目鱼肌（soleus, SOL）的最适频率相似，约为100Hz。间断强直收缩刺激条件下0.8%DC（Duty Cycle）以及30%DC的Pmin/Pmax值两组间未见统计学差异。完全强直收缩刺激条件下，CSNB大鼠收缩曲线到达峰值张力时程（time to peak tension，TPT）提前；但P30/P0比值两种大鼠间未见显著性差异。 4、Cacna1f突变大鼠各组织中钙通道蛋白的表达特征及骨骼肌中突出相关蛋白表达特征视网膜中，野生型大鼠与CSNB大鼠标记Cav1.4钙通道蛋白N端的抗体，在外网状层（outer plexi layer, OPL）、外界膜（outer segment, OS）、节细胞层（gamglion cell layer, GCL）均有表达，但在CSNB大鼠视网膜表达较弱；Cav1.4钙通道蛋白C端的抗体在CSNB视网膜上表达不明确。SYN在野生型大鼠视网膜的OPL中表达，而CSNB大鼠的这种点状表达较少。骨骼肌中，SYN与Cav1.4钙通道蛋白N端抗体染色，在CSNB大鼠骨骼肌表达较在野生型大鼠减少；Cav1.4钙通道蛋白C端染色在CSNB大鼠骨骼肌中表达不明确。通过Western Blot技术，SYN在野生型大鼠及CSNB大鼠SOL骨骼肌中表达都呈现出两个条带；分子量较大的SYN蛋白在CSNB大鼠表达较野生型大鼠明显下降，表达量约为对照组的三分之一（P 0.05）；小分子量SYN两种大鼠间差异不大；EDL中SYN表达不明确。脊髓灰质背根中Cav1.4在CSNB大鼠表达较野生型大鼠下降，两组大鼠钙通道蛋白在大脑均未见表达。结论 1.从睁眼后2天开始野生型大鼠与CSNB大鼠ERG差别增大，此前两者ERG表型相似。CSNB大鼠视觉发育最佳时期约在1月，13个月视觉完全衰退。CSNB大鼠视杆双极细胞树突发育进程异常；CSNB大鼠CAZ上突触相关蛋白表达下降，从而影响CAZ的功能。 2.证实CSNB大鼠杂交系F1代大鼠骨骼肌功能异常伴随CSNB疾病的ERG表型，为X连锁隐形遗传表型。 3. CSNB大鼠低频间断强直收缩功能未见改变，高频完全强直收缩功能改变；Cacna1f突变仅引起骨骼肌细胞膜功能的轻微异常。 4. Cacna1f突变确实引起CSNB大鼠骨骼肌上Cav1.4的异常表达，及影响慢肌CAZ上突触相关蛋白SYN的表达。以上结论初步验证了我们的假设：Cacna1f突变影响到骨骼肌NMJ的CAZ功能，从而导致突触信号传导阻滞，骨骼肌耐力下降；CAZ的功能改变导致视网膜及骨骼肌多系统功能发生变化。
[Abstract]:Congenital stationary night blindness (congenital stationary night blindness, CSNB) is a kind of hereditary retinal diseases, retinal rod cells showed abnormal function of the electroretinogram (electroretinogram, ERG) b wave decreased or disappeared, dark adaptation or loss of function is relatively poor, but usually not accompanied by the fundus changes, and patient do not feel their exercise capacity decreased. While some movement disorders is associated with CSNB disease phenotype, such as muscular dystrophy can be accompanied by abnormal visual system. The Duchenne muscular dystrophy (Duchenne muscular, dystrophy, DMD) with ERG waveform similarity and CSNB disease, but not retrieved CSNB disease associated with DMD reports of the disease we found and [1,2]. are building inbred an incomplete CSNB (CSNB2) [3,4] model rats, pathogenic genes for Cacna1f, genetic model for sex The genetic [5]; rat strains except the visual symptoms, previous studies also showed that the strains of rats may have abnormalities, such as pole test, rotarod test showed poor endurance (fatigue).Cacna1f gene mutation activity area transmission of neurotransmitters (active zone, CAZ) the ribbon body structure change (Experiment), can make the expression of synapse related proteins decreased, which block the visual signal from the photoreceptor cell transfer to two neurons, which may be one of the important mechanism leading to the visual phenotype. There is the neurotransmitter CAZ skeletal muscle neuromuscular junction, and also has the function similar to ribbon body structure compact body and synapse associated protein. Initially thought that the Cacna1f gene is only expressed in the retina, the calcium channel protein Cacna1f encoding also exists in skeletal muscle tissue in the [6,7 In view of CSNB. Rats skeletal muscle endurance adverse phenotype, we hypothesize that Cacna1f gene mutation may affect the neurotransmitter skeletal muscle nerve muscle joint release of the active region, the neurotransmitter release blocked signal block, resulting in neuromuscular function change. In order to verify the theory, this article conducts the research from the following four aspects: 1, the characteristics of development of structure and function of rat retinal changes of Cacna1f mutation; 2, verification of CSNB rat muscle endurance adverse genetic characteristics; 3, on the function of rat skeletal muscle changes the mutations of Cacna1f influence; 4, the characteristics of synaptic expression in rat skeletal muscle related protein Cacna1f mutation.
Materials and methods
The laboratory found and cultivation of inbred CSNB rats and wild-type SD rats of the same age, using visual electrophysiology techniques, to observe the development of CSNB rats retina changes of visual function in different periods; through the CSNB rats and control group rats to establish hybrid, F1 hybrid mice, genetic characteristics test card muscle endurance bad; the use of isolated soleus muscle strips, whether the systolic function of skeletal muscle of CSNB rats changed; using immunohistochemical technique, understanding the development of synaptic protein in CSNB rats and treated cells, and skeletal muscle, spinal cord, expression of calcium channel protein of the brain, and by laser confocal microscopy expression of protein was observed by western blotting; semi quantitative CSNB rat synaptosomal associated protein expression in characteristics of skeletal muscle.
