糖尿病性耳聋大鼠Connexin26、Connexin30表达与耳蜗病理学变化和发病机制探讨

发布时间：2018-02-07 15:21

本文关键词： 糖尿病1型2型血糖尿糖体重大鼠模型听性脑干反应(ABR) Cx26 Cx30 Western blot 耳聋毛细胞 HE染色免疫荧光染色共聚焦　出处：《河北医科大学》2013年博士论文　论文类型：学位论文

【摘要】：第一部分：复制糖尿病大鼠模型背景：随着我国国民经济的发展，人民生活水平的不断提高，生活方式的改变以及社会的老龄化，糖尿病的患病率逐渐增高，严重威胁我国人民的健康。糖尿病是一种胰岛素相对或绝对缺乏而使血糖水平异常增高、脂肪和蛋白质代谢紊乱的一种代谢性疾病，糖尿病的并发症之一是听力损害。糖尿病性听力下降严重影响着人们的生活质量，因此越来越受到各方面的高度重视。为研究糖尿病性听力下降的发病相关机制，必需建立适时的糖尿病动物模型，我们依据国内外公认的糖尿病动物模型建模标准，复制动物模型并监测其血糖、体重、尿糖等相关指标，为进一步研究提供实验和理论依据。方法：120只Wistar大鼠随机分为4组：1型糖尿病实验组（n=40）、2型糖尿病实验组（n=40）、1型糖尿病对照组（n=20），2型糖尿病对照组（n=20）。实验组的Wistar大鼠用两种不同饲养方法进行喂养。不同时间段取尾静脉全血用血糖仪测定血糖，并检测体重、尿糖等指标。每月对造模后糖尿病大鼠进行观察，包括精神状态、活动度、毛发光泽度、摄食、尿量、耳廓反射等情况。1型糖尿病大鼠随机血糖＞16.65mmol/L为成模标准；2型糖尿病大鼠随机空腹血糖≥7.8mmol/L为成模标准。结果：1实验动物一般情况两型实验组大鼠均呈现明显糖尿病症状，出现饮水、饮食、尿量增多及消瘦等症状，毛发发黄无光泽，精神萎靡，活动迟缓，双侧耳廓反应欠灵敏，耳镜检查外耳道清洁,正常。正常对照组大鼠无饮水、饮食、尿量增多及消瘦等糖尿病表现，生长及营养状况良好，双耳情况正常。 2实验动物血糖变化 1型糖尿病实验组大鼠在实验开始时（0月）随机血糖与对照组相比无统计学差异（P＞0.05），造模后1、2、3、4、5月血糖实验组与对照组相比升高显著(P＜0.01)。 2型糖尿病实验组大鼠在特殊饲料喂养8周后，空腹血糖与对照组相比明显升高(P＜0.05)，注射STZ后1个月至5个月，血糖升高显著（与对照组相比P0.01）。 3实验动物尿糖变化两型模型对照组实验期尿糖均阴性，两组实验组造模后尿糖均为(+++或++++)，符合糖尿病动物成模模型标准。 4实验动物体重变化 1型糖尿病实验组大鼠体重变化表现为0月时实验组与对照组无显著差异（P＞0.05），造模后1、2、3、4、5月实验组大鼠体重呈持续降低趋势，与对照组相比差异显著(P＜0.01)。 2型糖尿病实验组大鼠体重变化表现为特殊饲料喂养4周、8周时，实验组大鼠体重与对照相比显著增加（P0.01）。注射STZ后1个月，实验组大鼠体重比注射前有所降低（与特殊饲料喂养8周相比，（P0.05），与对照相比无显著差异（P＞0.05）；从成模后2月开始，实验组大鼠的体重呈下降趋势，与对照组相比差异显著(P0.01)。结论：本实验成功复制了近似于人类糖尿病症征的两种糖尿病大鼠模型，此两种模型建立方法简便易行，是糖尿病慢性并发症发病机制研究的理想动物模型。第二部分：糖尿病大鼠听功能变化与Cx26和Cx30在耳蜗中表达关系动态观察背景：耳聋是糖尿病慢性并发症之一。糖尿病性耳聋的主要特点是双侧的对称性神经性耳聋，表现为蜗性和或蜗后性耳聋，以高频听力下降为主，常伴有眩晕和前庭功能减退。国内外研究糖尿病性耳聋的发病机制主要从神经病变、微血管病变、血液流变学异常、免疫反应、遗传学等方面切入，但其发病机制至今尚不清楚,给干预或治疗糖尿病性耳聋带来了极大困难，这也是医学一直关注的问题。目前关于糖尿病内耳病变的报道多以外毛细胞散在缺失为主，其原因有待探讨。糖尿病患者的听力损害可能是耳蜗的微观结构和中枢听觉功能发生病变，因此，本研究将从宏观和微观两方面对此机理进行研究。连接蛋白是细胞间信号传递通道，属于缝隙连接，目前研究最多的连接蛋白是Cx26和Cx30。缝隙连接是细胞间通讯的结构基础，是细胞间电解质、第二信使、和代谢产物等信息传递的重要通道。内耳的缝隙连接除了输送营养物质、参与K+循环、交换代谢产物以外，还可能对内耳感觉上皮细胞的功能进行调控，维持内耳环境的稳定。K+是内耳淋巴液中产生听力的必不可少的离子，Cx26、Cx30又是K+转运的离子通道之一，因此Cx26，Cx30与K+是听力产生的必要条件。正常的哺乳动物耳蜗组织中存在Cx26及Cx30，维持耳蜗的正常功能。糖尿病性耳聋模型大鼠的耳蜗组织中Cx26及Cx30的表达情况如何？其表达量与听力下降是否有关？国内外尚无相关报道。因此，本研究采用糖尿病大鼠动物模型进行听性脑干反应（ABR）监测，探寻其听力功能动态变化特点。利用Western blot方法，检测Cx26和Cx30在糖尿病实验组大鼠中的表达量，，通过定量分析明确Cx26和Cx30表达量与动物听觉功能变化的相关关系，为探讨糖尿病性耳聋的发病机制提供分子生物学理论实验依据。