α晶体蛋白促进视网膜神经节细胞轴突再生的机制研究
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摘要
α晶体蛋白是晶状体来源的“神经保护物质”之一,它能有效地促进损伤后的视神经再生,但其作用机制不明。视神经损伤后RGCs的继发性死亡,是视神经难以再生的原因之一,但是,单纯保护RGCs存活并不能引起视神经的有效再生,因为损伤局部环境中的抑制性物质是阻碍视神经再生的另一关键因素。这些抑制性物质主要有髓鞘源性抑制物,胶质瘢痕来源的抑制物,以及生长锥导向抑制物。目前研究已证明:RhoA/Rock的表达与活性变化对中枢神经元轴突再生有着直接影响,是众多轴突再生抑制物信号传导的共同作用点。而α晶体蛋白不仅能促进RGCs存活,而且对RGCs轴突再生也具有明显促进作用。那么,阻碍轴突再生抑制物信号传导的关节点-----RhoA/Rock信号通路的传导,是否是α晶体蛋白促进视神经再生的机制?目前尚不清楚。
目的:通过在髓鞘抑制物质包被的培养板上培养RGCs,研究α晶体蛋白是否能拮抗髓鞘抑制物的作用而促进RGCs突起生长。而后分别通过在体视神经不全损伤大鼠模型和离体RGCs培养,研究α晶体蛋白对RhoA、Rock的表达和活化情况,其下游物质磷酸化情况,RGCs轴突再生及生长锥萎缩情况的影响。从而探讨RhoA/Rock信号通路在α晶体蛋白促进视神经再生中的作用。为视神经损伤和再生的基础研究提供理论依据,为临床治疗视神经损伤探索可行方法。
方法:1、从大鼠晶状体中提取混合晶体蛋白,用分子排阻凝胶色谱法纯化分离α晶体蛋白,肽指纹质谱法鉴定其纯度;从大鼠脑组织中提取髓鞘抑制物质,并对其成份及抑制神经元轴突生长的功能进行鉴定。2、在体实验:以成年Long Evans大鼠为研究对象,分为视神经损伤后玻璃体腔注射α晶体蛋白组、RhoA/Rock抑制剂组(阳性对照组),和牛血清白蛋白组(阴性对照组)。通过western blot分析统计视网膜中RhoA/Rock表达变化,亲和沉淀法研究RhoA活性变化,并进行统计学分析。同时视神经顺行示踪检测各组视神经再生长度。3、离体实验:在髓鞘抑制物包被的培养板上进行RGCs离体培养,分析比较培养1、3、5d,α晶体蛋白组与阳性和阴性对照组有突起的细胞数、最长突起长度及cofilin和MLC磷酸化程度的统计学差异。同时观察各组生长锥的萎缩情况。
结果:1、从大鼠晶状体中提取并纯化的α晶体蛋白经肽质纹质谱分析为alphaA-crystalline和alphaB-crystalline的混合物。western blot检测提取的髓鞘抑制物中含有髓鞘抑制物NogoA和MAG;在RGCs培养液中加入髓鞘抑制物1小时后,细胞生长状态差,出现细胞碎片,生长锥的膨大明显缩小,板足回退;2h后生长锥萎缩消失,RGCs突起断裂。在髓鞘抑制物质包被的培养板上培养RGCs,培养1d、2d、3d、5d,α晶体蛋白组有突起的细胞数均较牛血清白蛋白组增多,突起长度也明显长于牛血清白蛋白组(P<0.01)。
2、正常成年大鼠视网膜中,仅RGCs层可见少量RhoA和Rock表达;视神经损伤后1天,RhoA和Rock主要分布于RGCs层;损伤3天达内丛状层;损伤7天后,分布于RGCs、内丛状层、内核层及外丛状层。
3、视神经损伤后1、3、7天,RhoA和Rock的表达均高于正常组(P<0.01～0.05),并且活化的RhoA均较正常组明显增多(P<0.01)。
4、视神经损伤后,α晶体蛋白组、fasudil组及C3转移酶组,活化的RhoA均较牛血清白蛋白组明显减少(P<0.01～0.05)。但各组RhoA和Rock在视网膜中的表达量均无显著统计学差异。
5、视神经损伤后2周,牛血清白蛋白组仅可见极少量的神经纤维长入损伤区。而α晶体蛋白组及fasudil组,不仅损伤区可见较多的神经纤维长入,而且,神经纤维越过损伤区长入损伤区的远端。
6、在蛋白上样量基本一致的情况下,α晶体蛋白组和fasudil组磷酸化-cofilin和磷酸化-MLC占总cofilin和总MLC的比率,均明显少于牛血清白蛋白组(P<0.01),α晶体蛋白组和fasudil组比较无显著统计学差异。
7、在髓鞘抑制物质包被的培养板上培养RGCs,培养1、3、5d,α晶体蛋白组和fasudil组有突起的RGCs数目均明显多于牛血清白蛋白组(P<0.01),细胞最长突起长度也显著增长(P<0.01)。与α晶体蛋白组比较,培养1d时,有突起的细胞数fasudil组明显增多(P<0.01);培养3d和5d时,两组无显著统计学差异。但fasudil组细胞最长突起长度,于培养1、3、5d均较α晶体蛋白组明显增长(P<0.01)。
8、未加髓鞘的正常生长锥末端可见明显的膨大,并且可见较多的细小指状突起。加入髓鞘后,牛血清白蛋白组细胞突起末端膨大的生长锥消失,呈断裂状且未见细小分支;α晶体蛋白组和fasudil组细胞突起末端仍可见较小的膨大,并且可见少量的指状突起。
结论:1、视神经损伤可显著增加RhoA、Rock在视网膜中的表达量,扩大其在视网膜中的分布范围,并显著增强RhoA活性。RhoA/Rock在视神经损伤后再生过程中发挥重要的作用。
2、α晶体蛋白能拮抗髓鞘抑制物质的抑制作用促进RGCs轴突再生。
3、抑制RhoA的激活,阻碍髓鞘抑制物质的信号传导,防止生长锥的萎缩是α晶体蛋白促进RGCs轴突再生的机制之一,RhoA/Rock信号通路在α晶体蛋白促进RGCs轴突再生中发挥着重要的作用。
As one of lentogenic factors, alpha-crystallin can promote axon regeneration after optic nerve injury. So far, the mechanism is still poorly defined. Subsequent death of retinal ganglion cells(RGCs) was one reason of the failure in axon regeneration. However, promoting RGCs survival only can ont result in long axon regeneration. Recent research on molecular inhibition in central nervous microenviroment have indicated the inhibitory microenviroment plays an important role in the failure of regeneration. There are many inhibitors of regeneration, such as Nogo, myelin associated glycoprotein,oligodendrocyte myelin glcoprotein, chondroitin sulfate proteoglycans, ephexin and so on. Rencent studies on molecular inhibition in central nervous microenviroment have indicated a common crucial signaling event for all axonal inhibitors and repellents is the activation of RhoA/Rock signaling pathway. Alpha-crystallin can not only promote RGCs survival, but also stimulate axon regeneration. Then, can alpha-crystallin stimulate axon regeneration through RhoA/Rock signaling pathway?
