本文選題：聽覺剝奪 + Bcl-2��； 參考：《河北醫(yī)科大學》2015年碩士論文

【摘要】：目的:通過雙側(cè)耳蝸損毀術(shù)建立聽覺剝奪(Auditory Deprivation,AD)動物模型,研究SD大鼠在聽覺剝奪后Bcl-2、Bax、Fas在聽皮層的表達,以及腦苷肌肽注射液對Bcl-2、Bax、Fas表達變化的影響,了解聽覺剝奪后聽覺中樞可塑性變化以及腦苷肌肽注射液的作用,為聽力障礙患者的輔助治療提供動物實驗資料。方法:1選擇耳廓反射靈敏、無中耳疾患、生后3周齡的雄性清潔級健康SD大鼠,共64只,將其至于適宜的溫、濕度環(huán)境中飼養(yǎng)。按體重將其隨機分為8組(8只/組):7天對照組,7天手術(shù)組,14天對照組,14天手術(shù)組,14天治療組,28天對照組,28天手術(shù)組,28天治療組。手術(shù)組大鼠實施雙側(cè)耳蝸損毀術(shù),對照組大鼠只做耳后切口,治療組大鼠實施雙側(cè)耳蝸損毀術(shù)并每日腹腔注射腦苷肌肽注射液,劑量1 ml/kg,連續(xù)14或28天。2術(shù)后7天對各組大鼠進行聽性腦干反應(ABR)測試,然后在相同條件下飼養(yǎng)各組動物至對應的時間點后,每組隨機取4只大鼠的雙側(cè)大腦聽皮層做HE染色和免疫組化檢測,其余4只取聽皮層做實時熒光定量PCR和蛋白免疫印跡檢測。3用HE染色的方法觀察大鼠聽皮層部位形態(tài)學變化。4采用免疫組織化學方法測定大鼠聽皮層部位Bcl-2、Bax、Fas的表達,然后在光鏡下對棕色顆粒陽性細胞數(shù)目進行統(tǒng)計。5采用RT-PCR技術(shù)檢測大鼠聽皮層部位Bcl-2、Bax、Fas基因m RNA的表達。6采用Western Blot技術(shù)檢測大鼠聽皮層部位Bcl-2、Bax、Fas蛋白的表達變化。結(jié)果:1 ABR測試情況:對照組SD大鼠經(jīng)ABR測試在40SPL d B時出現(xiàn)重復波形,耳蝸損毀手術(shù)大鼠術(shù)后7天雙耳在90 SPL d B均不能引出重復波形,證明聽覺剝奪動物模型制作成功。2術(shù)后一般情況:術(shù)后第二天聽覺剝奪大鼠即能自主飲水攝食,爬行沿直線,拎尾懸空能保持平衡,但是與對照組相比行動仍顯遲緩。聽覺剝奪大鼠對外界聲音刺激不敏感,耳廓反射消失,而對照組耳廓反應靈敏。3 HE染色結(jié)果:手術(shù)組和治療組均出現(xiàn)不等量的凋亡細胞,體積縮小,顏色深染,胞漿著色淺淡,核固縮,染色質(zhì)凝集斷裂,核仁消失。治療組與相應的手術(shù)組相比,細胞凋亡現(xiàn)象減輕,胞漿紅染程度減輕,細胞核,染色質(zhì)聚集減少。實驗結(jié)果表明大鼠在聽覺剝奪后聽皮層出現(xiàn)凋亡現(xiàn)象,腦苷肌肽注射液可以減輕凋亡的程度。4免疫組化結(jié)果:各組各個時間點均有Bcl-2、Bax、Fas陽性細胞。手術(shù)組Bcl-2陽性細胞數(shù)明顯低于相應的對照組,治療組Bcl-2陽性細胞數(shù)顯著多于相應的手術(shù)組。而手術(shù)組Bax和Fas陽性細胞數(shù)顯著高于相應的對照組,治療組陽性細胞數(shù)顯著少于相應的手術(shù)組。實驗結(jié)果表明聽覺剝奪后大鼠聽皮層表達促凋亡基因Bax和Fas的細胞增多,表達抑凋亡基因Bcl-2的細胞減少,而腦苷肌肽注射液可以阻止這些變化。5蛋白免疫印跡檢測結(jié)果:手術(shù)組Bcl-2蛋白灰度比值顯著低于相應的對照組,治療組灰度比值顯著高于相應的手術(shù)組。而手術(shù)組Bax、Fas蛋白灰度比值顯著高于相應的對照組,治療組灰度比值顯著低于相應的手術(shù)組。實驗結(jié)果表明聽覺剝奪后大鼠聽皮層促凋亡基因蛋白Bax和Fas表達升高,抑凋亡蛋白Bcl-2表達降低,而腦苷肌肽注射液可以減輕這些變化的程度。6 RT-PCR檢測結(jié)果:手術(shù)組Bcl-2基因m RNA的表達顯著低于相應的對照組,治療組基因表達比相應的手術(shù)組顯著增強。而手術(shù)組Bax和Fas基因m RNA的表達顯著高于相應的對照組,治療組基因表達與相應的手術(shù)組比較顯著減弱。實驗結(jié)果表明聽覺剝奪后大鼠聽皮層促凋亡基因Bax和Fas的表達增強,抑凋亡基因Bcl-2的表達減弱,而腦苷肌肽注射液可以阻止這些變化。結(jié)論:1雙側(cè)耳蝸損毀術(shù)能夠建立穩(wěn)定性強的聽覺剝奪動物模型。2 Bcl-2、Bax、Fas這三個基因在大鼠的聽皮層均有表達。3 Bcl-2、Bax、Fas參與了聽覺剝奪后聽皮層神經(jīng)元凋亡的過程。4腦苷肌肽注射液通過抑制細胞凋亡的途徑實現(xiàn)對聽覺剝奪所致聽覺中樞損傷的保護作用。
[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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R965

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