本文選題：Nob1 + 大鼠��； 參考：《第四軍醫(yī)大學(xué)》2013年博士論文

【摘要】：背景 感音神經(jīng)性耳聾是導(dǎo)致言語(yǔ)交流障礙的一種常見(jiàn)疾病，嚴(yán)重影響患者的生活質(zhì)量。盡管目前臨床上治療感音神經(jīng)性耳聾的策略有很多，但治療效果不佳。明確耳蝸細(xì)胞損傷的分子調(diào)控網(wǎng)絡(luò)，尋找有效防治耳聾的目的基因，阻止誘導(dǎo)毛細(xì)胞凋亡的信號(hào)分子，成為未來(lái)耳聾基因治療新的方向。本研究團(tuán)隊(duì)首次發(fā)現(xiàn)Nob1在哺乳動(dòng)物受損耳蝸細(xì)胞中高表達(dá)，但這一表達(dá)變化的具體規(guī)律、機(jī)制及在感音神經(jīng)聾中所起的作用還不清楚。 目的 本研究主要探尋Nob1在出生后大鼠耳蝸內(nèi)的表達(dá)規(guī)律，擬利用基因轉(zhuǎn)染技術(shù)改變Nob1在耳蝸毛細(xì)胞中的表達(dá)，借助體內(nèi)外實(shí)驗(yàn)研究Nob1對(duì)耳蝸毛細(xì)胞生物學(xué)行為的影響及與耳蝸毛細(xì)胞損傷之間的關(guān)系，以期證明Nob1基因是一個(gè)新的候選耳聾基因，為深入理解耳蝸毛細(xì)胞損傷的分子事件，探討新的治療方法奠定基礎(chǔ)。 方法 通過(guò)免疫組織熒光技術(shù)、Weatern-blot及實(shí)時(shí)定量PCR技術(shù)，觀察Nob1在出生后大鼠耳蝸內(nèi)的表達(dá)規(guī)律。構(gòu)建含Nob1基因的慢病毒干擾載體（Nob1-RNAi-GFP-LV）以及腺病毒過(guò)表達(dá)載體（Ad5-Nob1-mCMV-EGFP），將病毒載體轉(zhuǎn)染至體外培養(yǎng)的HK-2細(xì)胞系，上調(diào)以及下調(diào)Nob1基因表達(dá)，探尋Nob1與凋亡之間的關(guān)系。在體外行離體耳蝸器官培養(yǎng)，利用順鉑誘導(dǎo)離體耳蝸毛細(xì)胞損傷后，觀察Nob1在毛細(xì)胞中的表達(dá)情況，并轉(zhuǎn)染慢病毒干擾載體（Nob1-RNAi-GFP-LV），用RT-PCR和Westernblot鑒定病毒的轉(zhuǎn)染及Nob1表達(dá)情況。最后在大鼠體內(nèi)進(jìn)一步驗(yàn)證Nob1在耳蝸毛細(xì)胞損傷中的作用，從耳蝸中階入路分別將Nob1-RNAi-GFP-LV干擾載體、慢病毒空載體和人工內(nèi)淋巴液注射入三組大鼠耳蝸，待RNAi發(fā)揮作用后，利用順鉑誘導(dǎo)大鼠耳蝸損傷，分別用ABR評(píng)估各組病毒載體注射前后大鼠聽(tīng)力變化；通過(guò)基底膜鋪片觀察毛細(xì)胞的形態(tài)變化，并采用全耳蝸毛細(xì)胞定量分析系統(tǒng)行耳蝸毛細(xì)胞計(jì)數(shù)，Western-blot及實(shí)時(shí)定量PCR方法進(jìn)一步明確相關(guān)分子的表達(dá)變化。 結(jié)果 Nob1在大鼠耳蝸毛細(xì)胞及螺旋神經(jīng)元中有表達(dá)，在細(xì)胞發(fā)育增殖分化高峰期比較弱，出生后隨著時(shí)間的增加而逐漸增強(qiáng)。當(dāng)毛細(xì)胞分化，內(nèi)外毛細(xì)胞形成，大、小上皮嵴開(kāi)始分化形成各種支持細(xì)胞時(shí)，Nob1表達(dá)較新生時(shí)期明顯增強(qiáng)。 我們成功構(gòu)建含Nob1基因的慢病毒干擾載體（Nob1-RNAi-GFP-LV）以及腺病毒過(guò)表達(dá)載體（Ad5-Nob1-mCMV-EGFP），在體外培養(yǎng)HK-2細(xì)胞系中成功上調(diào)及下調(diào)Nob1基因，通過(guò)流式細(xì)胞儀檢測(cè)到上調(diào)Nob1表達(dá)后的凋亡細(xì)胞數(shù)明顯高于下調(diào)Nob1基因表達(dá)組。 在體外培養(yǎng)的耳蝸器官組織中，通過(guò)低濃度順鉑成功誘導(dǎo)耳蝸毛細(xì)胞凋亡，發(fā)現(xiàn)Nob1在部分細(xì)胞核固縮的凋亡毛細(xì)胞細(xì)胞核中表達(dá)，出現(xiàn)核轉(zhuǎn)位現(xiàn)象。隨著順鉑作用時(shí)間的增加，毛細(xì)胞的損傷逐漸加重，Nob1表達(dá)也逐漸增高，Western-blot結(jié)果與免疫組織熒光結(jié)果一致。在體外培養(yǎng)的耳蝸器官組織中，慢病毒干擾載體無(wú)法轉(zhuǎn)染至耳蝸毛細(xì)胞，只能轉(zhuǎn)染至螺旋神經(jīng)元。通過(guò)耳蝸中階注射，將慢病毒干擾載體成功轉(zhuǎn)染至耳蝸外毛細(xì)胞、血管紋邊緣細(xì)胞及螺旋韌帶處。外淋巴內(nèi)可見(jiàn)少量慢病毒顆粒分布，而內(nèi)毛細(xì)胞處及螺旋神經(jīng)元處未見(jiàn)病毒分布。最后，在體內(nèi)慢病毒干擾可下調(diào)Nob1基因表達(dá)，再給予順鉑誘導(dǎo)耳蝸毛細(xì)胞損傷后，與對(duì)照組相比，下調(diào)Nob1基因組的大鼠耳蝸聽(tīng)力閾值降低，，耳蝸毛細(xì)胞損傷程度也降低。 結(jié)論 Nob1可能通過(guò)凋亡信號(hào)分子通路參與了耳蝸器官的形成，在順鉑誘導(dǎo)的耳蝸毛細(xì)胞損傷中，Nob1可能參與了耳蝸毛細(xì)胞的凋亡過(guò)程。抑制Nob1基因后可以通過(guò)凋亡信號(hào)通路，降低順鉑所誘導(dǎo)的大鼠聽(tīng)力損傷，從而減少耳蝸毛細(xì)胞的破壞。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R764.431

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