Result
1, the development characteristics of the structure and function of the retina of CSNB rats at different stages
In the open before 1 days, opening day, wild type and CSNB rats ERG, dark reaction is similar. 2 days of wild type rat eyes after rod reaction (rod ERG), the biggest mixed reaction (Max ERG) b wave, that reaction (Photopic ERG) OPs, wavelet latency amplitude increases; the previous shortened, 2 days after the waveform changes little, only the wave amplitude increases, latency prolonged.CSNB rats ERG waveform and waveform is similar to open eyes; twenty-sixth days after birth (Postnatal26, P26) CSNB Photopic ERG rats obviously. Flicker reaction of the two groups of rats like there is in the eyes.
The eyes of rats before and after development of visual function curve showed that from the day before to open P26, wild type and CSNB rats Max ERG a amplitude had no significant difference, but the latency was prolonged in CSNB rats (P0.05). Wild type Photopic ERG rats than in CSNB rats with high amplitude, short latency (P 0.01) were statistically significant. The visual function decline curve showed that the wild type rat Max ERG a Photopic ERG wave and b wave amplitude reached the maximum of 3 months, 17 months can still record.CSNB rat Max ERG a wave 21 to January to 13 month maximum amplitude, a wave disappeared; B P21 Photopic wave ERG, the maximum amplitude of 10 months in January, disappeared.
HE staining showed that two kinds of rat retina morphology development is similar. Synapse associated protein SYN and CtBP2 two CAZ in the rat retina in the eyes, no clear punctate expression, eyes after two kinds of protein in each period of CSNB rats compared with the expression of wild type rats decreased; both SYN and CtBP2 with each other, and the expression increased with the early development of the retina. Two rats the number of rod bipolar cells; open your eyes of two rat rod bipolar cell synapse number similarity; 2 days after opening, the rod bipolar cell synapse start than that of CSNB rats increased in wild-type rats.
2, genetic characteristics of muscular stamina in CSNB rats
After testing the behavior and visual electrophysiological techniques, in the hybrid F1 generation offspring, all female rats showed normal ERG male rats, and ERG showed CSNB phenotype; F1 generation male rats showed poor muscle endurance, climbing duration was significantly shortened in female rats (P 0.01) CSNB. Rat muscle hypoxi accompanied by CSNB visual disease phenotype, as recessive genetic characteristics.
Changes of muscle contractile function in 3, Cacna1f mutated rats
The wild type and CSNB rats soleus muscle (soleus, SOL) the optimum frequency is similar to that of about 100Hz. under the condition of intermittent tetanic stimulation of 0.8%DC (Duty Cycle) and 30%DC Pmin/Pmax there was no statistical difference between the two groups. Completely tetanic stimulation conditions, CSNB rats reached the peak tension time history curve of contraction (time to peak tension, TPT) in advance; but the ratio of P30/P0 two rats had no significant difference.
4, the expression of calcium channel protein in the tissues of Cacna1f mutant rats and the expression of protruding protein in skeletal muscle
In the retina, the wild type rats and CSNB rats labeled antibody Cav1.4 calcium channel protein N terminal, in the outer plexiform layer (outer plexi layer OPL (outer), segment, OS film outside the ganglion cell layer (gamglion), cell layer, GCL) were expressed, but weaker expression in the retina of CSNB rats; antibody of Cav1.4 calcium channel protein C terminal in the retina CSNB expression is not clear expression of.SYN in retina of rats in the wild type OPL, and this point CSNB expression in rat skeletal muscle. Less, SYN and Cav1.4 calcium channel protein N terminal antibody staining, than in wild-type rats reduced expression in skeletal muscle of CSNB rat; Cav1.4 calcium channel protein C expression was not clear in the end of the skeletal muscle of CSNB rats. The Western Blot technology, SOL SYN expression in skeletal muscle of wild type and CSNB rats are shown in the two bands; the larger molecular weight of SYN protein in CSNB rats compared to the wild The rats were significantly decreased, the expression level was about 1/3 in the control group (P 0.05); small molecular weight of two SYN rats were no significant difference between the expression of SYN in EDL; is not clear. The expression of Cav1.4 in spinal cord dorsal root in rats CSNB rats decreased compared to the wild type, the expression of the two groups of rats of calcium channel protein in the brain were seen.
conclusion
1. eyes from 2 days after the start of the wild type and CSNB rats ERG difference increases, the two had ERG phenotype similar to.CSNB rat visual development the best period of about 13 months in January, the development process of visual outrightrecession rod bipolar cell dendrites.CSNB rats CSNB rats CAZ anomaly; synapse related protein expression decreased which affect the function of CAZ.
2. it was confirmed that the dysfunction of skeletal muscle in F1 generation rats of CSNB hybrids was associated with the ERG phenotype of CSNB disease, which was a X linkage stealth hereditary phenotype.
3. CSNB rats had no change in low-frequency interrupted tetanic contractility and high frequency tetanic contractile function. Cacna1f mutation only caused slight abnormality in skeletal muscle cell membrane function.
4. Cacna1f mutation does cause abnormal expression in skeletal muscle of rats with CSNB Cav1.4, and the expression of synapse related protein SYN in slow muscle CAZ. The above conclusion confirms our initial hypothesis that Cacna1f mutations affect skeletal muscle NMJ CAZ function, resulting in synaptic signaling blocking, skeletal muscle endurance decreased; CAZ the function change leads to multi system function and retinal changes in skeletal muscle.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R774.1

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