方法：对照组和实验组大鼠均采用美国ICS-CHARTR型听性脑干监测仪分别进行听性脑干反应(ABR)测试，ABR阈值显著提高(≥40dBnHL)或波潜伏期及波间期延长作为入选糖尿病性耳聋的标准，进入下一步的实验观察。在预定的时间点将动物麻醉后断头，快速取出耳蜗，提取耳蜗蛋白，使用Western blot方法，检测Cx26和Cx30在耳蜗组织中的表达趋势，验证其表达是否与实验组动物听功能变化相一致。结果：11型糖尿病大鼠ABR指标变化主要是Ⅱ波、Ⅴ波潜伏期、Ⅰ-Ⅴ、Ⅲ-Ⅴ波间期的变化，造模后1个月对照组与实验组听力无明显差异（P＞0.05）；造模后2、3、4、5个月对照组与实验组Ⅱ波、Ⅴ波潜伏期有显著差异（P＜0.01）、Ⅰ-Ⅴ、Ⅲ-Ⅴ波间期（P＜0.05）有差异。 22型糖尿病大鼠ABR指标变化主要是Ⅲ波、Ⅴ波潜伏期、Ⅰ-Ⅲ、Ⅰ-Ⅴ波间期的变化，造模后1个月对照组与实验组听力无显著差异（P＞0.05）；造模后2、3、4、5个月对照组与实验组Ⅲ波、Ⅴ波潜伏期、Ⅰ-Ⅲ、Ⅰ-Ⅴ波间期（P＜0.01）具有显著差异。 32型糖尿病大鼠模型中，耳蜗Cx26、Cx30蛋白表达水平与对照组大鼠相比有显著下降，随着造模时间的延长，Cx26和Cx30蛋白表达逐渐降低，Cx26蛋白较Cx30蛋白变化更为明显。结论： 1两型糖尿病性聋大鼠从造模2个月开始，大鼠的听力出现下降。两型糖尿病大鼠都有不同程度的听力损害，脑干听诱发电位可作为早期诊断其听力下降的指标之一。 2从造模2个月开始，实验组耳蜗组织中的Cx26、Cx30蛋白的表达水平有改变，Cx26和Cx30蛋白表达均有下降趋势，Cx26和Cx30变化与听功能改变相一致。第三部分：糖尿病性耳聋大鼠Cx26、Cx30表达变化与耳蜗毛细胞损伤关系初步研究背景：随着糖尿病患者人数的增加，由糖尿病引起的听力损害的报道也逐渐增多。糖尿病听力损害起病隐匿，易与老年性耳聋混淆，待发现时常发展为不可逆病变，严重影响患者的生活质量。对糖尿病性耳聋发病机制的深入研究，是预防糖尿病性耳聋不可缺少的环节。在糖尿病性耳聋大鼠模型中，尤其是2型糖尿病性耳聋大鼠模型，随着时间的延长，听力损失越明显，影响听觉功能。维持耳蜗正常机械-电转换功能以及保证听力的正常传导作用的分子是缝隙连接蛋白，糖尿病性耳聋大鼠耳蜗中的连接蛋白，在成模后两个月发生变化，病变时间越长，其表达降低越显著。为进一步证实听力损失与耳蜗组织中的Cx26和Cx30相关性，本研究采用荧光免疫染色方法、HE染色，检测Cx26和Cx30在实验组大鼠中的分布是否与耳蜗组织的蛋白表达水平及听力损失相一致，并进行了全耳蜗铺片，观察毛细胞损失情况与听力损失的相关性。进一步探讨了Cx26、Cx30表达与糖尿病性耳聋的发病机制关系，从分子水平为糖尿病性聋的发病机制提供新的研究方向和理论实验依据。方法：大鼠麻醉后断头，取出耳蜗，固定、脱钙、石蜡包埋、切片。挑选结构完整耳蜗石蜡切片，分别进行HE染色及免疫组织化学染色，检测Cx26和Cx30在糖尿病性耳聋大鼠耳蜗的表达分布，激光共聚焦显微镜观察耳蜗的结构形态及Cx26和Cx30的表达情况是否与耳蜗组织的病理变化相一致，最后通过全耳蜗铺片来观察毛细胞损失情况与听力损失的相关性。结果：1两型糖尿病大鼠HE染色耳蜗侧壁组织、基底膜均出现实验组较正常组变薄，实验组大鼠耳蜗组织血管纹中管腔样结构与对照组耳蜗组织血管纹相比较出现减少，耳蜗侧壁及基底膜嗜伊红染色实验组较正常组深染，实验组与对照组大鼠耳蜗螺旋韧带中和螺旋神经节细胞数量比较有统计学差异（P㩳0.05），实验组大鼠耳蜗螺旋韧带中和螺旋神经节细胞数量少于对照组。 2两型糖尿病大鼠造模后1月实验组和对照组耳蜗内基底膜、血管纹和螺旋韧带中Cx26、Cx30表达无统计学差异（P㧐0.05），造模后2、3、4、5月实验组和对照组耳蜗内基底膜、血管纹和螺旋韧带中Cx26、Cx30表达水平显著低于对照组（P㩳0.05）。 3两型糖尿病大鼠模型造模后1月实验组和对照组大鼠耳蜗铺片比较，内外毛细胞均无损伤。 4两型糖尿病大鼠模型造模后2月实验组和对照组大鼠耳蜗铺片比较，实验组较对照组内、外毛细胞排列散乱，但无缺失；造模后3月实验组较正常组内、外毛细胞排列散乱，其程度较2月时严重，但内外毛细胞无缺失；造模后4、5月实验组较正常组内、外毛细胞排列散乱，并出现内外毛细胞的缺失。这些均与实验组耳蜗组织中的Cx26和Cx30蛋白表达水平较正常组在明显下降一致，与ABR听力测试结果相符合。结论： 1Cx26及Cx30是听力产生的必不可少的连接蛋白，推测糖尿病大鼠听力损害可能与Cx26、Cx30相关。 2两型型糖尿病大鼠造模后1月实验组和对照组耳蜗内基底膜、血管纹和螺旋韧带中Cx26、Cx30表达水平无化，造模后2、3、4、5月实验组和对照组耳蜗内基底膜、血管纹和螺旋韧带中Cx26、Cx30表达显著低于对照组。与听力变化较为一致。 3耳蜗内外毛细胞实验组较对照组排列散乱，随时间延长毛细胞损伤程度加重，与蛋白表达及听力测试结果均一致。两型糖尿病大鼠模型在造模后2月Cx26和Cx30在耳蜗组织中的表达较对照组均有降低，随时间延长其降低程度越显著。提示糖尿病性耳聋的听力减退可能与Cx26和Cx30表达降低有关。
