低频复合生理频率慢性电刺激对阻塞性睡眠呼吸暂停兔颏舌肌结构和功能影响的研究

发布时间：2018-06-09 17:54

本文选题：阻塞性睡眠呼吸暂停综合征 + 动物模型　；参考：《第三军医大学》2010年硕士论文

【摘要】： 阻塞性睡眠呼吸暂停综合征(Obstructive sleep apnea hypopnea syndrome OSAHS)是指在睡眠期间反复发生上气道阻塞并引起呼吸间歇性暂停的疾病,临床主要表现为睡眠打鼾、憋气,晨起头痛头晕,日间嗜睡乏力。由于在夜间睡眠中反复出现低氧血症和睡眠结构紊乱,导致OHSAS患者心脑血管疾病并发症增加。目前认为OSAHS是高血压心肌梗死和脑卒中等疾病的独立危险因素,严重影响人类的生活质量和生存时间,近年来已引起广泛重视。上气道解剖性狭窄和局部软组织易塌陷性是OSAHS发生的主要原因[1]。有研究发现OSAHS患者睡眠时呼吸道阻力比正常人睡眠时要高3倍[2]。上气道扩张肌收缩能力是决定OSAHS患者上气道开发程度的重要因素[3]。颏舌肌是主要的上气道扩张肌,正常收缩时舌体前移,可以阻止咽腔的缩小并对抗气流的阻力,维持气道开放。临床及基础研究证实[4,5],颏舌肌的功能异常可导致上气道塌陷,并且是引发OSAHS的最重要原因之一。因此,改善颏舌肌功能可成为有效、合理治疗OSAHS的关键。国内外研究发现[6,7,8],在夜间睡眠中呼吸暂停发生时,给予OSAHS患者颏舌肌急性电刺激,可促使颏舌肌的收缩,改善即刻的通气,终止呼吸暂停。但这种电刺激治疗模式系急性短期治疗,刺激频率是50Hz左右,不排除会损伤颏舌肌,且无长期治疗效果也无预防呼吸暂停事件发生的作用。因而,需要寻找一种更科学、有效的颏舌肌电刺激模型,实现对OSAHS颏舌肌肌纤维有宜的重构,防止上气道的塌陷和狭窄,改善OSAHS的通气,提高其远期生活质量。不同频率的电刺激可使骨骼肌产生不同的适应性变化,骨骼肌肌纤维类型可以发生转变,具有高度的可塑性[9]。研究发现[10],对新西兰白兔颏舌肌低频10Hz电刺激7天后,其颏舌肌I型肌纤维增加,颏舌肌抗疲劳能力得以增强。然而,如果电刺激后肌纤维I型纤维比例增加,随之而来的问题是肌肉收缩力量、速度及应急能力均会有所降低,显然这不是临床治疗OSAHS所需求的最佳肌纤维重构。以往我们在研究变频电刺激膈肌中发现[11],低频复合生理频率电刺激膈肌可以改善肌球蛋白比例和含量,既增强颏舌肌抗疲劳能力有增强收缩强度。据以上我们推测,低频复合生理频率慢性电刺激(low and physiological frequency chronic electrical stimulation,LPCES)可能是改善OSAHS颏舌肌肌纤维比例,增强肌肉抗疲劳能力和收缩力的最佳电刺激模式。目前,国内外研究尚无类似报道。本课题拟研究低频和低频复合生理频率电刺激对OSAHS兔颏舌肌肌纤维结构、能量代谢及力学特征的适应性变化特征和规律。论证阐明低频复合生理频率慢性电刺激(10+40HZ)防治颏舌肌疲劳的机理,为临床使用低频复合生理频率慢性电刺激颏舌肌治疗OSAHS提供理论依据。本课题主要包括以下内容: (一)阻塞性睡眠呼吸暂停兔模型的建立及模型的评价 1.采用低压缺氧法建立阻塞性睡眠呼吸暂停兔模型。 2.采用X线电子计算机断层扫描(computed tomography CT)观察阻塞性睡眠呼吸暂停兔模型上气道结构的变化。 3.采用Rm6240多道生理信号采集处理系统观察阻塞性睡眠呼吸暂停兔模型睡眠呼吸气流的变化。 4.观察阻塞性睡眠呼吸暂停兔模型血气分析的变化。 5.观察阻塞性睡眠呼吸暂停兔模型咽部及颏舌肌组织病理学的变化。 (二)低频复合生理频率慢性电刺激后阻塞性睡眠呼吸暂停兔颏舌肌肌球蛋白重链(myosin heavy chain MHC)亚型表达变化的实验观察 1.采用SDS-聚丙烯酰胺凝胶电泳(SDS-polyacrylamide gel electrophoresis SDS-PAGE)法测定,低频复合生理频率慢性电刺激后阻塞性睡眠呼吸暂停兔颏舌肌MHC四种亚型蛋白表达的变化。 2.采用半定量反转录聚合酶链式反应(Reverse transcripatase-polymerase Chainreaction RT-PCR)法测定,低频复合生理频率慢性电刺激后阻塞性睡眠呼吸暂停兔颏舌肌MHC四种亚型mRNA表达的变化 (三)低频复合生理频率慢性电刺激后阻塞性睡眠呼吸暂停兔颏舌肌力学特征与肌浆网Ca2+-ATP酶活性(sarcoplasmic reticulum Ca2+-ATPase SERCA)变化的实验观察 1.