Nob1在耳蜗毛细胞损伤中的作用研究

发布时间：2018-06-28 00:58

本文选题：Nob1 + 大鼠　；参考：《第四军医大学》2013年博士论文

【摘要】：背景感音神经性耳聋是导致言语交流障碍的一种常见疾病，严重影响患者的生活质量。尽管目前临床上治疗感音神经性耳聋的策略有很多，但治疗效果不佳。明确耳蜗细胞损伤的分子调控网络，寻找有效防治耳聋的目的基因，阻止诱导毛细胞凋亡的信号分子，成为未来耳聋基因治疗新的方向。本研究团队首次发现Nob1在哺乳动物受损耳蜗细胞中高表达，但这一表达变化的具体规律、机制及在感音神经聋中所起的作用还不清楚。目的本研究主要探寻Nob1在出生后大鼠耳蜗内的表达规律，拟利用基因转染技术改变Nob1在耳蜗毛细胞中的表达，借助体内外实验研究Nob1对耳蜗毛细胞生物学行为的影响及与耳蜗毛细胞损伤之间的关系，以期证明Nob1基因是一个新的候选耳聋基因，为深入理解耳蜗毛细胞损伤的分子事件，探讨新的治疗方法奠定基础。方法通过免疫组织荧光技术、Weatern-blot及实时定量PCR技术，观察Nob1在出生后大鼠耳蜗内的表达规律。构建含Nob1基因的慢病毒干扰载体（Nob1-RNAi-GFP-LV）以及腺病毒过表达载体（Ad5-Nob1-mCMV-EGFP），将病毒载体转染至体外培养的HK-2细胞系，上调以及下调Nob1基因表达，探寻Nob1与凋亡之间的关系。在体外行离体耳蜗器官培养，利用顺铂诱导离体耳蜗毛细胞损伤后，观察Nob1在毛细胞中的表达情况，并转染慢病毒干扰载体（Nob1-RNAi-GFP-LV），用RT-PCR和Westernblot鉴定病毒的转染及Nob1表达情况。最后在大鼠体内进一步验证Nob1在耳蜗毛细胞损伤中的作用，从耳蜗中阶入路分别将Nob1-RNAi-GFP-LV干扰载体、慢病毒空载体和人工内淋巴液注射入三组大鼠耳蜗，待RNAi发挥作用后，利用顺铂诱导大鼠耳蜗损伤，分别用ABR评估各组病毒载体注射前后大鼠听力变化；通过基底膜铺片观察毛细胞的形态变化，并采用全耳蜗毛细胞定量分析系统行耳蜗毛细胞计数，Western-blot及实时定量PCR方法进一步明确相关分子的表达变化。结果 Nob1在大鼠耳蜗毛细胞及螺旋神经元中有表达，在细胞发育增殖分化高峰期比较弱，出生后随着时间的增加而逐渐增强。当毛细胞分化，内外毛细胞形成，大、小上皮嵴开始分化形成各种支持细胞时，Nob1表达较新生时期明显增强。我们成功构建含Nob1基因的慢病毒干扰载体（Nob1-RNAi-GFP-LV）以及腺病毒过表达载体（Ad5-Nob1-mCMV-EGFP），在体外培养HK-2细胞系中成功上调及下调Nob1基因，通过流式细胞仪检测到上调Nob1表达后的凋亡细胞数明显高于下调Nob1基因表达组。在体外培养的耳蜗器官组织中，通过低浓度顺铂成功诱导耳蜗毛细胞凋亡，发现Nob1在部分细胞核固缩的凋亡毛细胞细胞核中表达，出现核转位现象。随着顺铂作用时间的增加，毛细胞的损伤逐渐加重，Nob1表达也逐渐增高，Western-blot结果与免疫组织荧光结果一致。在体外培养的耳蜗器官组织中，慢病毒干扰载体无法转染至耳蜗毛细胞，只能转染至螺旋神经元。通过耳蜗中阶注射，将慢病毒干扰载体成功转染至耳蜗外毛细胞、血管纹边缘细胞及螺旋韧带处。外淋巴内可见少量慢病毒颗粒分布，而内毛细胞处及螺旋神经元处未见病毒分布。最后，在体内慢病毒干扰可下调Nob1基因表达，再给予顺铂诱导耳蜗毛细胞损伤后，与对照组相比，下调Nob1基因组的大鼠耳蜗听力阈值降低，，耳蜗毛细胞损伤程度也降低。结论 Nob1可能通过凋亡信号分子通路参与了耳蜗器官的形成，在顺铂诱导的耳蜗毛细胞损伤中，Nob1可能参与了耳蜗毛细胞的凋亡过程。抑制Nob1基因后可以通过凋亡信号通路，降低顺铂所诱导的大鼠听力损伤，从而减少耳蜗毛细胞的破坏。
[Abstract]:background
Sensorineural deafness is a common disease causing speech communication disorders, which seriously affects the quality of life of the patients. Although there are many strategies for the treatment of sensorineural deafness, the therapeutic effect is poor. The signal molecules of apoptosis have become the new direction of the future deafness gene therapy. This research team has first found that Nob1 is highly expressed in the damaged cochlear cells of mammals, but the specific rules and mechanisms of this change and the role in the sensorineural deafness are not clear.
objective
This study seeks to explore the expression of Nob1 in the cochlea of the rat after birth, and to change the expression of Nob1 in the cochlear hair cells by gene transfection. The effects of Nob1 on the biological behavior of the cochlear hair cells and the relationship between the cochlear hair cells and the damage of the cochlear hair cells are studied in vitro and in vitro, in order to prove that the Nob1 gene is a new candidate. Deafness genes provide a basis for further understanding the molecular events of cochlear hair cell injury and exploring new therapies.
Method
The expression of Nob1 in the cochlea of postnatal rats was observed by immunofluorescence, Weatern-blot and real-time quantitative PCR. The Nob1 gene was constructed with the lentivirus interference carrier (Nob1-RNAi-GFP-LV) and adenovirus overexpressed vector (Ad5-Nob1-mCMV-EGFP), and the vector was transfected into the cultured HK-2 cell line in vitro and up and up. The expression of Nob1 gene was downregulated and the relationship between Nob1 and apoptosis was explored. The expression of Nob1 in hair cells was observed by cisplatin induced cochlear hair cell injury in vitro. The expression of Nob1 in hair cells was observed and transfected with lentivirus interference vector (Nob1-RNAi-GFP-LV). The transfection of the virus and the expression of Nob1 were identified