[Abstract]:Part one: replicating diabetic rat model
Background: with the development of our national economy, improve the people's living standards, changes in lifestyle and social aging, the prevalence of diabetes increased gradually, a serious threat to people's health in China. Diabetes is an absolute or relative lack of insulin to abnormally high blood sugar levels, a fat metabolic disease and protein metabolism disorders, one of the complications of diabetes is diabetic hearing impairment. The hearing loss seriously affects people's quality of life, so it attracts more and more attention from various aspects. For the onset mechanism of diabetes hearing loss, must establish the animal model of diabetes in time, based on our modeling standard animal model of diabetes at home and abroad accepted, to establish the animal model and monitor their blood glucose, body weight, urine and other related indicators, to provide experimental and theoretical basis for the further study.
Methods: 120 Wistar rats were randomly divided into 4 groups: type 1 diabetes mellitus group (n=40), type 2 diabetes mellitus group (n=40), type 1 diabetes control group (n=20), type 2 diabetes control group (n=20). Wistar rats in the experimental group with two different feeding methods of feeding. Different time from tail vein whole blood glucose was determined by blood glucose meter, and the body weight, glucose and other indicators. The observation of monthly DM rats, including mental state, activity, hair gloss, food intake, urine volume, and auricle reflex type.1 diabetic rats were randomized into model 16.65mmol/L blood sugar. Standard; type 2 diabetes mellitus rats were fasting blood glucose than 7.8mmol/L to become the standard model.
Results: 1 the general situation of experimental animals
