發(fā)布時(shí)間：2018-06-17 21:20

  本文選題：耳蝸 + 器官培養(yǎng)��； 參考：《華中科技大學(xué)》2016年博士論文

【摘要】：第一部分：SD大鼠乳鼠耳蝸器官體外培養(yǎng)技術(shù)目的：探討內(nèi)耳器官培養(yǎng)技術(shù)使毛細(xì)胞、螺旋神經(jīng)元及聽(tīng)神經(jīng)纖維在體外培養(yǎng)環(huán)境維持完整形態(tài),為進(jìn)一步開(kāi)展藥物的耳毒性及其保護(hù)性研究奠定了實(shí)驗(yàn)技術(shù)的基礎(chǔ)。方法：選用Sprague Dawley大鼠2-3天的新生乳鼠,解剖顯微鏡下解剖分離前庭器官膜、外側(cè)壁、基底膜。將基底膜分成三段,頂、中、底回三個(gè)節(jié)段,利用培養(yǎng)基的液體表面張力鋪放將基底膜和前庭器官等各個(gè)器官,平整向上平整鋪放在培養(yǎng)皿表面,培養(yǎng)48小時(shí)后觀察基底膜形態(tài)并計(jì)數(shù)毛細(xì)胞數(shù)量。結(jié)果：前庭毛細(xì)胞和耳蝸毛細(xì)胞靜纖毛整齊排列,無(wú)紊亂,前庭毛細(xì)胞排列整齊無(wú)缺失,基底膜三個(gè)節(jié)段的三排外毛細(xì)胞和一排內(nèi)毛細(xì)胞沿柯替氏器螺旋器(OC, Organ of Corti)整齊排列,無(wú)缺失。螺旋神經(jīng)結(jié)細(xì)胞排列密集,呈橢圓形,細(xì)胞體積大,胞核清晰,聽(tīng)神經(jīng)纖維排列整齊,無(wú)斷裂。結(jié)論：運(yùn)用液體表面張力貼壁方法,可以在體外環(huán)境下獲得觀察方便、形態(tài)結(jié)構(gòu)完整的的耳蝸器官和組織。第二部分：尼古丁對(duì)新生大鼠體外培養(yǎng)基底膜的毒性作用目的：建立尼古丁耳毒性的體外培養(yǎng)損傷模型,探討尼古丁在耳蝸毛細(xì)胞、螺旋神經(jīng)結(jié)細(xì)胞和神經(jīng)纖維及的毒性作用及劑量反應(yīng)關(guān)系。方法：分離培養(yǎng)2-3天SD大鼠耳蝸基底膜,按照不同作用濃度(0-100ng/ml)處理尼古丁24小時(shí)。對(duì)耳蝸毛細(xì)胞和螺旋神經(jīng)結(jié)及聽(tīng)神經(jīng)纖維絲采用免疫熒光染色進(jìn)行觀測(cè),分別對(duì)毛細(xì)胞,螺旋神經(jīng)纖維和神經(jīng)元計(jì)數(shù)。對(duì)毛細(xì)胞、支持細(xì)胞及毛細(xì)胞靜纖毛束的超微結(jié)構(gòu)使用掃描和透射電子顯微鏡觀察。結(jié)果：通過(guò)對(duì)不同尼古丁濃度處理的毛細(xì)胞計(jì)數(shù),毛細(xì)胞損失量隨尼古丁濃度的增加。尼古丁的內(nèi)、外毛細(xì)胞受到類似的損傷,毛細(xì)胞的損傷,且基底膜底回毛細(xì)胞較頂、中損傷嚴(yán)重。尼古丁的處理導(dǎo)致毛細(xì)胞胞漿空泡增多,靜纖毛束紊亂和缺失,線粒體數(shù)量減少、內(nèi)質(zhì)網(wǎng)腫脹和脫顆粒改變。螺旋神經(jīng)元和聽(tīng)神經(jīng)纖維未受到明顯損傷。結(jié)論：尼古丁主要損害體外培養(yǎng)毛細(xì)胞,尼古丁所造成的毛細(xì)胞損傷模式有耳蝸底回?cái)U(kuò)展到頂、中回,尼古丁耳毒性呈劑量依賴關(guān)系,但對(duì)螺旋神經(jīng)元和聽(tīng)神經(jīng)纖維無(wú)明顯損傷。第三部分：格爾德霉素衍生物17-DMAG通過(guò)上調(diào)HSP70對(duì)新生大鼠耳蝸尼古丁毒性保護(hù)作用目的：研究格爾德霉素衍生物17-DMAG在體外培養(yǎng)的環(huán)境下能否保護(hù)尼古丁引起的耳蝸毛細(xì)胞損傷。方法：分離培養(yǎng)2-3天SD大鼠耳蝸基底膜,篩選出具有培養(yǎng)能夠保護(hù)毛細(xì)胞的17-DMAG處理濃度,基底膜分為空白對(duì)照組、單獨(dú)尼古丁處理組和17-DMAG預(yù)處理5小時(shí)后尼古丁處理組。運(yùn)用ELISA和實(shí)時(shí)定量PCR檢測(cè)HSP70的蛋白和mRNA表達(dá)水平：通過(guò)熒光免疫組織化學(xué)技術(shù)檢測(cè)HSP70在離體培養(yǎng)基底膜的細(xì)胞定位,并對(duì)各組內(nèi)、外毛細(xì)胞進(jìn)行計(jì)數(shù)。結(jié)果：在體外培養(yǎng)基底膜,HSP70主要定位于體外培養(yǎng)基底膜周圍部分新生的支持細(xì)胞及內(nèi)、外毛細(xì)胞的胞漿內(nèi)。17-DMAG誘導(dǎo)HSP70的蛋白和mRNA水平的表達(dá)上調(diào)。經(jīng)過(guò)17-DMAG預(yù)處理5小時(shí)后尼古丁處理,通過(guò)對(duì)基底膜內(nèi)、外毛細(xì)胞計(jì)數(shù),預(yù)處理組毛細(xì)胞數(shù)量大于單獨(dú)尼古丁處理組,兩者有顯著性差異(P0.001)。17-DMAG能夠保護(hù)尼古丁對(duì)毛細(xì)胞的損傷。結(jié)論：17-DMAG可通過(guò)上調(diào)HSP70蛋白及mRNA的表達(dá)水平對(duì)新生大鼠耳蝸毛細(xì)胞尼古丁損傷保護(hù)作用。17-DMAG可作為保護(hù)尼古丁導(dǎo)致的毛細(xì)胞損傷的耳毒性保護(hù)劑。第四部分：鈣依賴性鉀通道BK在C57BL/6J小鼠耳蝸的年齡相關(guān)性表達(dá)及老年性聾的關(guān)系目的：研究C57BL/6J小鼠耳蝸鈣依賴性鉀通道BK年齡相關(guān)性表達(dá),探討B(tài)K通道與老年性聾的關(guān)系。方法：將小鼠按照年齡分為4、12、26和52周四個(gè)實(shí)驗(yàn)組。利用免疫熒光組化技術(shù)定位BK通道在各個(gè)年齡段的小鼠耳蝸表達(dá)部位,熒光實(shí)時(shí)定量PCR法和免疫印記法檢測(cè)BK通道在mRNA和蛋白水平的在不同周齡組的表達(dá)情況。結(jié)果：免疫熒光提示：在各年齡段的小鼠耳蝸內(nèi)BK主要定位在毛細(xì)胞、耳蝸外側(cè)壁和螺旋神經(jīng)元。實(shí)時(shí)定量PCR法和免疫印記法提示BK的蛋白及mRNA水平隨著年齡增加而逐漸減少。結(jié)論：BK通道在各組小鼠耳蝸中的蛋白及mRNA的表達(dá)量隨年齡增加而降低。年齡相關(guān)性BK表達(dá)減低與年齡相關(guān)性聽(tīng)力減退有關(guān)。本實(shí)驗(yàn)表明BK的表達(dá)減低可能與老年性聾的發(fā)病機(jī)制相關(guān)。
