本文選題：Rb + Raf-1　； 參考：《復(fù)旦大學(xué)》2013年博士論文

【摘要】：[目的] 由于衰老,噪音,耳毒性藥物等原因?qū)е碌拿?xì)胞的不可逆損傷和缺失是導(dǎo)致人類聽力下降和平衡功能障礙的首要因素。毛細(xì)胞再生重建聽覺器官結(jié)構(gòu)和功能仍然是聽力恢復(fù)的理想選擇。在小鼠胚胎期和新生早期選擇性敲除Rbl(retinoblastoma gene, Rb1)基因,能夠產(chǎn)生大量有功能的毛細(xì)胞,但是Rbl基因在毛細(xì)胞的長期存活中也具有重要的價值。因此,對pRb蛋白功能進(jìn)行功能調(diào)控比永久性敲除Rbl基因具有優(yōu)勢,能夠同時達(dá)到促進(jìn)細(xì)胞增殖和毛細(xì)胞長期存活的目的。pRb的功能狀態(tài)和磷酸化有關(guān),目前pRb在內(nèi)耳發(fā)育過程中的作用和磷酸化調(diào)控機(jī)制仍不清楚,此外pRb磷酸化在低等脊椎動物毛細(xì)胞再生過程中發(fā)揮的作用仍不明確。本研究將利用斑馬魚側(cè)線器神經(jīng)丘毛細(xì)胞損傷后再生的模型,以及雞胚耳囊體外培養(yǎng)模型,結(jié)合化學(xué)阻斷劑研究pRb在毛細(xì)胞損傷后再生過程中和內(nèi)耳早期發(fā)育過程中的價值,以及pRb磷酸化調(diào)控的信號通路,以期為pRb磷酸化調(diào)控促進(jìn)毛細(xì)胞再生奠定基礎(chǔ)。此外,通過構(gòu)建成年鼠耳蝸基底膜體外培養(yǎng)體系,為研究成年鼠聽覺上皮毛細(xì)胞再生提供模型支持。 [方法] 1.利用斑馬魚側(cè)線器神經(jīng)丘毛細(xì)胞再生模型,研究新霉素誘導(dǎo)的神經(jīng)丘毛細(xì)胞損傷和再生的機(jī)制,并通過RRD-251阻斷Rb-Raf-1相互作用介導(dǎo)的Rb磷酸化,研究其在新霉素誘導(dǎo)的毛細(xì)胞損傷后再生過程中的作用； 2.利用雞胚耳囊體外培養(yǎng)模型,通過RRD-251或者U0126,分別阻斷Rb-Raf-1相互作用介導(dǎo)的非細(xì)胞周期依賴性的和細(xì)胞周期依賴性的pRb磷酸化途徑,觀察對前體細(xì)胞增殖、凋亡及分化的影響。Western Blot以及qRT-PCR用于研究雞胚耳囊pRb, ERK的磷酸化狀態(tài),以及相關(guān)增殖基因的表達(dá)情況,進(jìn)一步揭示內(nèi)耳早期發(fā)育過程中pRb磷酸化調(diào)控途徑。 3.構(gòu)建成年鼠耳蝸基底膜與螺旋神經(jīng)節(jié)體外共培養(yǎng)的模型,研究培養(yǎng)過程中毛細(xì)胞和支持細(xì)胞的存活情況,支持細(xì)胞特性的維持和調(diào)整,支持細(xì)胞的增殖,并研究與增殖和再生密切相互的mTOR信號通路的活化情況；研究培養(yǎng)的上皮組織中毛細(xì)胞和螺旋神經(jīng)節(jié)軸突之間的連接,以及螺旋神經(jīng)節(jié)神經(jīng)元細(xì)胞的存活情況；此外用腺病毒感染培養(yǎng)的上皮組織,構(gòu)建基因轉(zhuǎn)染和異位表達(dá)模型。 [結(jié)果] 1.凋亡和壞死參與新霉素誘導(dǎo)的斑馬魚側(cè)線器神經(jīng)丘毛細(xì)胞的損傷過程,毛細(xì)胞再生早期(24h)轉(zhuǎn)分化和細(xì)胞增殖是新生毛細(xì)胞的來源,再生后期(24-48h)再生毛細(xì)胞主要來源于細(xì)胞增殖。RRD-251通過阻斷Rb-Raf-1之間的相互作用抑制支持細(xì)胞增殖達(dá)到抑制毛細(xì)胞再生的作用的,但不能夠抑制支持細(xì)胞向毛細(xì)胞的直接轉(zhuǎn)分化。提示Raf-1介導(dǎo)的pRb磷酸化在斑馬魚側(cè)線器毛細(xì)胞再生過程中發(fā)揮作用。 2.pRb磷酸化在雞胚內(nèi)耳早期發(fā)育過程中發(fā)揮重要的作用,阻斷pRb的磷酸化,前體細(xì)胞增殖減少,凋亡增加。其中成神經(jīng)細(xì)胞對RRD-251介導(dǎo)的非細(xì)胞依賴性的pRb磷酸化抑制更敏感,而感覺前體細(xì)胞對U0126介導(dǎo)的細(xì)胞周期依賴性的pRb磷酸化抑制更敏感；兩條調(diào)控pRb磷酸化的信號通路相對獨立,存在一定的疊加效應(yīng)；pRb磷酸化水平受到抑制后,內(nèi)耳早期發(fā)育過程中增殖相關(guān)基因表達(dá)水平全面下調(diào),但是下調(diào)的程度存在差異,其中Ccnb2,Ccnb3,Ccne2, Cdc2,Myb和Myc在RRD-251誘導(dǎo)的pRb的失活過程中表達(dá)下調(diào),而Ccnb3,Ccne2,Cdc2andMyc在U0126介導(dǎo)的pRb功能失活后表達(dá)下調(diào)；通過對Raf-1和Rbl基因表達(dá)水平的檢測發(fā)現(xiàn),Raf-1在RRD-251處理后表達(dá)下調(diào),而相對于對照組而言,Rbl的表達(dá)水平在U0126和RRD251處理之后都沒有顯著變化。 3.