Two types of experimental rats showed obvious symptoms of diabetes, drinking water, diet, urine volume increased and weight loss and other symptoms of yellow hair, no gloss, listlessness, slow reaction, bilateral ear less sensitive, ear canal clean and microscopic examination, normal. Rats in normal control group without drinking water, diet, urine volume increased and weight loss and other diabetic symptoms, growth and nutritional status, ears were normal.
2 blood glucose changes in experimental animals
In the experimental group of type 1 diabetes, there was no significant difference in the blood glucose at the beginning of the experiment (0 months) between the experimental group and the control group (P > 0.05). The blood glucose in the experimental group was significantly higher than that in the control group after 1,2,3,4,5 months (P < 0.01).
In type 2 diabetes mellitus group, fasting blood glucose increased significantly after 8 weeks of special feed (P < 0.05), and blood sugar increased significantly from 1 months to 5 months after STZ injection (P0.01 compared with control group).
3 experimental animal urine changes
Type two model control group the urine were negative, two groups of experimental group after modeling the urine were (+ + + + + + +, or) with diabetes animal model standard model.
4 body weight changes in experimental animals
The body weight changes in the experimental group of type 1 diabetes showed no significant difference between the experimental group and the control group at 0 months (P > 0.05). After modeling for 1,2,3,4,5 months, the body weight of the experimental group showed a decreasing trend, which was significantly different from that of the control group (P < 0.01).
Weight change in type 2 diabetic rats in the experimental group showed a special diet for 4 weeks, 8 weeks, body weight of rats in the experimental group compared with the control group increased significantly (P0.01). 1 months after STZ injection, body weight of rats in the experimental group was lower than that before injection (compared with the special diet for 8 weeks (P0.05). Compared with the control, no significant difference (P > 0.05); from the model after the beginning of February, the experimental group rats weight decreased, compared with the control group had significant difference (P0.01).
