枸橼酸西地那非对小鼠听力影响的实验研究
发布时间：2019-04-02 12:25
【摘要】：枸橼酸西地那非（Sildenafil citrate）是特异的5型磷酸二酯酶(type5phosphosdesterase, PDE5)磷酸二酯酶抑制剂，起先研发该类药物，目的用于治疗心血管方面疾病，而后发现在治疗勃起功能障碍（erectile dysfunction,ED）方面有显著效果。近年来随着枸橼酸西地那非被广泛使用，其药物副作用报道也增多，2007年美国FDA根据数十例临床案例的调查结果，首次报道该类药可导致药物性听力下降。为了研究这一问题，许多研究者进行了临床流行病学调查，显示5型磷酸二酯酶抑制剂类药物与听力突然丧失有着重要关联性，其中枸橼酸西地那非发病率最高，大剂量使用发病率则高达50％。由于用药不慎而致中毒性耳聋会给患者增加更多痛苦，因此，重视该类药物的耳毒性，研究这类药物耳毒性的发病机制具有重要价值，这可以指导临床医生正确使用该类药物。目前国内外对该类药物耳毒性的研究主要以回顾性病例分析总结为主，缺乏有效动物实验研究。 本课题拟通过动物实验，研究枸橼酸西地那非对小鼠听力的影响，建立昆明小鼠在体枸橼酸西地那非耳毒性模型，并探讨该药对听力损伤的可能机制，包括该药对耳蜗毛细胞形态、毛细胞离子通道和电生理特性的影响。为临床用药提供实验证据，指导临床正确使用该类药物。 研究目的： 1.通过灌胃给药，观察枸橼酸西地那非对小鼠听力的影响； 2.应用全细胞电流钳和细胞贴附电压钳技术，观察枸橼酸西地那非对小鼠耳蜗内 毛细胞电生理的影响，探讨其耳毒性机制。 材料和方法： 1.选择健康7周龄雄性昆明小鼠40只，随机分为4组，分别为枸橼酸西地那非低剂量组（0.1mg／(kg·d)、中剂量组（1mg／(kg·d)、高剂量组（10mg／(kg·d)）、空白对照组，检测每组于灌胃给药第0、1、5、10、20天的听性脑干反应阈值(Auditorybrain-stem response,ABR)，研究枸橼酸西地那非对小鼠听力的影响；采用异硫氰基荧光素(fluorescein isothiocyanate,FITC)标记的免疫荧光染色方法观察昆明小鼠耳蜗毛细胞的形态改变；并用高效液相色谱分析法(High performance liquid chromatography,HPLC)检测小鼠耳蜗内淋巴液枸橼酸西地那非浓度。 2.枸橼酸西地那非对小鼠耳蜗内毛细胞电生理影响的检测：分组如下：A组：正常组；B组：枸橼酸西地那非低剂量组。应用全细胞电流钳和细胞贴附电压钳技术，检测正常组和枸橼酸西地那非低剂量组内毛细胞(inner hair cell,IHC)的钙电流和膜电容的变化。 结果： 1. ABR的阈值结果显示：正常对照组20天内ABR阈值的波动较小，无明显变化；给药后1天，各组ABR阈值较正常对照组和喂药前无明显差异（未见明显统计学差异P0.05）；5天后西地那非给药组阈值则逐渐提高。西地那非各剂量组与空白对照组比较均有不同程度的听觉损伤，表现为ABR阈值上移，阈值上移程度与给药剂量有关，高剂量组较为明显，西地那非低剂量组较轻；各组随着给药时间延长，阈移逐渐增大。FITC荧光染色结果显示：绿色荧光为小鼠耳蜗毛细胞胞体，正常对照组小鼠耳蜗毛细胞即外毛细胞(outer hair cell,OHC)和内毛细胞(inner hair cell,IHC)形态正常，排列整齐，着色均匀、无缺失。西地那非低剂量组耳蜗损伤相对较轻，内、外毛细胞形态结构基本正常。西地那非中剂量组内毛细胞损伤较轻，3排外毛细胞损伤明显。西地那非高剂量组内、外毛细胞出现排列紊乱，细胞缺失，结构不完整，尤其是外毛细胞排列紊乱，甚至全部缺失。HPLC检测耳蜗内淋巴液结果显示：灌服一定剂量的枸橼酸西地那非后，能在小鼠耳蜗内淋巴液中检测到枸橼酸西地那非，出峰时间约在5.2min，且给药剂量增加，波峰增高，说明耳蜗内淋巴液中西地那非积聚浓度与给药剂量相关。 2.应用全细胞式膜片技术分别检测正常组和枸橼酸西地那非低剂量组耳蜗内毛细胞的钙电流和细胞膜电容，所得数据做两样本t检验。结果显示：所检测的钙电流为内向电流，，且成电压依赖性的激活。与正常组相比，枸橼酸西地那非使耳蜗内毛细胞钙电流内流减少，(P0．05，差异有统计学意义)，同时该药使内毛细胞的膜电容增量降低(P0．05，差异有统计学意义)。 结论： 1.枸橼酸西地那非可吸收入血液并能够通过血迷路屏障进入内耳，在内耳积聚；服用一定剂量的枸橼酸西地那非可以造成小鼠听力损伤。 2.枸橼酸西地那非可以造成耳蜗毛细胞的损伤，其损伤程度与使用剂量及时间有关。 3.枸橼酸西地那非使小鼠耳蜗内毛细胞的电生理发生改变，具体表现为内向钙电流和细胞膜电容减少，这可能是该药导致耳毒性的机制之一。
[Abstract]:Sildenafil citrate is a specific type 5 phosphodiesterase (PDE5) phosphodiesterase inhibitor, which is first developed for the treatment of cardiovascular diseases and is then found to have a significant effect in the treatment of erectile dysfunction (ED). As Sildenafil has been widely used in recent years, its drug-side effects have also increased. In 2007, the FDA reported for the first time the drug-induced hearing loss in response to the findings of several dozen clinical cases. In order to study this problem, a number of researchers conducted a clinical epidemiological survey showing an important link between the type 5 phosphodiesterase inhibitor and the sudden loss of hearing, with the highest incidence of sildenafil and a high dose of 50%. It is of great value to study the ototoxicity of the drug and to study the pathogenesis of the ototoxicity of the drug, which can guide the clinician to use the drug correctly. At present, the study on the ototoxicity of this kind of drug at home and abroad is mainly based on a retrospective case analysis and a lack of effective animal experiments. This topic is to study the effect of Sildenafil on the hearing of mice by animal experiments, and to establish a model of the toxicity of Sildenafil in the body of Kunming mice, and to explore the possible mechanism of the drug on hearing damage, including the effect of the drug on the hair cell shape of the cochlea. The Shadow of the Ion Channels and Electrophysiological Properties of the Hair Cells and Hair Cells In response, the clinical medication is provided with experimental evidence to guide the correct use of this type of drug A. Research Objective:1. To observe the effect of Sildenafil on mice by intragastric administration. 