[Abstract]:The first part: in vitro culture of the cochlear organs of SD rats: To explore the internal ear organ culture technology to make the hair cells, spiral neurons and auditory nerve fibers in the culture environment to maintain a complete form in vitro, which lays the foundation for the further development of the ototoxicity and protective research of the drug. Methods: Sprague Dawl Ey neonatal rats for 2-3 days, dissected the vestibular organ membrane, the lateral wall and the basement membrane under anatomical microscope. The basement membrane was divided into three segments, the top, middle and bottom three segments. The basal membrane and the vestibule organs were laid out by the liquid surface tension of the medium. The surface of the basement membrane and the vestibule organs were leveled up and laid on the surface of the culture dish. After 48 hours of culture, the observation was observed. Results: the vestibular hair cells and cochlear hair cells were arranged neatly, without disorder, the vestibular hair cells were arranged neatly and without deletion, and the three outer hair cells and a row of inner hair cells in the three segments of the basement membrane were arranged along the cocot spiral (OC, Organ of Corti), and the spiral nerve was fine. The cell line is dense, elliptical, large cell volume, clear cell nucleus, neatly arrayed nerve fibers, without breakage. Conclusion: using liquid surface tension adherence method, the cochlear organs and tissues can be observed conveniently and completely in the external environment. Second parts: nicotine poison in the basement membrane of newborn rats in vitro Objective: to establish a toxicity model of nicolin in vitro, to explore the toxicity and dose response relationship of nicotine in cochlear hair cells, spiral neurojunction cells and nerve fibers. Methods: the cochlear basement membrane of 2-3 days SD rats was isolated and cultured for 24 hours according to different concentration (0-100ng/ml). The snail hair cells and the spiral nerve fibers and the auditory nerve fibers were observed by immunofluorescence staining, respectively, on the hair cells, the spiral nerve fibers and the neurons. The ultrastructure of the hair cells, the support cells and the hair cell static cilium were observed by scanning and transmission electron microscopy. The number of hair cells and the loss of hair cells increased with the concentration of nicotine. Inside the nicotine, the outer hair cells were damaged, the hair cells were damaged, and the basal membrane bottom hair cells were more severe. The nicotine treatment resulted in the increase of vacuoles in the hair cells, the disturbance and loss of the static cilium, the decrease of mitochondria, and the swelling of the endoplasmic reticulum. Conclusion: nicotine mainly damages the cultured hair cells in vitro, and the model of hair cell damage caused by nicotine is a dose dependent relationship between the bottom of the cochlea and the middle gyrus, and the nicotine ototoxicity is in a dose-dependent manner, but there is no obvious damage to the spiral neurons and the auditory nerve fibers. The third part: the purpose of the protective effect