Conclusion: the two diabetic rat models similar to human diabetes symptoms were successfully replicated in the experiment. The two models are simple and convenient, and are ideal animal models for studying the pathogenesis of diabetic chronic complications.
The second part: the dynamic observation of the relationship between the changes of the auditory function of diabetic rats and the expression of Cx26 and Cx30 in the cochlea
Background: deafness is one of the chronic complications of diabetes. The main characteristics of diabetic deafness is the symmetry of bilateral nerve deafness, cochlear and performance or retrocochlear deafness, with high-frequency hearing loss, often accompanied by dizziness and vestibular dysfunction. The pathogenesis of diabetic deafness at home and abroad mainly from neuropathy, micro vascular disease, abnormal blood rheology, immune response, genetics and other aspects, but its pathogenesis is still unclear, brought great difficulties to the intervention or treatment of diabetic deafness, which is the medicine has been a concern. At present about diabetic inner ear pathological reports outside hair cells scattered in the absence of the main, to causes of hearing impairment. Patients with diabetes may be the micro structure and the central auditory function of cochlear lesions, therefore, this study will be two from the macro and micro aspects of this machine Make a research on the connection. Protein is the signal transmission path between cells belonging to gap junctions, one of the most connected protein is Cx26 and Cx30. is the structural basis of gap junction intercellular communication, one cell electrolyte, second messenger and metabolites information important channel. The inner gap junctions in addition to transporting nutrients in K+, circulation, exchange of metabolites, may also be the inner ear sensory epithelial cell function regulation, maintain the stability of the.K+ environment is essential for inner ear hearing ear ion, Cx26, and Cx30 is one of the K+ ion channel transport in the Cx26, Cx30 and K+ is a necessary condition for the hearing.
The presence of Cx26 and Cx30 in normal mammalian cochlea, maintain the normal function of the cochlea. How the expression of Cx26 and Cx30 in the cochlear tissue of diabetic deafness rats in? And the expression level of hearing loss is about? There is no relevant reports at home and abroad. Therefore, this study used the animal model of diabetic rat auditory brainstem reaction monitoring (ABR), to explore the dynamic changes of hearing function. By using Western blot method, the expression detection of Cx26 and Cx30 in diabetic rats in the experimental group, through quantitative analysis of the expression of Cx30 and Cx26 clear correlation and dynamic changes of auditory function, provide experimental basis for the theory of molecular biology to explore the pathogenesis of diabetes deafness.
Methods: the control group and the experimental groups of rats were ICS-CHARTR respectively monitor auditory brainstem auditory brainstem response (ABR) test, the ABR threshold was significantly increased (more than 40dBnHL) or latencies and wave interval prolongation was selected as diabetic deafness, enter into the next experiment. The breakage in the predetermined anesthesia the time will come out quickly after the animal cochlea and cochlear protein extraction, using Western blot method, the expression of Cx26 and Cx30 in the detection of trends in cochlear tissues, verify whether the expression and experimental group animal auditory function changes consistent.
Results: the changes of ABR index in type 11 diabetic rats is mainly II wave, V wave latency, I-V, changes of III-V wave interval, made 1 months after the experimental group and the control group hearing had no significant difference (P > 0.05); model 2,3,4,5 months after the experimental group and the control group II V wave wave, the incubation period had significant difference (P < 0.01), I-V, III-V wave interval (P < 0.05) difference.
The change of ABR index in type 22 diabetic rats is mainly III wave, V wave latency of I-III, changes of I-V wave interval model, 1 months after the experimental group and the control group hearing no significant difference (P > 0.05); after modeling 2,3,4,5 months the control group and the experimental group III wave. V wave latency of I-III, I-V wave interval (P < 0.01) has significant differences.
In type 32 diabetic rats, the expression level of Cx26 and Cx30 protein in cochlea decreased significantly compared with control rats. With the prolongation of modeling time, the expression of Cx26 and Cx30 decreased gradually, and Cx26 protein changed more significantly than Cx30 protein.
Conclusion:
1, type two diabetic deafness rats started hearing loss for 2 months from model building. All types two diabetic rats had different degrees of hearing loss. Brainstem auditory evoked potentials could be used as early diagnosis.
