MicroRNA-30b调控Sema3A在视神经损伤修复中的作用及机制研究

发布时间：2018-06-14 08:03

  本文选题：视神经 + 视网膜神经节细胞　； 参考：《第三军医大学》2015年博士论文

【摘要】：研究背景和意义视神经是由视网膜神经节细胞(retinal ganglioncells,RGCs)的轴突汇聚而成,RGCs的状态一定程度上反应了视神经的功能。作为中枢神经的一部分,视神经伤后恢复亦成为研究的热点和难点。众多的调控因素使视神经损伤后再生修复变得异常困难、复杂。损伤区抑制性蛋白的存在、胶质瘢痕形成以及神经营养因子缺乏等因素是中枢神经再生障碍的主要原因,其中抑制性因素处于重要地位。Sema3A作为重要的轴突抑制物,在神经损伤后诱导生长锥塌陷,阻止神经修复,扮演着重要的调控角色,受到广泛的关注。神经纤毛蛋白(Neuropilin1,NRP1)和丛状蛋白(Plexin A1-A4,PlexA1-A4)被证实为Sema3A的共受体,与Sema3A结合后发挥对神经生长的抑制作用,当配体Sema3A不存在时,Plex以一种自我抑制的状态存在,共受体NRP1与Plex结合使得这种抑制状态更加稳定,在Sema3A与NRP1结合后,Plex的构象发生了改变,从自我抑制状态释放,进一步与Sema3A、NRP1结合,启动下游的信号传导,最终引起神经生长锥塌陷,抑制轴突延伸。p38丝裂原活化蛋白激酶(mitogen activated protein kinases p38MAPK)和caspase-3信号通路参与了多种细胞凋亡进程。文献报道,Sema3A能激活p38MAPK,使其磷酸化后将促进神经祖细胞凋亡。而且,Sema3A还能减少巨噬细胞对髓磷脂的吞噬作用,抑制中枢神经髓鞘再生,妨碍神经营养因子运输,阻止损伤神经恢复。microRNA(mi RNA)是一种独特的内源性小分子,它通过与靶mRNA的3’UTR的互补配对抑制靶基因活性或使其降解,导致相应蛋白表达水平降低,从而调节细胞的增殖、分化与凋亡,在生物体生长发育的不同阶段发挥不同功能,实现其生物调节作用。而且,许多mi RNAs在哺乳动物大脑、视网膜中特异性存在并且在其各个发育阶段动态表达,表明了这些miRNAs与哺乳动物神经发育密切相关。这些研究结果为找寻抑制Sema3A表达的mi RNAs、促进损伤视神经修复提供了新的思路。目的:通过对视神经损伤后视网膜中Sema3A及其下游信号通路的研究,初步探讨Sema3A抑制视神经再生的作用机制,明确Sema3A是mi R-30b的靶基因,证实mi R-30b可通过调控sema3a表达并抑制其信号通路、发挥保护视网膜神经节细胞、促进轴突生长的作用,为临床治疗视神经损伤提供新的靶点和思路。方法:1、建立sd大鼠视神经钳夹损伤模型,于正常及伤后第1、3、7、14、21d行qrt-pcr和westernblotting检测,观察视网膜中mir-30b、sema3a、nrp1、plexa1、p-p38mapk、和activecaspase-3表达变化,免疫组织化学观察正常及伤后第7d视网膜上sema3a的表达情况。2、双荧光素酶报告基因系统验证mir-30b与sema3a3’utr结合并抑制其表达;将重组腺相关病毒(raav)包装的mir-30bmimic、mir-30binhibitor、mirnanc和pbs分别转染到体外培养的rgcs;培养第7d,westernblotting检测sema3a、nrp1、plexa1、p-p38mapk和activecaspase-3表达情况;制作sd大鼠视神经损伤模型,将raav-mir-30bmimic、raav-mir-30binhibitor、raav-mirnanc和pbs分别注射入损伤眼玻璃体腔内,分别于伤后3、7d用qrt-pcr和westernblotting检测视网膜中mir-30b、sema3a的表达量。3、构建以sema3a为靶标的sirna,lipofectamine2000将其转染到体外培养的rgcs,westernblotting检测sema3a表达量,选取最强抑制效果的sirna,再将其转染到体外培养的rgcs,培养第5d再分别转染raav-mir-30bmimic和raav-mir-30binhibitor,培养第9dwesternblotting再检测nrp1和p-p38mapk的表达情况,初步明确mir-30b调控sema3a的信号通路。4、将raav包装的mir-30bmimic、mir-30binhibitor、mirnanc和pbs分别转染到体外培养的rgcs;培养第5d流式细胞仪检测其凋亡率,培养第7d采用gap-43着染,免疫荧光显微镜观察各组rgcs轴突长度情况。5、制作sd大鼠视神经钳夹伤模型,将raav-mir-30bmimic、raav-mir-30binhibitor、raav-mirnanc和pbs分别注射入损伤眼玻璃体腔内,利用荧光金逆行标记观察伤后7、14、28d时4组rgcs存活率变化;闪光视觉诱发电位检测伤后28d各组视功能恢复情况。结果:1、sd大鼠视神经钳夹伤后,视网膜中mir-30b表达先升高后下降,伤后3d达高峰,伤后7d又逐渐下降,视网膜中sema3a、nrp1、plexa1、p-p38mapk和activecaspase-3也先升高后下降,伤后7d达高峰;免疫组化检测发现,sema3a表达在正常视网膜中内核层和节细胞层,伤后7d,节细胞层和内核层的阳性细胞明显增多。2、双荧光素酶报告基因系统检测发现处理组相对荧光素酶活性明显低于对照组和突变组,mi R-30b与Sema3A的结合位点为:UGUUUACA;转染miR-30b到体外培养的RGCs,mi R-30b mimic组中Sema3A、NRP1、PlexA1、p-p38MAPK和active caspase-3表达量最低,而mi R-30b inhibit组最高;大鼠视神经钳夹伤后,玻璃体腔内分别注射r AAV-miR-30b mimic、rAAV-mi R-30b inhibitor、rAAV-mi RNA NC和PBS,伤后3d和7d,视网膜中,miR-30b表达量在mimic组最高、inhibitor组最低,而Sema3A正好相反。3、在体外培养的RGCs中,si RNA2组内Sema3A蛋白表达量最低,用siRNA2敲低Sema3A后,再分别转染r AAV-mi R-30b mimic和r AAV-miR-30b inhibitor,两组中Sema3A、NRP1和p-p38MAPK表达量无明显差异。4、转染mi R-30b到体外培养的RGCs,流式细胞仪检测发现mi R-30b mimic组RGCs凋亡率最低,mi R-30b inhibit组最高,RGCs轴突长度mi R-30b mimic组最长,mi R-30b inhibit组最短。5、大鼠视神经钳夹伤后,玻璃体腔内分别注射rAAV-mi R-30b mimic、r AAV-miR-30b inhibitor、rAAV-mi RNA NC和PBS,虽然,伤后四组RGCs存活率都不同程度下降,但是在伤后7、14、28d,mimic组的RGCs存活率都最高,inhibitor组都最低;FVEP显示,mimic组中P1波幅也是最宽,inhibitor组最窄。全文结论1、离体及在体实验证实,Sema3A是miR-30b的靶基因;2、miR-30b可通过抑制Sema3A表达改变p-p38MAPK和active caspase-3的水平,减少RGCs凋亡,提高视神经损伤后RGCs存活率;3、miR-30b可通过抑制Sema3A表达改变NRP1、Plex A1的水平,促进RGCs轴突生长,并加快视神经损伤后视功能恢复,这可能是mi R-30b促进损伤后视神经修复的主要分子机制。