by RT-PCR and Westernblot. Finally, the role of Nob1 in the cochlear hair cell injury was further verified in the rat. The Nob1-RNAi-GFP-LV interference carrier, the lentivirus empty carrier and the artificial internal lymph fluid were injected into the cochlea of three groups from the cochlear step approach. After RNAi, the cochlear injury was induced by cisplatin, and the virus vectors were evaluated by ABR, respectively. The changes in the hearing of the rats were observed before and after the injection, and the morphological changes of the hair cells were observed through the basement membrane paving. The cochlear hair cell count was counted by the whole cochlear hair cell quantitative analysis system. The expression of the related molecules was further determined by Western-blot and real-time quantitative PCR.
Result
The expression of Nob1 in the cochlear hair cells and spiral neurons in the rat is weak at the peak period of proliferation and differentiation, and gradually increases with the increase of time. When the hair cells differentiate, the internal and external hair cells are formed, the large and small epithelial crista begins to differentiate into various supporting cells, the expression of Nob1 is obviously enhanced in the new period.
We successfully constructed the Nob1 gene lentivirus interference vector (Nob1-RNAi-GFP-LV) and adenovirus overexpression vector (Ad5-Nob1-mCMV-EGFP). The Nob1 gene was up-regulated and down regulated in the cultured HK-2 cell lines in vitro. The number of dead cells after the up-regulated Nob1 expression was significantly higher than that of the down regulated Nob1 gene expression group by flow cytometry.
In the cochlear organ tissue cultured in vitro, the apoptosis of cochlear hair cells was induced by low concentration of cisplatin. It was found that Nob1 was expressed in the nuclei of the apoptotic cell nuclei of the nucleus retraction of the nucleus, and the nuclear translocation appeared. With the increase of cisplatin, the damage of hair cells increased gradually, and the expression of Nob1 increased gradually, and the result of Western-blot was increased. In the cochlear organ tissue cultured in vitro, the lentivirus interference carrier can not be transfected into the cochlear hair cells and can only be transfected into the spiral neurons. Through the middle order injection of the cochlea, the lentivirus interference carrier is successfully transfected into the outer hair cells of the cochlea, the marginal cells of the vascular pattern and the spiral ligament. A small amount of lentivirus particles were distributed, but no virus distribution was found at the inner hair cells and the spiral neurons. Finally, the Nob1 gene expression was down regulated by the lentivirus interference in the body and the cochlear hair cell injury induced by cisplatin, compared with the control group, the auditory threshold of the cochlea was reduced and the damage degree of the cochlear hair cells decreased as compared with the control group.
conclusion
Nob1 may participate in the formation of the cochlear organs through the apoptotic signaling pathway. In cisplatin induced cochlear hair cell damage, Nob1 may participate in the apoptosis process of the cochlear hair cells. After inhibiting the Nob1 gene, the apoptosis signal pathway can be used to reduce the damage of the cochlear hair cells induced by cisplatin, which reduces the damage of the cochlear hair cells.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R764.431

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