糖尿病大鼠螺旋神经节细胞和蜗核神经元自噬与凋亡的时序性变化与机制探讨

发布时间：2018-03-04 14:30

本文选题：糖尿病　切入点：听力损害　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：目的和意义糖尿病对听觉系统可造成不同程度的损害,目前发生机制尚不明确。研究发现,自噬和凋亡与糖尿病及其并发症,如糖尿病肾病、糖尿病神经病变、糖尿病视网膜病变等有关并起重要作用,然而在糖尿病听力损害的研究鲜见报道。我们推断自噬在糖尿病听力损害中也同样发挥作用。本实验建立糖尿病大鼠模型,研究糖尿病对听觉系统的时序性病理改变,探讨自噬和凋亡是否参与高血糖对螺旋神经节细胞和蜗核神经元的毒性过程及相关信号通路,为研究糖尿病听力损害的发病机制提供实验依据。方法制备糖尿病模型,在糖尿病第4、8、12周末,检测大鼠畸变产物耳声发射(distortion product otoacoustic emision,DPOAE)和听性脑干反应(auditory brainstem response,ABR)。观察各组糖尿病大鼠螺旋神经节细胞(spiral ganglion cells,SGCs)的形态学变化,进行SGCs计数。透射电镜观察蜗神经、坐骨神经、SGCs和蜗核神经元的超微结构。采用免疫组化法检测蜗核胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)的表达,免疫组化和免疫印迹法检测SGCs和蜗核神经元LC3、Beclin-1、Bcl-2、bax和Cleaved Caspase-3蛋白的表达,并进行相关性分析。结果1.成功制备糖尿病大鼠模型,造模成功率72.50%,死亡率23.57%。2.听功能检测结果:DM12W组大鼠DPOAE各频率幅值均出现明显下降。DM8W、DM12W组ABR的反应阈均升高,DM12W组各波潜伏期及波间期延长。3.光镜下见对照组与DM4W组SGCs与蜗核神经元大体形态正常;DM8W、DM12W组SGCs与蜗核神经元细胞密度降低,SGCs计数明显减少(P0.05)。4.超微结构改变:蜗神经在DM8W、DM12W组有髓神经纤维减少,髓鞘萎缩、板层分离、断裂;轴索萎缩,胞浆内可见自噬溶酶体。坐骨神经在DM4W组可见部分髓鞘分离;DM8W、DM12W组髓鞘断裂、分离多见,出现脱髓鞘,轴索萎缩、破裂。DM4W组SGCs内线粒体出现少部分肿胀,DM8W、DM12W组线粒体肿胀、空泡化,自噬体及溶酶体增多,髓鞘板层分离。蜗核神经元在DM4W组出现髓鞘板层松弛或断裂,DM8W、DM12W组自噬体和溶酶体逐渐增多,髓鞘变形、板层分离、断裂。5.SGCs自噬和凋亡蛋白的表达:免疫组化检测发现Beclin-1、LC3、Bax与Cleaved Caspase-3在糖尿病组阳性表达多见,对照组少见,Bcl-2则相反。Western blotting检测:各糖尿病组SGCs Beclin-1的表达均显著上调(P0.05)。DM8W 和 DM12W 组 Cleaved-LC3、Bax 与 Cleaved Caspase-3 的表达明显高于对照组(P0.05)。Bcl-2的表达在DM8W组和DM12W组显著下降。6.蜗核神经元蛋白的表达:免疫组化检测发现糖尿病组GFAP阳性表达的星形胶质细胞增多。Beclin-1、LC3、Bax与Cleaved Caspase-3在各糖尿病组阳性表达多见,对照组少见,Bcl-2则相反。Western blotting检测Beclin-1、Bax在各糖尿病组表达升高,Cleaved-LC3蛋白在DM8W和DM12W组的表达明显升高。DM4W 组和 DM8W 组 Cleaved Caspase-3 表达上调。Bcl-2 在 DM8W 组和DM12W组表达降低。结论1.持续性高血糖对SGCs、蜗神经和蜗核神经元造成慢性损害。2.高血糖可促进耳蜗核的星形胶质细胞增生活化。3.自噬和凋亡参与了高血糖对SGCs和蜗核神经元的神经毒性过程。4.SGCs和蜗核的自噬和凋亡水平随糖尿病病程延长而增强。5.自噬可能通过Bcl-2/Beclin-1通路参与高血糖对SGCs和蜗核的毒性过程。
[Abstract]:The purpose and significance of diabetes can cause different damage to the auditory system, the mechanism is not clear. The study found that autophagy and apoptosis in diabetes and its complications, such as diabetic nephropathy, diabetic neuropathy, diabetic retinopathy and other relevant and plays an important role, but in diabetic hearing impairment has not been reported. We conclude that autophagy in diabetic hearing impairment also play a role. In this experiment, establish the model of diabetic rats, the pathological changes of diabetes on the timing of the auditory system, to investigate whether autophagy and apoptosis involved in high glucose toxicity of spiral ganglion cells and cochlear nucleus neurons and the related signaling pathways, to provide the experimental basis for the study of the pathogenesis of diabetic hearing impairment the model of diabetes. Preparation methods, in diabetes after 4,8,12 weeks, the detection of distortion product otoacoustic emission (distortion rats Product otoacoustic emision, DPOAE) and auditory brainstem response (auditory brainstem, response, ABR). The observation group of spiral ganglion cells in diabetic rats (spiral ganglion, cells, SGCs) the morphological changes of the SGCs count. Observation of cochlear nerve, sciatic nerve ultrastructure by transmission electron microscope, SGCs and cochlear nucleus neurons by immunohistochemistry. Detection of cochlear nucleus glial fibrillary acidic protein (glial fibrillary