枸櫞酸西地那非對小鼠聽力影響的實驗研究
發(fā)布時間：2019-04-02 12:25
【摘要】：枸櫞酸西地那非（Sildenafil citrate）是特異的5型磷酸二酯酶(type5phosphosdesterase, PDE5)磷酸二酯酶抑制劑，起先研發(fā)該類藥物，目的用于治療心血管方面疾病，而后發(fā)現(xiàn)在治療勃起功能障礙（erectile dysfunction,ED）方面有顯著效果。近年來隨著枸櫞酸西地那非被廣泛使用，其藥物副作用報道也增多，2007年美國FDA根據(jù)數(shù)十例臨床案例的調(diào)查結(jié)果，首次報道該類藥可導(dǎo)致藥物性聽力下降。為了研究這一問題，許多研究者進行了臨床流行病學(xué)調(diào)查，顯示5型磷酸二酯酶抑制劑類藥物與聽力突然喪失有著重要關(guān)聯(lián)性，其中枸櫞酸西地那非發(fā)病率最高，大劑量使用發(fā)病率則高達50％。由于用藥不慎而致中毒性耳聾會給患者增加更多痛苦，因此，重視該類藥物的耳毒性，研究這類藥物耳毒性的發(fā)病機制具有重要價值，這可以指導(dǎo)臨床醫(yī)生正確使用該類藥物。目前國內(nèi)外對該類藥物耳毒性的研究主要以回顧性病例分析總結(jié)為主，缺乏有效動物實驗研究。 本課題擬通過動物實驗，研究枸櫞酸西地那非對小鼠聽力的影響，建立昆明小鼠在體枸櫞酸西地那非耳毒性模型，并探討該藥對聽力損傷的可能機制，包括該藥對耳蝸毛細胞形態(tài)、毛細胞離子通道和電生理特性的影響。為臨床用藥提供實驗證據(jù)，指導(dǎo)臨床正確使用該類藥物。 研究目的： 1.通過灌胃給藥，觀察枸櫞酸西地那非對小鼠聽力的影響； 2.應(yīng)用全細胞電流鉗和細胞貼附電壓鉗技術(shù)，觀察枸櫞酸西地那非對小鼠耳蝸內(nèi) 毛細胞電生理的影響，探討其耳毒性機制。 材料和方法： 1.選擇健康7周齡雄性昆明小鼠40只，隨機分為4組，分別為枸櫞酸西地那非低劑量組（0.1mg／(kg·d)、中劑量組（1mg／(kg·d)、高劑量組（10mg／(kg·d)）、空白對照組，檢測每組于灌胃給藥第0、1、5、10、20天的聽性腦干反應(yīng)閾值(Auditorybrain-stem response,ABR)，研究枸櫞酸西地那非對小鼠聽力的影響；采用異硫氰基熒光素(fluorescein isothiocyanate,FITC)標(biāo)記的免疫熒光染色方法觀察昆明小鼠耳蝸毛細胞的形態(tài)改變；并用高效液相色譜分析法(High performance liquid chromatography,HPLC)檢測小鼠耳蝸內(nèi)淋巴液枸櫞酸西地那非濃度。 2.枸櫞酸西地那非對小鼠耳蝸內(nèi)毛細胞電生理影響的檢測：分組如下：A組：正常組；B組：枸櫞酸西地那非低劑量組。應(yīng)用全細胞電流鉗和細胞貼附電壓鉗技術(shù)，檢測正常組和枸櫞酸西地那非低劑量組內(nèi)毛細胞(inner hair cell,IHC)的鈣電流和膜電容的變化。 結(jié)果： 1. ABR的閾值結(jié)果顯示：正常對照組20天內(nèi)ABR閾值的波動較小，無明顯變化；給藥后1天，各組ABR閾值較正常對照組和喂藥前無明顯差異（未見明顯統(tǒng)計學(xué)差異P0.05）；5天后西地那非給藥組閾值則逐漸提高。西地那非各劑量組與空白對照組比較均有不同程度的聽覺損傷，表現(xiàn)為ABR閾值上移，閾值上移程度與給藥劑量有關(guān)，高劑量組較為明顯，西地那非低劑量組較輕；各組隨著給藥時間延長，閾移逐漸增大。FITC熒光染色結(jié)果顯示：綠色熒光為小鼠耳蝸毛細胞胞體，正常對照組小鼠耳蝸毛細胞即外毛細胞(outer hair cell,OHC)和內(nèi)毛細胞(inner hair cell,IHC)形態(tài)正常，排列整齊，著色均勻、無缺失。西地那非低劑量組耳蝸損傷相對較輕，內(nèi)、外毛細胞形態(tài)結(jié)構(gòu)基本正常。西地那非中劑量組內(nèi)毛細胞損傷較輕，3排外毛細胞損傷明顯。西地那非高劑量組內(nèi)、外毛細胞出現(xiàn)排列紊亂，細胞缺失，結(jié)構(gòu)不完整，尤其是外毛細胞排列紊亂，甚至全部缺失。HPLC檢測耳蝸內(nèi)淋巴液結(jié)果顯示：灌服一定劑量的枸櫞酸西地那非后，能在小鼠耳蝸內(nèi)淋巴液中檢測到枸櫞酸西地那非，出峰時間約在5.2min，且給藥劑量增加，波峰增高，說明耳蝸內(nèi)淋巴液中西地那非積聚濃度與給藥劑量相關(guān)。 2.應(yīng)用全細胞式膜片技術(shù)分別檢測正常組和枸櫞酸西地那非低劑量組耳蝸內(nèi)毛細胞的鈣電流和細胞膜電容，所得數(shù)據(jù)做兩樣本t檢驗。結(jié)果顯示：所檢測的鈣電流為內(nèi)向電流，，且成電壓依賴性的激活。與正常組相比，枸櫞酸西地那非使耳蝸內(nèi)毛細胞鈣電流內(nèi)流減少，(P0．05，差異有統(tǒng)計學(xué)意義)，同時該藥使內(nèi)毛細胞的膜電容增量降低(P0．05，差異有統(tǒng)計學(xué)意義)。 結(jié)論： 1.枸櫞酸西地那非可吸收入血液并能夠通過血迷路屏障進入內(nèi)耳，在內(nèi)耳積聚；服用一定劑量的枸櫞酸西地那非可以造成小鼠聽力損傷。 2.枸櫞酸西地那非可以造成耳蝸毛細胞的損傷，其損傷程度與使用劑量及時間有關(guān)。 3.枸櫞酸西地那非使小鼠耳蝸內(nèi)毛細胞的電生理發(fā)生改變，具體表現(xiàn)為內(nèi)向鈣電流和細胞膜電容減少，這可能是該藥導(dǎo)致耳毒性的機制之一。
[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
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R965
本文編號：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
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R965
【引證文獻】
相關(guān)碩士學(xué)位論文 前2條
1 顧翔;AP-2蛋白在小鼠耳蝸毛細胞的表達及其調(diào)控突觸囊泡胞吞的實驗研究[D];第三軍醫(yī)大學(xué);2015年
2 陳知己;小鼠內(nèi)毛細胞CME相關(guān)蛋白Myosin6-Dab2的表達研究[D];第三軍醫(yī)大學(xué);2015年
本文編號：2452562
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