RIP3调控螺旋神经元程序性坏死的机制研究

Irreversible damage of spiral ganglion neurons(SGNs) is one of the most important.reasons of sensorineural hearing loss.The mechanisms of SGNs apoptosis or necrosis.have not been clearly demonstrated. Therefore,it may be a promising method to.prevent sensorineural hearing loss by protecting SGNs from stress damage and.sencondary injuries. Unfortunately, there are not such effective methods.being found despite of the exploration and efforts. "Necroptosis", which is also.called programmed necrosis, has been brought forward as a third pathway of cell.death except for apoptosis and necrosis. RIP3 is thought to be the key point of.the necroptosis.The formation of RIP3 compplex would activate the downstream.molecules of the signaling pathway and initiate the autophagy program of cells..Our previous results have already showed that the expression of RIP3 in SGNs of.rat after birth gradually increased with the development,and that elevated.expression of RIP3 in ouabain induced SGNs damage. We also found that the.application of necrosis pathway inhibitor Nec-1 can effectively reduce the.expression of RIP3,and protect the SGNs and the hearing of rats. In this study, we.would establish models of SGNs damage, focus on the observation of the.distinguishexpression of RIP3 in different injuries of SGNs,and the role of RIP3.in necroptosis process of SGNs, explore the possible hearing protection role of.RIP3sigalling pathway in SGNs necroptosis.And we up and down regulate the.expression of RIP3 by using the adenoviral transfection technology to find a new.target of mediating cell death pathway. It is planned to explore the protection.mechanisms of RIP3 during the damage of SGNs, thereby finding a brand-new method.to block the pathway of programmed necrosis and then treat sensorineural hearing.loss.

螺旋神经元损伤是感音神经性聋的重要病因之一,其致伤机制目前仍不清楚。近年来，“程序性坏死”作为细胞死亡的第三种形式被提出，在该死亡通路中RIP3被视为关键分子。本课题组前期研究结果显示RIP3表达量在大鼠出生后随发育而逐渐增多，在药物致聋大鼠螺旋神经元损伤模型中RIP3表达明显增高，而应用程序性坏死通路抑制剂Nec-1可有效减少RIP3的表达，明显降低螺旋神经元损伤数目，并对大鼠听力起到一定保护作用。因此，本课题拟在上述基础上，通过建立体内外螺旋神经元损伤模型，观察RIP3的表达变化情况；同时，应用腺病毒转染相关技术，上/下调RIP3的表达，观察程序性坏死通路对螺旋神经元损伤的影响，并研究RIP3在螺旋神经元程序性坏死过程中的关键作用及其对程序性坏死通路的调控机制，观察在损伤因素作用下阻断该通路对螺旋神经元和听力的保护作用，以期为预防螺旋神经元损伤、减少感音神经性聋的发生发展探寻新的途径。

螺旋神经元损伤是感音神经性聋的重要病因之一,近年来，“程序性坏死”作为细胞死亡的第三种形式被提出，在该死亡通路中RIP3被视为关键分子。本课题组前期研究结果显示RIP3表达量在大鼠出生后随发育而逐渐增多，在药物致聋大鼠螺旋神经元损伤模型中RIP3表达明显增高，而应用程序性坏死通路抑制剂Nec-1可有效减少RIP3的表达，明显降低螺旋神经元损伤数目，并对大鼠听力起到一定保护作用。因此，本课题在上述基础上，通过建立体内外螺旋神经元损伤模型，观察RIP3的表达变化情况；同时，应用腺病毒转染相关技术，上/下调RIP3的表达，观察程序性坏死通路对螺旋神经元损伤的影响，我们将Ouabain应用于胎鼠SGNs中，并分别注射于Sprague-Dawley大鼠体内和体外构建损伤模型。流式细胞术和MTT法测定sgn坏死率。western blotting和免疫荧光检测RIP3的蛋白水平和磷酸化水平。用HE染色和免疫荧光法观察SGNs损伤情况。采用听性脑干反应(ABR)法和畸变产物耳声发射(DPOAE)法评价大鼠的听力功能。 Ouabain引起体内SGNs的剂量依赖性坏死和耳蜗轴SGNs的显著丢失。RIP3和pRIP3在促进SGNs损伤的同时上调，体外过表达RIP3可促进Ouabain链诱导的SGNs坏死，而坏死statin-1可抑制这种坏死。RIP3敲低抑制了Ouabain链诱导的坏死，降低了MLKL的磷酸化水平，但RIP3对MLKL水平无依赖性作用。在体内抑制RIP3可降低ABR阈值变化，促进DPOAE振幅，从而保护大鼠免受Ouabain诱导的听力损伤，而过表达RIP3可增强Ouabain诱导的听力损伤，而这种损伤可被necrostatin-1部分逆转。当RIP3过表达时，SGNs密度降低，pRIP3上调，而当RIP3沉默时则相反。因此，RIP3在介导瓦萨链诱导的SGNs损伤的坏死中起重要作用。临床上以RIP3为靶点，可预防SGNs死亡，最终帮助治疗感音神经性听力损失。