of germycin derivative 17-DMAG on the cochlear nicotine toxicity of newborn rats by up regulation of HSP70: To study the protection of the hair cell damage caused by nicotine in the culture of 17-DMAG in vitro. Methods: to isolate the basilar membrane of the cochlea of the 2-3 days of cultured SD rats and screen out the issue. The 17-DMAG treatment concentration was cultivated to protect the hair cells. The basal membrane was divided into blank control group, the nicotine treatment group and the nicotine treatment group were pretreated with 17-DMAG for 5 hours. The protein and mRNA expression level of HSP70 were detected by ELISA and real-time quantitative PCR: the detection of HSP70 in the basement membrane in vitro by fluorescent immunohistochemical technique Results: the basal membrane was cultured in vitro, and HSP70 was mainly located in some new supporting cells around the basement membrane in vitro, and the expression of HSP70 protein and mRNA was up regulated by.17-DMAG in the cytoplasm of outer hair cells. After 17-DMAG preconditioning, 5 hours later nikopin was used. By counting the outer hair cells in the basement membrane, the number of hair cells in the pretreated group was greater than that of the single nicotine treatment group. There was a significant difference (P0.001).17-DMAG to protect the damage of nicotine on the hair cells. Conclusion: 17-DMAG can protect the nicotine damage of the cochlear hair cells of newborn rats by up regulation of HSP70 protein and mRNA. Protective effect of.17-DMAG can be used as an otoprotective agent for the protection of hair cell damage caused by nicotine. The fourth part: the relationship between calcium dependent potassium channel BK in the age related expression of C57BL/6J mouse cochlea and the relationship between senile deafness: To study the BK age related expression of calcium dependent potassium channel in the cochlea of C57BL/6J mice and to explore the BK channel and the elderly The relationship of sexual deafness. Methods: the mice were divided into 4,12,26 and 52 experimental groups according to age. The expression of BK channel in the cochlea of mice at all ages was located by immunofluorescence technology. The expression of BK channel at mRNA and protein levels in different weeks of age was detected by real time fluorescence quantitative PCR and immuno imprint. The immunofluorescence showed that BK was mainly located in the hair cells, the lateral wall of the cochlea and the spiral neurons in the cochlea of all ages. The real-time quantitative PCR method and the immuno imprint method suggested that the protein and mRNA level of BK gradually decreased with age. Conclusion: the expression of protein and mRNA in the cochlea of mice in each group increases with age. The decrease of age related BK expression is associated with age related hearing loss. This experiment shows that the decrease of BK expression may be associated with the pathogenesis of senile deafness.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R764

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