在體外培養(yǎng)過程中,外毛細(xì)胞在150min內(nèi)很快死亡,應(yīng)用壞死抑制劑Necrostatin-1能夠延緩?fù)饷?xì)胞的死亡過程,但是不能逆轉(zhuǎn)死亡命運；內(nèi)毛細(xì)胞在培養(yǎng)的過程中逐漸死亡,仍有部分可以存活14d以上,表達(dá)Myo7a和Espin等毛細(xì)胞標(biāo)記；部分存活的內(nèi)毛細(xì)胞與螺旋神經(jīng)節(jié)神經(jīng)元突觸之間的連接仍然存在；支持細(xì)胞在培養(yǎng)的過程中存活良好,培養(yǎng)21天后,仍有大量支持細(xì)胞存活,特別是頂圈和中頂圈,在長期培養(yǎng)過程中支持細(xì)胞數(shù)目相對穩(wěn)定；支持細(xì)胞仍然表達(dá)Sox-2, Jagged-1等支持細(xì)胞特異性的標(biāo)記,能夠被病毒感染,并穩(wěn)定表達(dá)外源性基因；此外與增殖再生密切相關(guān)的mTOR信號通路全面活化,但細(xì)胞增殖實驗顯示,培養(yǎng)中的支持細(xì)胞仍處于增殖靜止?fàn)顟B(tài)。 [結(jié)論] 1.小分子阻斷劑RRD-251能夠阻斷Raf-1介導(dǎo)的pRb磷酸化,從而抑制新霉素誘導(dǎo)的斑馬魚神經(jīng)丘毛細(xì)胞損傷后的增殖及再生； 2.在雞胚耳囊早期發(fā)育階段pRb高磷酸化狀態(tài)對于前體細(xì)胞增殖、凋亡和發(fā)育具有重要意義；存在細(xì)胞周期依賴性和細(xì)胞周期非依賴性兩條相對獨立的pRb磷酸化機(jī)制,為進(jìn)一步pRb磷酸化調(diào)控促進(jìn)細(xì)胞增殖和毛細(xì)胞再生奠定了基礎(chǔ)； 3.我們建立了良好的成年鼠耳蝸培養(yǎng)體系及病毒轉(zhuǎn)染模型,使體外成年鼠毛細(xì)胞再生藥物的篩查及基因干預(yù)成為可能。
[Abstract]:[Objective]
Irreversible damage and loss of hair cells caused by aging, noise, ototoxic drugs and other causes are the primary factors that lead to human hearing loss and balance dysfunction. Regenerating and reconstructing auditory organs and functions of hair cells is still an ideal choice for hearing recovery. Rbl (retinobla Stoma gene, Rb1) genes can produce a large number of functional hair cells, but the Rbl gene is also of great value in the long-term survival of hair cells. Therefore, functional regulation of pRb protein function is superior to the permanent knockout of Rbl gene, and can simultaneously achieve the function of.PRb, which promotes cell proliferation and long-term survival of hair cells. Status is related to phosphorylation. The role of pRb in the development of the inner ear and the regulatory mechanism of phosphorylation are still unclear. In addition, the role of pRb phosphorylation in the process of hair cell regeneration in lower vertebrates is not clear. This study will use the model of regeneration after the injury of the chick hair cells in the zebrafish side line, and the external of the chicken embryo sac. The value of pRb in the process of regeneration and early development of inner ear after the injury of hair cells and the signal pathway of pRb phosphorylation regulation are studied in combination with chemical blockers, in order to lay the foundation for the regulation of pRb phosphorylation to promote the regeneration of hair cells. In addition, the adult rat cochlear basement membrane in vitro culture system is constructed to study adult. Rat auditory hair cell regeneration provides model support.
