大鼠听觉剥夺后Bcl-2、Bax、Fas在听皮层的表达变化及脑苷肌肽注射液对其变化的影响

发布时间：2018-07-03 13:40

本文选题：听觉剥夺 + Bcl-2　；参考：《河北医科大学》2015年硕士论文

【摘要】：目的:通过双侧耳蜗损毁术建立听觉剥夺(Auditory Deprivation,AD)动物模型,研究SD大鼠在听觉剥夺后Bcl-2、Bax、Fas在听皮层的表达,以及脑苷肌肽注射液对Bcl-2、Bax、Fas表达变化的影响,了解听觉剥夺后听觉中枢可塑性变化以及脑苷肌肽注射液的作用,为听力障碍患者的辅助治疗提供动物实验资料。方法:1选择耳廓反射灵敏、无中耳疾患、生后3周龄的雄性清洁级健康SD大鼠,共64只,将其至于适宜的温、湿度环境中饲养。按体重将其随机分为8组(8只/组):7天对照组,7天手术组,14天对照组,14天手术组,14天治疗组,28天对照组,28天手术组,28天治疗组。手术组大鼠实施双侧耳蜗损毁术,对照组大鼠只做耳后切口,治疗组大鼠实施双侧耳蜗损毁术并每日腹腔注射脑苷肌肽注射液,剂量1 ml/kg,连续14或28天。2术后7天对各组大鼠进行听性脑干反应(ABR)测试,然后在相同条件下饲养各组动物至对应的时间点后,每组随机取4只大鼠的双侧大脑听皮层做HE染色和免疫组化检测,其余4只取听皮层做实时荧光定量PCR和蛋白免疫印迹检测。3用HE染色的方法观察大鼠听皮层部位形态学变化。4采用免疫组织化学方法测定大鼠听皮层部位Bcl-2、Bax、Fas的表达,然后在光镜下对棕色颗粒阳性细胞数目进行统计。5采用RT-PCR技术检测大鼠听皮层部位Bcl-2、Bax、Fas基因m RNA的表达。6采用Western Blot技术检测大鼠听皮层部位Bcl-2、Bax、Fas蛋白的表达变化。结果:1 ABR测试情况:对照组SD大鼠经ABR测试在40SPL d B时出现重复波形,耳蜗损毁手术大鼠术后7天双耳在90 SPL d B均不能引出重复波形,证明听觉剥夺动物模型制作成功。2术后一般情况:术后第二天听觉剥夺大鼠即能自主饮水摄食,爬行沿直线,拎尾悬空能保持平衡,但是与对照组相比行动仍显迟缓。听觉剥夺大鼠对外界声音刺激不敏感,耳廓反射消失,而对照组耳廓反应灵敏。3 HE染色结果:手术组和治疗组均出现不等量的凋亡细胞,体积缩小,颜色深染,胞浆着色浅淡,核固缩,染色质凝集断裂,核仁消失。治疗组与相应的手术组相比,细胞凋亡现象减轻,胞浆红染程度减轻,细胞核,染色质聚集减少。实验结果表明大鼠在听觉剥夺后听皮层出现凋亡现象,脑苷肌肽注射液可以减轻凋亡的程度。4免疫组化结果:各组各个时间点均有Bcl-2、Bax、Fas阳性细胞。手术组Bcl-2阳性细胞数明显低于相应的对照组,治疗组Bcl-2阳性细胞数显著多于相应的手术组。而手术组Bax和Fas阳性细胞数显著高于相应的对照组,治疗组阳性细胞数显著少于相应的手术组。实验结果表明听觉剥夺后大鼠听皮层表达促凋亡基因Bax和Fas的细胞增多,表达抑凋亡基因Bcl-2的细胞减少,而脑苷肌肽注射液可以阻止这些变化。5蛋白免疫印迹检测结果:手术组Bcl-2蛋白灰度比值显著低于相应的对照组,治疗组灰度比值显著高于相应的手术组。而手术组Bax、Fas蛋白灰度比值显著高于相应的对照组,治疗组灰度比值显著低于相应的手术组。实验结果表明听觉剥夺后大鼠听皮层促凋亡基因蛋白Bax和Fas表达升高,抑凋亡蛋白Bcl-2表达降低,而脑苷肌肽注射液可以减轻这些变化的程度。6 RT-PCR检测结果:手术组Bcl-2基因m RNA的表达显著低于相应的对照组,治疗组基因表达比相应的手术组显著增强。而手术组Bax和Fas基因m RNA的表达显著高于相应的对照组,治疗组基因表达与相应的手术组比较显著减弱。实验结果表明听觉剥夺后大鼠听皮层促凋亡基因Bax和Fas的表达增强,抑凋亡基因Bcl-2的表达减弱,而脑苷肌肽注射液可以阻止这些变化。结论:1双侧耳蜗损毁术能够建立稳定性强的听觉剥夺动物模型。2 Bcl-2、Bax、Fas这三个基因在大鼠的听皮层均有表达。3 Bcl-2、Bax、Fas参与了听觉剥夺后听皮层神经元凋亡的过程。4脑苷肌肽注射液通过抑制细胞凋亡的途径实现对听觉剥夺所致听觉中枢损伤的保护作用。
[Abstract]:Objective: to establish Auditory Deprivation (AD) animal model by bilateral cochlear damage and study the expression of Bcl-2, Bax, Fas in auditory cortex after hearing deprivation, and the influence of Cattle Encephalon Glycoside and Ignotin Injection on the changes of Bcl-2, Bax, Fas expression after hearing deprivation in SD rats, and to understand the plasticity of auditory center after hearing deprivation and Cattle Encephalon Glycoside and Ignotin Injection. To provide animal experimental data for the adjuvant therapy of hearing impaired patients. Methods: 1 male clean SD rats with sensitive auricle reflex, no middle ear disease and 3 weeks of age after birth were selected, and 64 were bred in a suitable temperature and humidity environment. According to weight, they were divided into 8 groups (8 / group): 7 days control group, 7 day operation group, 14 days. The control group, the 14 day operation group, the 14 day treatment group, the 28 day control group, the 28 day operation group and the 28 day treatment group. The rats in the operation group carried out bilateral cochlear damage, the control group only did the post ear incision, the rats in the treatment group carried out bilateral cochlear damage and intraperitoneal injection of Cattle Encephalon Glycoside and Ignotin Injection daily, the dose of 1 ml/kg, and 14 or 28 days after the operation for 7 days after the.2 operation. The rats were tested with auditory brainstem response (ABR), and then the animals were fed to the corresponding time points under the same conditions. The bilateral cerebral auditory cortex of 4 rats in each group was randomly selected to do HE staining and immunohistochemical detection. The other 4 auditory cortex was measured by real-time fluorescence quantitative PCR and egg white immunoblotting detection.3 with HE staining method. The morphological changes of auditory cortex site.4 were measured by immunohistochemistry. The expression of Bcl-2, Bax, Fas in the auditory cortex of rats was measured, and then the number of brown granules positive cells was counted under light microscope..5 was used to detect the expression of Bcl-2, Bax and Fas m RNA in the auditory cortex of rats by RT-PCR technology. The changes in the expression of Bcl-2, Bax and Fas protein in the layer. Results: 1 ABR test: the SD rats in the control group were repeated by