acidic protein, GFAP) expression and detection of SGCs and cochlear nucleus LC3, immunohistochemistry and Western blotting of Beclin-1, Bcl-2, Bax and Cleaved expression of Caspase-3 protein, and correlation analysis. The results of 1. successful preparation of diabetic rat model, rats the mortality rate was 72.50%, 23.57%.2. auditory function test results: DM12W rats of DPOAE group were significantly decreased the amplitude of.DM8W, DM12W group ABR response threshold was increased, the latency of DM12W group Wave and prolongation of.3. under the light microscope, the control group and the DM4W group of SGCs and cochlear nucleus neurons in normal morphology; DM8W, DM12W group SGCs and cochlear nucleus cell density decreased and SGCs count was significantly reduced (P0.05): the ultrastructures of.4. cochlear nerve in DM8W, DM12W group of myelinated nerve fibers decreased, myelin sheath atrophy. Lamellar separation, fracture; axonal atrophy in the cytoplasm of autolysosomes. Sciatic nerve separation in DM4W group visible part of myelin sheath; DM8W, DM12W group of myelin fracture, separation, emergence of demyelination, axonal atrophy, rupture of mitochondria in group.DM4W SGCs is a small part of the DM8W DM12W group, swelling, swelling of mitochondria. Vacuolar autophagosomes and lysosomes, lamellar myelin. Isolated cochlear nucleus neurons appeared lamellar myelin loose or broken, in the DM4W DM8W group, DM12W group of autophagosome and lysosome increased gradually, myelin lamellar separation, fracture deformation, autophagy and apoptosis protein.5.SGCs Expression: immunohistochemistry showed that Beclin-1, LC3, Bax and Cleaved Caspase-3 are more common in diabetes mellitus group positive control group, while Bcl-2 is rare,.Western blotting detection: the expression of SGCs Beclin-1 in each diabetic group were significantly up-regulated (P0.05.DM8W) and DM12W Cleaved-LC3 group, the expression of Bax and Cleaved were significantly higher than the control group (Caspase-3 P0.05) the expression of.Bcl-2 in DM8W group and DM12W group were.6. expression in cochlear nucleus protein decreased: immunohistochemical detection showed that the diabetic group GFAP positive astrocytes increased.Beclin-1, LC3, Bax and Cleaved Caspase-3 is more common in the diabetes group positive control group, while Bcl-2 is rare,.Western blotting detection Beclin-1 the expression of Bax in the diabetic group, increased expression of Cleaved-LC3 protein in DM8W and DM12W group were significantly increased in.DM4W group and DM8W group Cleaved Caspase-3 expression. The expression of Bcl-2 decreased in DM8W group and DM12W group. Conclusion 1. sustained high blood glucose on SGCs, cochlear nerve and cochlear nucleus neurons damage caused by chronic hyperglycemia can promote.2. cochlear nucleus astrocytes increased autophagy and apoptosis of life.3. in high blood glucose on SGCs and cochlear nucleus neurons in cochlear nucleus and.4.SGCs toxicity autophagy and apoptosis level increases with the duration of diabetes increased.5. through Bcl-2/Beclin-1 pathway involved in autophagy may be high blood glucose on SGCs and cochlear nucleus toxicity.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2;R764

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