[method]
1. the mechanism of injury and regeneration of nerve colliculus cells induced by neomycin was studied by using the regeneration model of the zebrafish collateral nerve colliculus hair cells, and the effect of Rb phosphorylation mediated by Rb-Raf-1 interaction was blocked by RRD-251, and the role of it in the regeneration of neomycin induced hair cell injury was studied.
2. using an in vitro culture model of chicken embryo sac in vitro, the non cell cycle dependent and cell cycle dependent pRb phosphorylation pathways mediated by Rb-Raf-1 interaction were blocked by RRD-251 or U0126, and the effects of.Western Blot on the proliferation, apoptosis and differentiation of precursor cells were observed and qRT-PCR was used to study pRb of chicken embryo sac, ERK phosphoric acid. The expression of pRb and the related genes were further revealed in the early development of inner ear.
3. the model of cochlear basal membrane and spiral ganglion co culture of adult rat cochlea was constructed, the survival of hair cells and support cells in the process of culture were studied, the maintenance and adjustment of cell characteristics were supported and the proliferation of cells were supported. The activation of mTOR signaling pathway closely related to proliferation and regeneration was studied, and the cultured epithelial tissue was studied. The connection between the hair cells and the axons of the spiral ganglion, and the survival of the spiral ganglion neurons, and the construction of the gene transfection and ectopic expression model with the epithelial tissue cultured by adenovirus.
[results]
1. apoptosis and necrosis participate in the damage process of neomycin induced zebrafish collateral hair cells in the zebrafish side line. In the early stage of hair cell regeneration (24h), transdifferentiation and cell proliferation are the source of new hair cells, and the regenerated later (24-48h) regenerated hair cells mainly originate in cell proliferation.RRD-251 by blocking the interaction between Rb-Raf-1 and inhibiting the support of the cells. Cell proliferation inhibits the regeneration of hair cells, but does not inhibit direct transdifferentiation of support cells to hair cells. It suggests that Raf-1 mediated phosphorylation of pRb plays a role in the regeneration of zebrafish side line hair cells.
2.pRb phosphorylation plays an important role in the early development of the inner ear of chicken embryo, blocking the phosphorylation of pRb, reducing the proliferation of precursor cells and increasing apoptosis, in which the neurocells are more sensitive to the inhibition of RRD-251 mediated non cellular pRb phosphorylation, and the cell cycle dependent pRb phosphorylation of the sensory precursor cells is mediated by U0126. The system is more sensitive; the two signal pathways regulating the phosphorylation of pRb are relatively independent and have a certain superposition effect. After the phosphorylation level of pRb is inhibited, the expression level of the proliferation related genes in the early development of the inner ear is down completely, but the degree of down regulation is different, in which Ccnb2, Ccnb3, Ccne2, Cdc2, Myb and Myc are in RRD-251 induced pRb. In the process of inactivation, the expression of Ccnb3, Ccne2, and Cdc2andMyc were down regulated after the U0126 mediated pRb function inactivation. Through the detection of Raf-1 and Rbl gene expression levels, Raf-1 was down regulated after RRD-251 treatment. Compared with the control group, the expression level of Rbl was not significantly changed after U0126 and RRD251 treatment.
3. in the process of culture in vitro, the outer hair cells die quickly in 150min, and the necrosis inhibitor Necrostatin-1 can delay the death process of the outer hair cells, but it can not reverse the death fate. The inner hair cells die gradually in the process of culture, and some of them can survive 14d to express Myo7a and Espin and other hair cell markers. The synaptic connections between the surviving internal hair cells and the spiral ganglion neurons still exist, and the support cells survive well during the culture process. After 21 days of culture, a large number of support cells still survive, especially the top and middle top rings. The number of supporting cells is relatively stable during the long period of culture, and the support cells still express Sox-2, Jagged- 1 and other support cell specific markers can be infected by the virus and express the exogenous gene stably; in addition, the mTOR signaling pathway, which is closely related to proliferation and regeneration, is fully activated, but the cell proliferation experiment shows that the support cells in the culture are still in the static state of proliferation.
[Conclusion]
1. small molecule blocker RRD-251 can block the phosphorylation of pRb mediated by Raf-1, thus inhibiting the proliferation and regeneration of neomycin induced injury of zebrafish nerve colliculus.
2. the high phosphorylation of pRb in the early development stage of the chicken embryo sac is of great significance to the proliferation, apoptosis and development of the precursor cells. There are two relative independent pRb phosphorylation mechanisms of cell cycle dependence and cell cycle non dependence, which lays the foundation for further pRb phosphorylation to promote the proliferation of fine cell and the regeneration of hair cells.
3. we have established a good adult rat cochlear culture system and a virus transfection model, so that it is possible to screen and intervene in the regenerative drugs of adult rat hair cells in vitro.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R764;R-332

【共引文獻(xiàn)】

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