2. The application of all-cell current clamp and cell-attached electric pressure-clamp technique to observe the effect of the acid in the western region The effect of selenium on the electrophysiology of hair cells in the cochlea of mice to study its ototoxicity Materials and Methods:1. Forty healthy 7-week-old male Kunming mice were randomly divided into 4 groups. The low-dose group (0.1 mg/ (kg 路 d), medium-dose group (1 mg/ (kg 路 d) and high-dose group (10 mg/ kg 路 d) were selected as the low-dose group (0.1 mg/ (kg 路 d), middle-dose group (1 mg/ (kg 路 d) and high-dose group (10 mg/ kg 路 d). (kg 路 d), blank control group, and the auditory brainstem response threshold (ABR) in group 0,1,5,10 and 20 of each group were detected, and the effect of Sildenafil on the hearing of mice was studied. The morphology of the hair cells in the cochlea of the mice was changed, and the cochlea of the mice was detected by high performance liquid chromatography (HPLC). Sildenafil concentration of the internal lymph fluid.2. Detection of the electrophysiologic effects of sildenafil on the hair cells in the cochlea of mice: Group A: normal group Group B: Sildenafil, a low-dose group of sildenafil, was used to detect the inside hair cells of the low-dose group of sildenafil in the normal group and the low-dose group of Sildenafil. IHC Results:1. The threshold of ABR threshold showed that the fluctuation of ABR threshold in the normal control group was small and there was no significant change. The threshold of ABR in each group was less than that of the normal control group and the control group. Significant difference (no significant difference between P0.05) After 5 days, the threshold of sildenafil treatment group was increased gradually. There were different levels of hearing damage in the group of sildenafil and the blank control group, which showed that the threshold of ABR was higher, the degree of the shift was related to the dose of administration, and the high-dose group was more obvious. The results of FITC fluorescence staining showed that the green fluorescence was the cell body of the cochlea hair cell of the mouse, and the outer hair cells (OHC) and inner hair cells (IHC) of the cochlea hair cells of the normal control group were the outer hair cells (OHC) and inner hair cells (IHC). ) Normal shape, regular arrangement, uniform color and no deletion. Cochlear injury in low-dose group of sildenafil The morphology of the inner and outer hair cells was generally normal in a relatively light, inner and outer hair cell, and the dose group in sildenafil In the group of high dose of sildenafil, the outer hair cells were in disorder, the cells were absent, and the structure was incomplete. In particular, the external hair cell arrangement disorder, or even the whole deletion, was detected by HPLC. The results of the detection of the lymph fluid in the cochlea showed that after the administration of a certain dose of sildenafil, it was found that the peak time of sildenafil was detected in the lymph fluid in the cochlea of the mouse, and the peak-out time was about 5.2 min, and the dose of the drug is increased, the peak is increased, and the inside of the cochlea is explained. in that lymph fluid, the accumulation concentration of sildenafil is related to the administration dose.2. the total