ABR test at 40SPL D B, and 7 days after the operation of the cochlear damage operation rats, the double ears in 90 SPL D B could not lead to the repetition of the waveform, which proved that the hearing deprivation animal model was successful after the operation: second days of hearing after the operation. The rats were deprived of their own drinking water, crawling along the line, and holding the tail in the air to keep the balance, but the action was still slow compared with the control group. The auditory deprivation rats were not sensitive to the external sound stimulation and the auricle reflex disappeared, while the control group's auricle reaction was sensitive.3 HE staining results: both the hands group and the treatment group had the apoptotic cells in the unequal amount. In the treatment group, the apoptosis phenomenon was reduced, the cytoplasmic red dye was reduced, the cell nucleus and the chromatin aggregation decreased. The experimental results showed that the rats were apoptotic in the auditory cortex after the deprivation of auditory sensation, and the Cattle Encephalon Glycoside and Ignotin Injection was found to be apoptotic in the rat cortex after hearing deprivation. The degree of apoptosis was reduced by.4 immunohistochemistry: Bcl-2, Bax, Fas positive cells were found at each time point. The number of Bcl-2 positive cells in the operation group was significantly lower than that in the corresponding control group. The number of Bcl-2 positive cells in the treatment group was significantly more than that of the corresponding operation group. The number of Bax and Fas positive cells in the operation group was significantly higher than that of the corresponding control group, the treatment group was significantly higher. The number of positive cells was significantly less than that in the corresponding surgical group. The results showed that the number of apoptotic gene Bax and Fas in the auditory cortex was increased and the cells expressing the apoptosis inhibiting gene Bcl-2 were decreased after hearing deprivation, and Cattle Encephalon Glycoside and Ignotin Injection could prevent these changes of.5 protein immunoblotting detection results: the ratio of the gray level of Bcl-2 protein in the operation group was significant. The grayscale ratio of the treatment group was significantly higher than that of the corresponding operation group. The gray ratio of Bax, Fas protein in the operation group was significantly higher than that in the corresponding control group. The gray ratio of the treatment group was significantly lower than that of the corresponding operation group. The experimental results showed that the expression of Bax and Fas in the auditory cortex of the auditory cortex was increased and the expression of Fas was increased. The expression of dead protein Bcl-2 decreased, while Cattle Encephalon Glycoside and Ignotin Injection could reduce the degree of.6 RT-PCR detection of these changes: the expression of Bcl-2 gene m RNA in the operation group was significantly lower than that in the corresponding control group. The expression of gene expression in the treatment group was significantly higher than that in the corresponding operation group. The expression of M RNA of the Bax and Fas genes in the operation group was significantly higher than that of the corresponding control group. The results showed that the expression of Bax and Fas in the auditory cortex was enhanced and the expression of Bcl-2 was weakened after the hearing deprivation, while Cattle Encephalon Glycoside and Ignotin Injection could prevent these changes. Conclusion: 1 bilateral cochlear damage can establish a stable and strong auditory peeling. The three genes,.2 Bcl-2, Bax, Fas, expressed.3 Bcl-2 in the auditory cortex of rats, Bax, and Fas participated in the apoptosis of auditory cortex neurons after hearing deprivation..4 Cattle Encephalon Glycoside and Ignotin Injection could protect the auditory center induced by auditory deprivation by inhibiting the apoptosis pathway.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R965

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