[Abstract]:The study background and significance optic nerve are formed by the axons of the retinal ganglion cells (retinal GanglionCells, RGCs). The state of the RGCs reacts to the function of the optic nerve to some extent. As a part of the central nervous system, the recovery of the optic nerve after injury is also a hot and difficult point. The existence of inhibitory proteins, the formation of glial scar and the lack of neurotrophic factors are the main causes of central nerve regeneration. The inhibitory factors are in the important position of.Sema3A as an important axon inhibitor, inducing conical collapse and blocking the nerve after nerve injury. Repair, playing an important regulatory role, is widely concerned. Neuropilin1 (NRP1) and Plexin A1-A4 (PlexA1-A4) are proved to be common receptors of Sema3A, and they are combined with Sema3A to play an inhibitory effect on nerve growth. When the ligand Sema3A does not exist, Plex is present in a state of self inhibition and co receptor. The combination of NRP1 and Plex makes this inhibition more stable. After the combination of Sema3A and NRP1, the conformation of Plex is changed, released from the state of self inhibition, further combining with Sema3A, NRP1, and starting downstream signal conduction, eventually causing the collapse of the nerve growth cone, and inhibiting the axon to extend.P38 mitogen activated protein kinase (mitogen activated PR). Otein kinases p38MAPK) and caspase-3 signaling pathway participate in a variety of apoptotic processes. It is reported that Sema3A can activate p38MAPK and promote the apoptosis of neural progenitor cells after phosphorylation. Moreover, Sema3A can reduce macrophage phagocytosis of myelin, inhibit the regeneration of myelin sheath, obstruct the transport of neurotrophic factors and prevent the transport of neurotrophic factors. The injured nerve recovery.MicroRNA (MI RNA) is a unique endogenous small molecule. It inhibits the activity or degradation of the target gene through the complementary pairing with the 3 'UTR of the target mRNA, resulting in the reduction of the expression level of the corresponding protein, thus regulating cell proliferation, differentiation and apoptosis, and exerting different functions in different stages of growth and development of raw materials. Moreover, many mi RNAs are specific in the mammalian brain and in the retina and are expressed dynamically at their various developmental stages, indicating that these miRNAs are closely related to the development of mammalian nerve. These results provide a new idea for the search for the MI RNAs that inhibits the expression of Sema3A and to the repair of the repair of the optic nerve. Objective: through the study of the Sema3A and its downstream signal pathway in the retina after optic nerve injury, the mechanism of Sema3A inhibition of optic nerve regeneration was preliminarily investigated, and Sema3A was a target gene for MI R-30b. It was confirmed that MI R-30b could protect retinal ganglion cells and promote axonal growth by regulating Sema3A expression and inhibiting its signal pathway. In order to provide new targets and ideas for clinical treatment of optic nerve injury. Methods: 1, a SD rat model of optic nerve clamp injury was established, and qRT-PCR and westernblotting were detected in 1,3,7,14,21d after normal and post injury. The changes of mir-30b, Sema3A, Nrp1, plexa1, P-P38MAPK, activecaspase-3 expression in the retina were observed, and the immunohistochemical observation was observed. The expression of Sema3A on the retina of 7D after normal and post injury.2, the dual luciferase reporter gene system verified that mir-30b was combined with sema3a3 'UTR to inhibit its expression, and the mir-30bmimic, mir-30binhibitor, mirnanc and PBS, packaged in recombinant adeno-related virus (rAAV), were transfected to the extracellular RGCs. The expression of Nrp1, plexa1, P-P38MAPK and activecaspase-3, the model of the optic nerve injury in SD rats was made, and raav-mir-30bmimic, raav-mir-30binhibitor, raav-mirnanc and PBS were injected into the vitreous cavity of the injured eye respectively. The expression of the retina in the retina was detected by