cell membrane technique is applied to detect the hair cells in the cochlea of the low-dose group of the normal group and the low-dose group of Sildenafil The calcium current and the cell membrane capacitance, and the obtained data were tested with two samples. The results showed that the results were as follows: In comparison with the normal group, the calcium current of the inner hair cells in the cochlea was reduced by Sildenafil (P0.05). volume increment Conclusion:1. Sildenafil can be absorbed into the blood and can enter the inner ear through the blood-labyrinth barrier and accumulate in the inner ear. ; Sildenafil with a certain dose can cause hearing damage in mice. 3. Sildenafil in the cochlea of the cochlea of the cochlea of the cochlea of the cochlea of the cochlea of the cochlea of the cochlea is changed, as shown in the following
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965
本文编号：2452562
[Abstract]:Sildenafil citrate is a specific type 5 phosphodiesterase (PDE5) phosphodiesterase inhibitor, which is first developed for the treatment of cardiovascular diseases and is then found to have a significant effect in the treatment of erectile dysfunction (ED). As Sildenafil has been widely used in recent years, its drug-side effects have also increased. In 2007, the FDA reported for the first time the drug-induced hearing loss in response to the findings of several dozen clinical cases. In order to study this problem, a number of researchers conducted a clinical epidemiological survey showing an important link between the type 5 phosphodiesterase inhibitor and the sudden loss of hearing, with the highest incidence of sildenafil and a high dose of 50%. It is of great value to study the ototoxicity of the drug and to study the pathogenesis of the ototoxicity of the drug, which can guide the clinician to use the drug correctly. At present, the study on the ototoxicity of this kind of drug at home and abroad is mainly based on a retrospective case analysis and a lack of effective animal experiments. This topic is to study the effect of Sildenafil on the hearing of mice by animal experiments, and to establish a model of the toxicity of Sildenafil in the body of Kunming mice, and to explore the possible mechanism of the drug on hearing damage, including the effect of the drug on the hair cell shape of the cochlea. The Shadow of the Ion Channels and Electrophysiological Properties of the Hair Cells and Hair Cells In response, the clinical medication is provided with experimental evidence to guide the correct use of this type of drug A. Research Objective:1. To observe the effect of Sildenafil on mice by intragastric administration. 2. The application of all-cell current clamp and cell-attached electric pressure-clamp technique to observe the effect of the acid in the western region The effect of selenium on the electrophysiology of hair cells in the cochlea of mice to study its ototoxicity Materials and Methods:1. Forty healthy 7-week-old male Kunming mice were randomly divided into 4 groups. The low-dose group (0.1 mg/ (kg 路 d), medium-dose group (1 mg/ (kg 路 d) and high-dose group (10 mg/ kg 路 d) were selected as the low-dose group (0.1 mg/ (kg 路 d), middle-dose group (1 mg/ (kg 路 d) and high-dose group (10 mg/ kg 路 d). (kg 路 d), blank control group, and the auditory brainstem response threshold (ABR) in group 0,1,5,10 and 20 of each group were detected, and the effect of Sildenafil on the hearing of mice was studied. The morphology of the hair cells in the cochlea of the mice was