采用Rm6240多道生理信号采集处理系统测定低频复合生理频率慢性电刺激后阻塞性睡眠呼吸暂停兔颏舌肌力学特征的改变。 2.采用南京建成生物工程研究所提供的Ca2+-ATP酶测试盒测定低频复合生理频率电刺激后阻塞性睡眠呼吸暂停兔颏舌肌SERCA活性的改变。主要结果: 1.阻塞性睡眠呼吸暂停兔模型组上气道CT扫描结果显示:舌骨水平的咽后壁增厚(P 0.01)、前后径减小(P 0.05)、截面积减小(P 0.01);软腭中点水平软腭增厚(P 0.05),上下径减小(P0.05)。 2.阻塞性睡眠呼吸暂停兔模型组较对照组睡眠期间的呼吸暂停低通气指数(apnea hypopnea index,AHI)明显升高(P0.01)、呼吸频率加快(P 0.01)、呼吸时比增加(P0.01)、平均呼吸深度增加(P0.05)。 3.阻塞性睡眠呼吸暂停兔模型组较对照组动脉血氧分压(PaO2)降低(P0.01)、二氧化碳分压(PaCO2)增高(P0.05)。 4.阻塞性睡眠呼吸暂停兔模型组织病理检测显示:光镜下可见咽壁黏膜上皮组织增厚并角化,黏膜下层组织腺体增生;颏舌肌肌纤维萎缩、有溶解断裂现象。 5.在SDS- PAGE分析颏舌肌MHC亚型比例实验中,10Hz电刺激组及(10+40)Hz电刺激组颏舌肌MHCⅠ亚型比例增加(P 0.05),MHCⅡa亚型减少(P 0.05);(10+40)Hz电刺激组变化的趋势更加明显。 6.在RT-PCR法分析颏舌肌MHC亚型mRNA表达实验中,10Hz电刺激组MHCⅠ亚型mRNA表达量稍有上升,但差异不显著(P 0.05);(10+40)Hz电刺激组MHCⅠ亚型mRNA表达量显著上升(P 0.05);MHCⅡa,MHCⅡb及MHCⅡx三种亚型mRNA表达无显著差异。 7.在观察颏舌肌力学特征的实验中,10Hz组颏舌肌抗疲劳能力明显提高(P0.05),但收缩力改善不明显(P 0.05),(10+40)Hz组颏舌肌肌收缩力和抗疲劳能力都明显提高(P0.05)。 8.在观察颏舌肌SERCA活性的实验中,10Hz组颏舌肌SERCA活性明显降低(P0.05)、(10+40)Hz组颏舌肌SERCA活性稍增高(P0.05)。全文结论 1.慢性低压缺氧方法可以建立阻塞性睡眠呼吸暂停综合征兔模型,其病理生理改变与阻塞性睡眠呼吸暂停综合征患者相似。 2.低频复合生理频率慢性电刺激可使阻塞性睡眠呼吸暂停兔颏舌肌MHC由Ⅱa型向Ⅰ型转化,即“由快向慢”转化,颏舌肌肌纤维结构上的变化是其功能改善的基础。 3.低频复合生理频率慢性电刺激可提高阻塞性睡眠呼吸暂停兔颏舌肌抗疲劳能力及收缩力,并增强阻塞性睡眠呼吸暂停兔颏舌肌SERCA活性、改善兴奋-收缩偶联。提示低频复合生理频率慢性电刺激是对OSAHS患者治疗的较好频率模式。
[Abstract]:Obstructive sleep apnea syndrome ( Obstructive Sleep Apnea Syndrome ) is a disease caused by repeated upper airway obstruction and intermittent apnea during sleep . The clinical manifestations are sleep snoring , breath hold , morning headache , dizziness , daytime drowsiness and fatigue . As a result of the repeated occurrence of hypoxia and sleep disorder during the night sleep , the cardiovascular and cerebrovascular disease complications of OHSAS patients are increased . Currently , it is considered that the obstructive sleep apnea syndrome is an independent risk factor for hypertension , myocardial infarction and stroke , which seriously affects the quality of life and the survival time of human beings , and has attracted extensive attention in recent years .

