尼古丁在新生大鼠耳蜗毒性机制及17-DMAG的保护作用

发布时间：2018-06-17 21:20

本文选题：耳蜗 + 器官培养　；参考：《华中科技大学》2016年博士论文

【摘要】：第一部分：SD大鼠乳鼠耳蜗器官体外培养技术目的：探讨内耳器官培养技术使毛细胞、螺旋神经元及听神经纤维在体外培养环境维持完整形态,为进一步开展药物的耳毒性及其保护性研究奠定了实验技术的基础。方法：选用Sprague Dawley大鼠2-3天的新生乳鼠,解剖显微镜下解剖分离前庭器官膜、外侧壁、基底膜。将基底膜分成三段,顶、中、底回三个节段,利用培养基的液体表面张力铺放将基底膜和前庭器官等各个器官,平整向上平整铺放在培养皿表面,培养48小时后观察基底膜形态并计数毛细胞数量。结果：前庭毛细胞和耳蜗毛细胞静纤毛整齐排列,无紊乱,前庭毛细胞排列整齐无缺失,基底膜三个节段的三排外毛细胞和一排内毛细胞沿柯替氏器螺旋器(OC, Organ of Corti)整齐排列,无缺失。螺旋神经结细胞排列密集,呈椭圆形,细胞体积大,胞核清晰,听神经纤维排列整齐,无断裂。结论：运用液体表面张力贴壁方法,可以在体外环境下获得观察方便、形态结构完整的的耳蜗器官和组织。第二部分：尼古丁对新生大鼠体外培养基底膜的毒性作用目的：建立尼古丁耳毒性的体外培养损伤模型,探讨尼古丁在耳蜗毛细胞、螺旋神经结细胞和神经纤维及的毒性作用及剂量反应关系。方法：分离培养2-3天SD大鼠耳蜗基底膜,按照不同作用浓度(0-100ng/ml)处理尼古丁24小时。对耳蜗毛细胞和螺旋神经结及听神经纤维丝采用免疫荧光染色进行观测,分别对毛细胞,螺旋神经纤维和神经元计数。对毛细胞、支持细胞及毛细胞静纤毛束的超微结构使用扫描和透射电子显微镜观察。结果：通过对不同尼古丁浓度处理的毛细胞计数,毛细胞损失量随尼古丁浓度的增加。尼古丁的内、外毛细胞受到类似的损伤,毛细胞的损伤,且基底膜底回毛细胞较顶、中损伤严重。尼古丁的处理导致毛细胞胞浆空泡增多,静纤毛束紊乱和缺失,线粒体数量减少、内质网肿胀和脱颗粒改变。螺旋神经元和听神经纤维未受到明显损伤。结论：尼古丁主要损害体外培养毛细胞,尼古丁所造成的毛细胞损伤模式有耳蜗底回扩展到顶、中回,尼古丁耳毒性呈剂量依赖关系,但对螺旋神经元和听神经纤维无明显损伤。第三部分：格尔德霉素衍生物17-DMAG通过上调HSP70对新生大鼠耳蜗尼古丁毒性保护作用目的：研究格尔德霉素衍生物17-DMAG在体外培养的环境下能否保护尼古丁引起的耳蜗毛细胞损伤。方法：分离培养2-3天SD大鼠耳蜗基底膜,筛选出具有培养能够保护毛细胞的17-DMAG处理浓度,基底膜分为空白对照组、单独尼古丁处理组和17-DMAG预处理5小时后尼古丁处理组。运用ELISA和实时定量PCR检测HSP70的蛋白和mRNA表达水平：通过荧光免疫组织化学技术检测HSP70在离体培养基底膜的细胞定位,并对各组内、外毛细胞进行计数。结果：在体外培养基底膜,HSP70主要定位于体外培养基底膜周围部分新生的支持细胞及内、外毛细胞的胞浆内。17-DMAG诱导HSP70的蛋白和mRNA水平的表达上调。经过17-DMAG预处理5小时后尼古丁处理,通过对基底膜内、外毛细胞计数,预处理组毛细胞数量大于单独尼古丁处理组,两者有显著性差异(P0.001)。17-DMAG能够保护尼古丁对毛细胞的损伤。结论：17-DMAG可通过上调HSP70蛋白及mRNA的表达水平对新生大鼠耳蜗毛细胞尼古丁损伤保护作用。17-DMAG可作为保护尼古丁导致的毛细胞损伤的耳毒性保护剂。第四部分：钙依赖性钾通道BK在C57BL/6J小鼠耳蜗的年龄相关性表达及老年性聋的关系目的：研究C57BL/6J小鼠耳蜗钙依赖性钾通道BK年龄相关性表达,探讨BK通道与老年性聋的关系。方法：将小鼠按照年龄分为4、12、26和52周四个实验组。利用免疫荧光组化技术定位BK通道在各个年龄段的小鼠耳蜗表达部位,荧光实时定量PCR法和免疫印记法检测BK通道在mRNA和蛋白水平的在不同周龄组的表达情况。结果：免疫荧光提示：在各年龄段的小鼠耳蜗内BK主要定位在毛细胞、耳蜗外侧壁和螺旋神经元。实时定量PCR法和免疫印记法提示BK的蛋白及mRNA水平随着年龄增加而逐渐减少。结论：BK通道在各组小鼠耳蜗中的蛋白及mRNA的表达量随年龄增加而降低。年龄相关性BK表达减低与年龄相关性听力减退有关。本实验表明BK的表达减低可能与老年性聋的发病机制相关。
[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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R764

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