changed, and the cochlea of the mice was detected by high performance liquid chromatography (HPLC). Sildenafil concentration of the internal lymph fluid.2. Detection of the electrophysiologic effects of sildenafil on the hair cells in the cochlea of mice: Group A: normal group Group B: Sildenafil, a low-dose group of sildenafil, was used to detect the inside hair cells of the low-dose group of sildenafil in the normal group and the low-dose group of Sildenafil. IHC Results:1. The threshold of ABR threshold showed that the fluctuation of ABR threshold in the normal control group was small and there was no significant change. The threshold of ABR in each group was less than that of the normal control group and the control group. Significant difference (no significant difference between P0.05) After 5 days, the threshold of sildenafil treatment group was increased gradually. There were different levels of hearing damage in the group of sildenafil and the blank control group, which showed that the threshold of ABR was higher, the degree of the shift was related to the dose of administration, and the high-dose group was more obvious. The results of FITC fluorescence staining showed that the green fluorescence was the cell body of the cochlea hair cell of the mouse, and the outer hair cells (OHC) and inner hair cells (IHC) of the cochlea hair cells of the normal control group were the outer hair cells (OHC) and inner hair cells (IHC). ) Normal shape, regular arrangement, uniform color and no deletion. Cochlear injury in low-dose group of sildenafil The morphology of the inner and outer hair cells was generally normal in a relatively light, inner and outer hair cell, and the dose group in sildenafil In the group of high dose of sildenafil, the outer hair cells were in disorder, the cells were absent, and the structure was incomplete. In particular, the external hair cell arrangement disorder, or even the whole deletion, was detected by HPLC. The results of the detection of the lymph fluid in the cochlea showed that after the administration of a certain dose of sildenafil, it was found that the peak time of sildenafil was detected in the lymph fluid in the cochlea of the mouse, and the peak-out time was about 5.2 min, and the dose of the drug is increased, the peak is increased, and the inside of the cochlea is explained. in that lymph fluid, the accumulation concentration of sildenafil is related to the administration dose.2. the total cell membrane technique is applied to detect the hair cells in the cochlea of the low-dose group of the normal group and the low-dose group of Sildenafil The calcium current and the cell membrane capacitance, and the obtained data were tested with two samples. The results showed that the results were as follows: In comparison with the normal group, the calcium current of the inner hair cells in the cochlea was reduced by Sildenafil (P0.05). volume increment Conclusion:1. Sildenafil can be absorbed into the blood and can enter the inner ear through the blood-labyrinth barrier and accumulate in the inner ear. ; Sildenafil with a certain dose can cause hearing damage in mice. 3. Sildenafil in the cochlea of the cochlea of the cochlea of the cochlea of the cochlea of the cochlea of the cochlea of the cochlea is changed, as shown in the following
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965
【引证文献】
相关硕士学位论文 前2条
1 顾翔;AP-2蛋白在小鼠耳蜗毛细胞的表达及其调控突触囊泡胞吞的实验研究[D];第三军医大学;2015年
2 陈知己;小鼠内毛细胞CME相关蛋白Myosin6-Dab2的表达研究[D];第三军医大学;2015年
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