水杨酸钠通过激活caspase-3诱导豚鼠耳蜗螺旋神经节细胞凋亡的实验研究

发布时间：2018-04-20 09:17

本文选题：水杨酸钠 + caspase-3　；参考：《广西医科大学》2010年硕士论文

【摘要】： 目的研究水杨酸钠(sodium salicylate, SS)诱导豚鼠耳蜗螺旋神经节细胞(spiral ganglion neuron, SGN)凋亡及凋亡的机制。方法40只成年花色雄性黑目豚鼠随机分为4组,每组10只。A组:空白对照组;B组:人工外淋巴液(artificial perilymph, APL)组(左耳经圆窗龛注入APL后未给SS);C组:SS(400 mg·kg~(-1)·d~(-1))+APL组(左耳经圆窗龛注入APL后经腹腔SS给药);D组:SS(400 mg·kg~(-1)·d~(-1))+zDEVD-FMK组(左耳经圆窗龛注入zDEVD-FMK后经腹腔SS给药)。所有动物均于给药前后分别进行听性脑干反应(auditory brainstem response, ABR)阈值测试。10 % SS(溶于APL配制而成)均采用腹腔注射给药方式,400 mg·kg~(-1)·d~(-1),并于连续给药10天后即时处死动物取出耳蜗,采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling, TUNEL)检测耳蜗SGN凋亡细胞,免疫组织化学染色方法检测SGN内激活型caspase-3蛋白表达情况,透射电镜(transmission electron microscopy, TEM)观察SGN超微结构变化。结果ABR测试结果显示A、B两组动物听阈均无明显变化,C组动物给药后听阈提高,与A、B两组比较有显著性差异(p0.01,p0.01),D组动物听阈较C组降低(p0.05);TUNEL标记显示A、B两组动物耳蜗中均未见SGN凋亡细胞,亦无激活型caspase-3蛋白表达,而C组则出现较多TUNEL阳性着色SGN,并伴有激活型caspase-3表达的增高,D组较C组TUNEL阳性着色SGN减少,激活型caspase-3表达亦降低,A、B两组与C、D两组比较差异均有显著统计学意义(p0.01,p0.01),而C、D两组比较亦有显著差别(p0.01);透射电镜观察结果显示A、B两组SGN超微结构正常,C组SGN伴有明显细胞凋亡形态学特征,D组SGN超微结构未见明显凋亡征象。结论①SS可诱导豚鼠耳蜗SGN发生凋亡;②SGN凋亡与caspase-3激活有关;③zDEVD-FMK可在一定程度上拮抗caspase-3的激活,并阻断其引起的SGN凋亡。
[Abstract]:Objective to investigate the mechanism of sodium salicylate (SS) induced apoptosis of spiral ganglion cells (SGNN) in guinea pig cochlea. Methods Forty adult male black guinea pigs were randomly divided into 4 groups. 10 rats in each group: control group B: artificial perilymphs (left ear after injection of APL through a round window niche without APL) APL group (left ear was injected with APL through a circular window niche and intraperitoneal administration of SS: 400 mg / kg) zDEVD-FMK group (left ear group: 400 mg / kg ~ (-1)) zDEVD-FMK group (left ear: n = 400 mg / kg 路kg ~ (-1)) / d ~ (-1) / d ~ (-1) / d ~ (-1) / d ~ (-1) / d ~ (-1) / d ~ (-1) / d ~ (-1) / d ~ (-1) / d ~ (-1) / d ~ (-1) / d ~ (-1) / d ~ (-1) / d ~ (-1). ZDEVD-FMK was injected through a round window niche and then intraperitoneally administered with SS. All animals were given auditory brainstem response (ABRs) threshold test. 10% SS (dissolved in APL) was administered intraperitoneally with 400 mg / kg ~ (-1) DX respectively. Cochlea were removed immediately after 10 days of continuous administration, and cochlea were taken out immediately after 10 days of continuous administration. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (Tunel) was used to detect SGN apoptotic cells in cochlea, and the expression of activated caspase-3 protein in SGN was detected by immunohistochemical staining. The ultrastructural changes of SGN were observed by transmission electron microscopy (TM). Results the results of ABR test showed that there was no significant change in hearing threshold in both groups. There was a significant difference between group A and group A (P 0.01). Compared with group C, the auditory threshold of group D was significantly lower than that of group C. No apoptotic SGN cells were found in cochlea of group A and B, and no expression of activated caspase-3 protein was found in the cochlea of the two groups. In group C, there were more TUNEL positive staining SGNs, and increased expression of activated caspase-3 in group D was lower than that in group C in TUNEL positive staining SGN. The expression of activated caspase-3 was also decreased in both groups. There was a significant difference in the expression of activated caspase-3 between group A and C, and the difference between group C and group C was significant (p 0.01), and there was also a significant difference between group C and group C (P 0.01). Transmission electron microscopy showed that the ultrastructure of SGN in group A B was normal and that in group C was significantly higher than that in group C (P < 0.05). The ultrastructure of SGN in group D showed no obvious signs of apoptosis. Conclusion 1SS can induce apoptosis of SGN in guinea pig cochlea. The apoptosis of 2SGN is related to the activation of caspase-3. 1SS can antagonize the activation of caspase-3 to some extent and block the apoptosis of SGN induced by 1SS.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R764

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