pRb磷酸化调控与内耳发育及毛细胞再生

发布时间：2018-04-22 19:13

本文选题：Rb + Raf-1　；参考：《复旦大学》2013年博士论文

【摘要】：[目的] 由于衰老,噪音,耳毒性药物等原因导致的毛细胞的不可逆损伤和缺失是导致人类听力下降和平衡功能障碍的首要因素。毛细胞再生重建听觉器官结构和功能仍然是听力恢复的理想选择。在小鼠胚胎期和新生早期选择性敲除Rbl(retinoblastoma gene, Rb1)基因,能够产生大量有功能的毛细胞,但是Rbl基因在毛细胞的长期存活中也具有重要的价值。因此,对pRb蛋白功能进行功能调控比永久性敲除Rbl基因具有优势,能够同时达到促进细胞增殖和毛细胞长期存活的目的。pRb的功能状态和磷酸化有关,目前pRb在内耳发育过程中的作用和磷酸化调控机制仍不清楚,此外pRb磷酸化在低等脊椎动物毛细胞再生过程中发挥的作用仍不明确。本研究将利用斑马鱼侧线器神经丘毛细胞损伤后再生的模型,以及鸡胚耳囊体外培养模型,结合化学阻断剂研究pRb在毛细胞损伤后再生过程中和内耳早期发育过程中的价值,以及pRb磷酸化调控的信号通路,以期为pRb磷酸化调控促进毛细胞再生奠定基础。此外,通过构建成年鼠耳蜗基底膜体外培养体系,为研究成年鼠听觉上皮毛细胞再生提供模型支持。 [方法] 1.利用斑马鱼侧线器神经丘毛细胞再生模型,研究新霉素诱导的神经丘毛细胞损伤和再生的机制,并通过RRD-251阻断Rb-Raf-1相互作用介导的Rb磷酸化,研究其在新霉素诱导的毛细胞损伤后再生过程中的作用； 2.利用鸡胚耳囊体外培养模型,通过RRD-251或者U0126,分别阻断Rb-Raf-1相互作用介导的非细胞周期依赖性的和细胞周期依赖性的pRb磷酸化途径,观察对前体细胞增殖、凋亡及分化的影响。Western Blot以及qRT-PCR用于研究鸡胚耳囊pRb, ERK的磷酸化状态,以及相关增殖基因的表达情况,进一步揭示内耳早期发育过程中pRb磷酸化调控途径。 3.构建成年鼠耳蜗基底膜与螺旋神经节体外共培养的模型,研究培养过程中毛细胞和支持细胞的存活情况,支持细胞特性的维持和调整,支持细胞的增殖,并研究与增殖和再生密切相互的mTOR信号通路的活化情况；研究培养的上皮组织中毛细胞和螺旋神经节轴突之间的连接,以及螺旋神经节神经元细胞的存活情况；此外用腺病毒感染培养的上皮组织,构建基因转染和异位表达模型。 [结果] 1.凋亡和坏死参与新霉素诱导的斑马鱼侧线器神经丘毛细胞的损伤过程,毛细胞再生早期(24h)转分化和细胞增殖是新生毛细胞的来源,再生后期(24-48h)再生毛细胞主要来源于细胞增殖。RRD-251通过阻断Rb-Raf-1之间的相互作用抑制支持细胞增殖达到抑制毛细胞再生的作用的,但不能够抑制支持细胞向毛细胞的直接转分化。提示Raf-1介导的pRb磷酸化在斑马鱼侧线器毛细胞再生过程中发挥作用。 2.pRb磷酸化在鸡胚内耳早期发育过程中发挥重要的作用,阻断pRb的磷酸化,前体细胞增殖减少,凋亡增加。其中成神经细胞对RRD-251介导的非细胞依赖性的pRb磷酸化抑制更敏感,而感觉前体细胞对U0126介导的细胞周期依赖性的pRb磷酸化抑制更敏感；两条调控pRb磷酸化的信号通路相对独立,存在一定的叠加效应；pRb磷酸化水平受到抑制后,内耳早期发育过程中增殖相关基因表达水平全面下调,但是下调的程度存在差异,其中Ccnb2,Ccnb3,Ccne2, Cdc2,Myb和Myc在RRD-251诱导的pRb的失活过程中表达下调,而Ccnb3,Ccne2,Cdc2andMyc在U0126介导的pRb功能失活后表达下调；通过对Raf-1和Rbl基因表达水平的检测发现,Raf-1在RRD-251处理后表达下调,而相对于对照组而言,Rbl的表达水平在U0126和RRD251处理之后都没有显著变化。 3.在体外培养过程中,外毛细胞在150min内很快死亡,应用坏死抑制剂Necrostatin-1能够延缓外毛细胞的死亡过程,但是不能逆转死亡命运；内毛细胞在培养的过程中逐渐死亡,仍有部分可以存活14d以上,表达Myo7a和Espin等毛细胞标记；部分存活的内毛细胞与螺旋神经节神经元突触之间的连接仍然存在；支持细胞在培养的过程中存活良好,培养21天后,仍有大量支持细胞存活,特别是顶圈和中顶圈,在长期培养过程中支持细胞数目相对稳定；支持细胞仍然表达Sox-2, Jagged-1等支持细胞特异性的标记,能够被病毒感染,并稳定表达外源性基因；此外与增殖再生密切相关的mTOR信号通路全面活化,但细胞增殖实验显示,培养中的支持细胞仍处于增殖静止状态。 [结论] 1.小分子阻断剂RRD-251能够阻断Raf-1介导的pRb磷酸化,从而抑制新霉素诱导的斑马鱼神经丘毛细胞损伤后的增殖及再生； 2.在鸡胚耳囊早期发育阶段pRb高磷酸化状态对于前体细胞增殖、凋亡和发育具有重要意义；存在细胞周期依赖性和细胞周期非依赖性两条相对独立的pRb磷酸化机制,为进一步pRb磷酸化调控促进细胞增殖和毛细胞再生奠定了基础； 3.我们建立了良好的成年鼠耳蜗培养体系及病毒转染模型,使体外成年鼠毛细胞再生药物的筛查及基因干预成为可能。
[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.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R764;R-332

【共引文献】