One of the indicators of its hearing loss.
2, from the 2 months after modeling, the expression level of Cx26 and Cx30 protein in the cochlea tissue of experimental group changed, and the expression of Cx26 and Cx30 decreased. The change of Cx26 and Cx30 was consistent with the change of auditory function.
The third part: a preliminary study on the relationship between the changes of Cx26, Cx30 expression and cochlear hair cell damage in diabetic deafness rats
Background: with the increase in the number of patients with diabetes, hearing impairment caused by diabetes are increasingly reported. Diabetic hearing impairment onset occult, easily confused with senile deafness, often to be found for the development of irreversible lesions, seriously affecting the quality of life of patients. Further research on diabetes deafness pathogenesis, prevention diabetes deafness is an indispensable link.
In the model of diabetic deafness rats, especially the model of type 2 diabetic deafness rats, with the extension of time, the hearing loss is more obvious, effect of auditory function. To maintain the molecular normal cochlea mechano electrical conversion function and ensure the normal hearing conduction is the gap junction protein, connexin in diabetic rat cochlear deafness the change in the two months after modeling, the longer the disease, its expression decreased more significantly. In order to further confirm the Cx26 and Cx30 correlation between hearing loss and cochlear tissues, this study used immunofluorescence staining, HE staining, the distribution of detection of Cx26 and Cx30 in experimental rats with cochlear tissue protein expression level and hearing loss are consistent, and the whole cochlea preparation and observation of the relationship between hearing loss of hair cell loss. To further explore the expression of Cx30 and Cx26, diabetes mellitus The pathogenesis of deafness provides new research direction and theoretical experimental basis from molecular level to the pathogenesis of diabetic deafness.
Methods: the rats were anesthetized and decapitated, remove the cochlea, fixed, decalcified, paraffin embedded, sliced. Select the structural integrity of cochlear paraffin sections, HE staining and immunohistochemical staining respectively, to detect Cx26 and Cx30 expression in diabetic rat cochlear deafness distribution and expression were observed by confocal cochlear morphology and structure Cx26 and Cx30 microscopy and pathological changes of cochlea is consistent, correlation finally through the whole cochlear sections to observe the situation and hearing loss of hair cell loss.
Results: 1 type two diabetic rats HE staining of cochlear lateral wall tissue, basement membrane appeared in the experimental group compared with the normal group, thinning, lumen like structure stria cochlea tissue in the experimental group rats compared with control group, the cochlea stria appears to reduce the lateral wall of the cochlea and basement membrane eosinophilic staining in experimental group compared with normal group anachromasis, experimental group and control group, the number of rat cochlea spiral ligament and spiral ganglion cells were statistically significant difference (P? 0.05), the number of spiral ligament rats and spiral ganglion cells than the control group.
2 rats of type two diabetes model in January after the experimental group and the control group in the cochlear basilar membrane Cx26, stria vascularis and spiral ligament, the expression of Cx30 had no significant difference (P? 0.05), modeling 2,3,4,5 months after the experimental group and the control group in the cochlear basilar membrane Cx26, stria vascularis and spiral ligament, the expression level of Cx30 was significantly lower than the control group (P? 0.05).
3 the model of type two diabetic rat model was made in the experimental group and the control group was compared with the cochlear paving in the control group. There was no damage to the inner and outer hair cells.
4 type two diabetic rat model after the model February experimental group and control group rats cochlea preparation, experimental group than in the control group, the outer hair cells arranged in disorder, but no lack of modeling after March; the experimental group compared with the normal group, the outer hair cells were irregular and scattered, the degree is serious in February, but the outer hair cell missing; modeling 4,5 months after the experimental group compared with the normal group, the outer hair cells were irregular and scattered, and lack of inner and outer hair cells. These were the experimental group and the cochlear tissue Cx26 and the expression level of Cx30 protein decreased significantly compared with normal group in uniform, consistent with the ABR listening test results.
Conclusion:
1Cx26 and Cx30 are essential connexin in hearing generation. It is presumed that hearing impairment in diabetic rats may be associated with Cx26, Cx30

【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R587.2;R764.43

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