丁酸钠对庆大霉素耳毒性保护机制的体内研究

发布时间：2019-06-04 09:15

【摘要】：实验一：凝胶海绵圆窗龛局部给药对正常豚鼠听力的影响目的：研究经手术，圆窗龛局部放置凝胶海绵的给药方式对正常豚鼠的听性脑干反应（AuditorybrainstemresponseABR）阈值的影响。方法：5只健康红目白化豚鼠，单耳手术。显微钻打开听泡,暴露圆窗,取0.3mm~3凝胶海绵吸取10ul外淋巴液放置于圆窗龛。术后立即行ABR检测。了解通过手术，圆窗龛局部放置凝胶海绵的给药方式对正常豚鼠ABR阈值的影响。结果：配对T检验：显示手术前后ABR的反应阈值有统计学差异（P0.01）。结论：本实验证实了，圆窗龛放置凝胶海绵的给药方式，因为改变了正常的传导方式，故而影响了正常豚鼠的ABR反应阈值。所以，在接下来的实验中，ABR阈值的检测结果的比较要考虑到给药方式本身的影响。实验二：丁酸钠圆窗给药毒性研究目的：了解组蛋白去乙酰化酶抑制剂（HDACi）丁酸钠（NaB）圆窗龛给药对毛细胞有无毒性作用。方法:5只红目白化豚鼠单耳手术，圆窗龛放置0.3mm~3凝胶海绵，吸附10ul丁酸钠NaB(100mg/ml)，局部放置14天。而后行ABR检测、基底膜铺片、扫描电镜。观察丁酸钠对毛细胞有无毒性作用。结果：单因素方差分析显示：给药后ABR阈值与给药后-给药前的ABR阈移，与单纯手术组相比无统计学意义（P0.05）。全基底膜铺片显示：毛细胞无缺失。扫描电镜显示：纤毛形态正常。结论：丁酸钠对正常豚鼠的听力，以及毛细胞无损伤作用。本实验证实圆窗膜龛放置丁酸钠无毒性损伤作用。故可采用圆窗膜给药的方式进一步验证其是否对庆大霉素耳毒性具有保护作用。实验三：丁酸钠对庆大霉素耳毒性的保护作用目的：研究丁酸钠对庆大霉素耳毒性的保护作用。方法：10只雄性红目白化豚鼠，实验期间，可以自由摄取食物与水。经ABR检测听力正常后，双耳手术。通过手术方法向置放在豚鼠右耳圆窗龛内约0.3mm~3的明胶海绵注入10ul丁酸钠（100mg/ml）溶液；向置放在豚鼠左耳圆窗龛内约0.3mm~3的明胶海绵注入10ul外淋巴液作为空白对照。3天后肌注庆大霉素（200mg/kg.day连续5天）.休息3天行ABR检测、基底膜铺片染色、扫描电镜观察、毛细胞缺失率计数及免疫荧光染色观察丁酸钠是否对庆大霉素耳毒性具有保护作用。结果：配对T检验显示：丁酸钠组ABR阈值与外淋巴液组相比较有统计学差异（P0.01)。基底膜铺片，鬼笔环肽染色示：第二转丁酸钠组毛细胞缺失较外淋巴液组少。扫描电镜观察：对照组毛细胞缺失严重，特别是第二、三层外毛细胞。残存的毛细胞纤毛肿胀、倒伏，排列紊乱，失去毛细胞之间的正常连接。同时，内毛细胞亦有缺失，伴排列紊乱，纤毛倒伏、肿胀。给予保护剂丁酸钠后外毛细胞缺失明显减少，纤毛肿胀也较对照组减轻。毛细胞缺失率计数显示：对照组，可见外毛细胞距顶转约10-40%即有减少，距顶转约55%开始明显减少，距顶转约70%基本无外毛细胞存在。内毛细胞距顶转约60%开始减少，70%处明显减少，80%处基本无内毛细胞存在。给予保护剂丁酸钠组可见外毛细胞距顶转约55-65%即有减少，距顶转约70%开始明显减少，距顶转约80%基本无外毛细胞存在。内毛细胞距顶转约70%开始减少，75%处明显减少，80%处基本无内毛细胞存在。免疫荧光检测示:1.正常豚鼠圆窗龛给予丁酸钠（NaB）作用后，HDAC1的表达减低。 2.随着庆大霉素作用时间的延长，HDAC1的荧光亮度增加，表达量增高。3.形态异常的核，HDAC1的表达是增加的。4.庆大霉素作用后5天，出现核固缩、溶解、消失。HDAC1由于是核表达，故表现出同样的变化。5.给予丁酸钠组庆大霉素作用后5天。虽然也有核的溶解、消失，但多数核的形态正常。且HDAC1的荧光亮度较对照组低，表达量较对照组减少。结论：组蛋白去乙酰化酶抑制剂，，丁酸钠（NaB）对庆大霉素耳毒性具有保护作用。并且此保护作用是通过抑制组蛋白的去乙酰化水平而实现的。
[Abstract]:Objective: To study the effect of local administration of gel sponge round window niche on the hearing of the normal guinea pig: study the effect of the administration of local gel sponge on the threshold of the auditory brainstem response (ABR) of the normal guinea pig. Method:5 healthy red eyes albino guinea pigs, single ear Intraoperative. The micro-drill opened the sound bulb and exposed the round window, and the 0.3 mm ~ 3 gel sponge was used to draw the 10 ul of the external lymph fluid to the round window. niches. The ABR test was performed immediately after the operation Measurement. See the shadow of the normal guinea pig's ABR threshold by means of the procedure and the local placement of the gel sponge in the round window niche. Response: paired T-test: There was a statistical difference in the response threshold of the ABR before and after the operation (P0. 01). Conclusion: The experimental results confirm that the round window niche is used to place the gel sponge, because the normal conduction mode is changed, thus the AB of the normal guinea pig is affected. R reaction threshold. Therefore, in the next experiment, the comparison of the detection results of the ABR threshold should take into account the mode of administration The effect of sodium butyrate on its own. The purpose of the study on the toxicity of round-window administration: to understand the administration of histone to the niches (NaB) round-window niche of histone-deethanolase inhibitor (HDACi). The hair cells were non-toxic. Methods:5 eyes with albino guinea pigs were operated in a single ear, 0.3 mm ~ 3 gel sponge was placed in the round window niche, and NaB (100 mg/ kg) was adsorbed. ml), localized for 14 days, followed by an ABR test And scanning electron microscope to observe the butyric acid. The effect of sodium on hair cells was non-toxic. Results: The single-factor analysis of variance showed that the ABR threshold after administration and the ABR threshold after administration-predose were compared to the simple surgical group. No statistical significance (P0.05). The hair cells showed no missing hair cells. Scanning electron microscope (SEM) showed that the ciliated form was normal. The hearing of the mouse, as well as the damage of the hair cell, confirmed the