qRT-PCR and westernblotting, respectively. For the target siRNA, lipofectamine2000 transfected it into the cultured RGCs, westernblotting detected the Sema3A expression, selected the strongest inhibitory effect siRNA, then transfected it into the cultured RGCs, cultured 5D and then transfected raav-mir-30bmimic and raav-mir-30binhibitor respectively. The expression of.4 was preliminarily defined by mir-30b, which was used to regulate the Sema3A, mir-30bmimic, mir-30binhibitor, mirnanc and PBS were transfected to RGCs in vitro, respectively. The apoptosis rate was detected by the flow cytometry. The GAP-43 staining was used in the culture 7d, and the length of the axon was observed by the immunofluorescence microscope. D rat model of optic nerve clamp injury, raav-mir-30bmimic, raav-mir-30binhibitor, raav-mirnanc and PBS were injected into the vitreous cavity of the injured eye respectively. The changes of the 4 groups of RGCs survival rates were observed by the fluorescent gold retrograde labeling, and the visual evoked potential of the 28d in each group after injury was detected by the flash visual evoked potential. Results: 1, SD rats were deity. After the clamp injury, the expression of mir-30b in the retina increased first and then decreased, and the 3D reached the peak after injury, and the 7d decreased gradually after injury. The Sema3A, Nrp1, plexa1, P-P38MAPK and activecaspase-3 in the retina increased first and then declined, and the 7d reached the peak. The immunohistochemical detection found that the Sema3A table reached the core layer and the ganglion layer in the normal retina, 7d after injury, slender after injury. The positive cells in the cell layer and the core layer were significantly increased by.2. The double luciferase reporter gene system detection showed that the relative luciferase activity of the treatment group was significantly lower than the control group and the mutation group. The binding site of MI R-30b and Sema3A was UGUUUACA, RGCs in the transfected miR-30b to the in vitro culture, Sema3A in the MI R-30b mimic group. The expression of Caspase-3 was the lowest, and the highest in the MI R-30b inhibit group. After the clamp injury of the optic nerve in the rat, the intravitreal R AAV-miR-30b mimic, rAAV-mi R-30b inhibitor, rAAV-mi RNA, and the retina were the highest and the lowest in the retina. In s, the expression of Sema3A protein in group Si RNA2 is the lowest. After siRNA2 knocking down Sema3A, R AAV-mi R-30b mimic and R AAV-miR-30b are respectively transfected. The 0b inhibit group was the highest, the RGCs axon length mi R-30b mimic group was the longest and the MI R-30b inhibit group was the shortest.5. After the optic nerve clamp injury in the rat, the vitreous cavity was injected respectively with rAAV-mi R-30b. Although, the survival rate of the four groups after injury decreased in varying degrees. The survival rate of Cs was the highest and that of group inhibitor was the lowest; FVEP showed that P1 amplitude in group mimic was also the most wide and inhibitor group was the narrowest. Conclusion 1, in vitro and in vivo experiments, Sema3A is the target gene of miR-30b; 2, miR-30b can be inhibited by Sema3A expression to change p-p38MAPK and active levels to reduce apoptosis and improve the optic nerve injury. The survival rate of Cs; 3, miR-30b can promote the growth of RGCs axon by inhibiting the expression of NRP1 and Plex A1 by inhibiting the expression of Sema3A, and accelerating the recovery of visual function after optic nerve injury, which may be the main molecular mechanism of MI R-30b to promote the repair of optic nerve after injury.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R774.6

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