The main reason why the upper airway anatomic stenosis and local soft tissue collapse is the main cause of the occurrence of obstructive sleep apnea . The results showed that the respiratory tract resistance was 3 times higher than that of normal subjects during sleep . The ability of the upper airway to expand muscle contraction is an important factor influencing the degree of airway development in patients with obstructive sleep apnea . The genioglossus is the main upper airway extension muscle , the tongue moves forward during normal contraction , which can prevent the pharyngeal cavity from shrinking and resist the flow resistance and maintain the airway open .

Therefore , it is necessary to find a more scientific and effective electrical stimulation model of the chin tongue muscle , which can improve the ventilation and improve the long - term quality of life .

Different frequency electric stimulation can make skeletal muscle produce different adaptability , skeletal muscle fiber types can change , and have high plasticity . It was found that the muscle contraction strength , the speed and the emergency ability were decreased after 7 days of 10 Hz electrical stimulation . However , it was found that low and physiological frequency chronic electrical stimulation ( LPCES ) might be the best mode of electrical stimulation to improve the muscle fiber ratio , muscle fatigue resistance and contraction force . At present , there are no similar reports at home and abroad .

This study intends to study the adaptive changes in the structure , energy metabolism and mechanical characteristics of the chin - glossus muscle fibers in obstructive sleep apnea syndrome by low - frequency and low - frequency composite physiological frequency electrical stimulation . It is demonstrated that the mechanism of chronic electrical stimulation ( 10 + 40HZ ) of low - frequency composite physiological frequency can be used to control the fatigue of the chin - glossus muscle , which provides a theoretical basis for the clinical use of low - frequency composite physiological frequency chronic electrical stimulation of the chin - glossus muscle .

This topic mainly includes the following :

( 1 ) Establishment and evaluation of model of obstructive sleep apnea rabbit model

1 . The model of obstructive sleep apnea rabbit was established by low pressure hypoxia .

2 . X - ray computed tomography ( computed tomography ) was used to observe the changes of airway structure in obstructive sleep apnea - rabbit model .

3 . Rm6240 multichannel physiological signal acquisition and processing system was used to observe the changes of sleep - breathing gas flow in obstructive sleep apnea - rabbit model .

4 . Observe the changes of blood gas analysis in obstructive sleep apnea rabbit model .

5 . To observe the pathological changes of pharyngeal and genioglossus in obstructive sleep apnea .

( 2 ) Experimental observation on the expression of myosin heavy chain MHC ( MHC ) subtype in patients with obstructive sleep apnea after chronic electrical stimulation of low - frequency composite physiological frequency

1 . SDS - polyacrylamide gel electrophoresis ( SDS - PAGE ) method was used to determine the changes of the expression of MHC four isoforms in the submental tongue muscle of rabbits after chronic electrical stimulation with low frequency compound physiological frequency .

2 . The changes of the mRNA expression of MHC four isoforms in the chin tongue muscle of rabbits after chronic electrical stimulation with low frequency and complex physiological frequencies were determined by reverse transcriptase polymerase chain reaction ( RT - PCR ) .