circle The window film niche is used for the non-toxic damage of sodium butyrate, so that the round window membrane can be used for further verifying that it is No protective effect on the ototoxicity of gentamicin. The protective effect of sodium butyrate on the ototoxicity of gentamicin : To study the protective effect of sodium butyrate on the ototoxicity of gentamicin. in that albino guinea pig, free uptake during the course of the experiment The food and water were tested by the ABR. After the hearing was normal, the two-ear operation was performed. The solution of 10 ul of sodium butyrate (100 mg/ ml) was injected into the gelatin sponge placed in the round window niche of the right ear of the guinea pig through the surgical method, and 10 ul of the external lymph fluid was injected into the gelatin sponge placed in the round window niche of the left ear of the guinea pig to be used as the blank control. After 3 days, the gentamicin (200 mg/ < Chunk> kg. day 5 consecutive days). ABR detection, basement membrane spreading, scanning electron microscopy, hair cell loss rate count and immunofluorescence staining for 3 days of rest. The protective effect of sodium butyrate on the ototoxicity of gentamicin was observed. Results: The test of paired T-test showed that the ABR threshold of sodium butyrate group There was a significant difference between the value and the external lymph group (P0.01). The results showed that the hair cells of the second sodium butyrate group were less than that of the external lymph fluid group. The hair cells of the control group were missing, especially the second and the three outer hair cells. The remaining hair cells were cilia. Swelling, lodging, disorder, and loss of normal connections between hair cells. And the inner hair cells are also deleted, and the hair cells are arranged in a disorder, the cilia lodging and the swelling are arranged, and sodium butyrate is administered. The loss of hair cells in the outer hair cells was significantly reduced, and the cilia swelling was also reduced in the control group. The counting of the loss rate of the hair cells showed that the control group, the visible outer hair cell was about 10-40% from the top to the top, and the distance from the top to the top was about 55%. There was a significant reduction in the beginning, about 70% from the top and about 70% in the absence of an outer hair cell. The inner hair cell started to decrease from the top to about 60%. There is a significant reduction in 70%, and there is no internal hair cell at 80%. It can be seen that the hair cell is reduced from the top to about 55-65% from the top to about 70% from the top. There was a significant reduction in the onset of the primary hair cell from the top to about 80%. The inner hair cell was reduced from the top to about 70%. at 75%, There are basically no internal hair cells at 80%. Immunofluorescence detection:1. The normal guinea pig round window niche The expression of HDAC1 decreased after the effect of sodium butyrate (NaB). The increase of the fluorescence intensity of HDAC1 and the increase of the expression of HDAC1 were increased. High.3. The expression of HDAC1 is increased in the nucleus with abnormal morphology. .4. After 5 days after the effect of gentamycin, the presence of the nuclear solid, the dissolution and the elimination Loss. HDAC1 is a core expression, so the table the same changes were shown.5. The effect of gentamicin in the sodium butyrate group After 5 days, although there was also the dissolution of the nucleus, it disappeared, but the majority of the nuclei were in normal form. The fluorescence intensity of HDAC1 was lower than that of the control group. The expression of HDAC1 was lower than that in the control group. The preparation and sodium butyrate (NaB) have a protective effect on the ototoxicity of gentamicin.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R764

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