( 3 ) Experimental observation on the changes of the mechanical characteristics and Ca2 + - ATPase activity of the muscle sarcoplasmic reticulum ( Ca 2 + - ATPase SERCA ) in patients with obstructive sleep apnea after chronic electrical stimulation of low - frequency composite physiological frequency

1 . Using Rm6240 multi - channel physiological signal acquisition and processing system to measure the changes of the mechanical characteristics of the chin - glossus muscle of rabbits after chronic electrical stimulation with low - frequency composite physiological frequency .

2 . Using the Ca2 + - ATPase test kit provided by Nanjing Institute of Biological Engineering to measure the changes of SERCA activity of the chin tongue muscle of rabbits after low - frequency compound physiological frequency electrical stimulation .

Main results :

1 . The results of CT scan on the upper airway of obstructive sleep apnea rabbit model showed that the thickness of posterior pharyngeal wall increased ( P 0.01 ) , the anterior and posterior diameter decreased ( P 0.05 ) , the cross - sectional area decreased ( P 0.01 ) , and the soft palate increased significantly ( P 0.05 ) and the upper and lower diameters decreased ( P0.05 ) .

2 . Obstructive sleep apnea rabbits model group was significantly higher than that in the control group during sleep ( P0.01 ) , the respiratory rate increased ( P0.01 ) , the respiration time ratio increased ( P0.01 ) , and the average respiration depth increased ( P0.05 ) .

3 . Compared with the control group , the obstructive sleep apnea rabbits model group was lower than the control group ( P0.01 ) , and the carbon dioxide partial pressure ( PaCO 2 ) was increased ( P0.05 ) .

4 . The pathological examination of obstructive sleep apnea rabbit model showed that the epithelium of the pharyngeal wall was thickened and keratose under the light microscope , the glandular hyperplasia of the submucous glands of the mucous membrane was observed , and the muscular fibers of the genioglossus muscle atrophy and the phenomenon of dissolution and fracture was observed .

5 . After SDS - PAGE analysis , the proportion of MHC鈪爋f the genioglossus in 10 Hz electric stimulation group and ( 10 + 40 ) Hz electric stimulation group increased ( P 0.05 ) , the MHC 鈪 subtype decreased ( P 0.05 ) , and ( 10 + 40 ) Hz electric stimulation group changed more clearly .

6 . In the experiment of RT - PCR , the expression of MHC class I mRNA in 10Hz electrical stimulation group was slightly increased , but the difference was not significant ( P 0.05 ) ; ( 10 + 40 ) Hz electrical stimulation group MHC class I mRNA expression increased significantly ( P 0.05 ) ; there was no significant difference between MHC 鈪 , MHC 鈪 and MHC 鈪 mRNA expression .

7 . In the experiment of observing the mechanical characteristics of the genioglossus , the fatigue resistance of the genioglossus in 10 Hz group was obviously improved ( P0.05 ) , but the improvement of the contraction force was not obvious ( P 0.05 ) , and the contraction force and fatigue resistance of the genioglossus muscle in the ( 10 + 40 ) Hz group were obviously improved ( P0.05 ) .

8 . In the experiment of observation of SERCA activity , the SERCA activity of the genioglossus muscle of 10Hz group was significantly decreased ( P0.05 ) , and the activity of SERCA in the genioglossus of the ( 10 + 40 ) Hz group was slightly increased ( P0.05 ) .

full - text conclusion

1 . The model of obstructive sleep apnea syndrome can be established by chronic low pressure hypoxia , and its pathological changes are similar to those of obstructive sleep apnea syndrome .

2 . Chronic electrical stimulation of low - frequency composite physiological frequency can cause obstructive sleep apnea to change MHC from type 鈪 to type I , that is , " slow to slow " transformation , and changes in the structure of the muscle fiber of the genioglossus muscle fibers are the basis of its function improvement .

3 . Chronic electrical stimulation of low frequency compound physiological frequency can improve the fatigue resistance and contraction force of the chin tongue muscle in obstructive sleep apnea , and enhance the SERCA activity of the chin tongue in the obstructive sleep apnea and improve the excitation - contraction coupling . It is suggested that the chronic electrical stimulation of low frequency composite physiological frequency is a good frequency mode for the treatment of patients with obstructive sleep apnea .
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R766

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