Objective: To study the antagonism effect of alpha-crystaliin on myelin and stimulate RGCs axon regeneration . And to study whether alpha-crystallin can stimulate axon regeneration through RhoA/Rock signaling pathway or not by analysing the expression and activation of RhoA and Rock, the phosphorylation of cofilin and myosin light chain(MLC), RGCs axon regeneration and cone growth collapse in vivo and in vitro.
Methods: 1. Extracted and identified the myelin and alpha-crystallin from rats. 2. Alpha-crystallin, bovine serum albumin(BSA) and the inhibitor of RhoA/Rock were injected into vitreous cavity respectively. The expression of RhoA and Rock were analysed by western blot and the activation of RhoA assayed by affinity precipitation. At the same time, the regeneration length of optic nerve were measured by anterograde tracing using cholera toxin subunit B (CTB). 3. RGCs were cultured on myelin-coated dishes with DMEM containing alpha-crystallin , BSA, and the inhibitor of RhoA/Rock. The density of RGCs with neurite and the longest neurite of the cells were measured on day 1,,3 and 5. The phosphorylation of cofilin and MLC were assayed by western blot. And the morphology of growth cone was observed by scanning electron microscope.
Results: 1. Peptide mass fingerprinting analysis showed that alpha-crystallin was composed of alphaA-crystallineand alphaB-crystalline. The results of western blot showed that myelin contained NogoA and myelin associated glycoprotein.And myelin could induced RGCs growth cone retraction.
2. In normal retina, RhoA and Rock were only distributed in RGCs layer. One day after optic nerve injury, the distribution of RhoA and Rock was the same as that in normal retina. After 3 days, RhoA and Rock existed in both the RGCs and inner plexiform layers. Its immunoreactivity was abundant in RGCs layers, inner plexiform layers, inner nuclear layers and outer plexiform layers 7 days after optic nerve injury.
3. Compared to the normal retina, the expression of RhoA and Rock and RhoA activation were enhanced significantly 1, 3 and 7 days after optic nerve injury.
4. Compared to BSA, treatment of alpha-crystallin, C3 transferase and fasudil resulted in significant decrease in GTP-RhoA after optic nerve injury. However, the expression of RhoA and Rock can not be decrease.
5. Tow weeks after optic nerve injury, a little RGCs axon regenerated into crushed region. Whereas, a lot of axons regrowed across the crushed region and entered the distal optic nerve after treated with alpha-crystallin or fasudil.
6. Compared to BSA, alpha-crystallin and fasudi resulted in significant decrease in phosphorylation of cofilin and MLC. And the phosphorylation of cofilin and MLC were similar between alpha-crystallin and fasudil.
7. Compared to the BSA control group, alpha-crystallin and fasudil could increase the density of RGCs with neurite significantly after cultured 1, 3 and 5 days. Alpha-crystallin and fasudil could also promote neurite outgrowth in comparison with the BSA.
8. In normal RGCs axon, growth cone, filopodia and lamellipodia can be seen. The growth cone disappeared after treated with BSA and myelin, and no filopodia and lamellipodia can be seen. However, alpha-crystaliin and fasudil could antagonize myelin and inhibite growth cone retraction in a certain extent. Although, the filopodia and lamellipodia were not very typical, same processes can be seen at the end of the axon after treated with alpha-crystallin and fasudil.
Conclusions: 1. The expression of RhoA and Rock was enhanced significantly, and the disstribution was extended after optic nerve injury. RhoA/Rock was involved in optic nerve degeneration.
2. Alpha-crystaliin could antagonize myelin and stimulate RGCs axon regeneration.
3. Inhibiting RhoA activation was one of the mechanisms of alpha-crystallin stimulating RGCs axon regeration. Alpha-crystallin could stimulate axon regeneration through RhoA/Rock signaling pathway.
目的:通过在髓鞘抑制物质包被的培养板上培养RGCs,研究α晶体蛋白是否能拮抗髓鞘抑制物的作用而促进RGCs突起生长。而后分别通过在体视神经不全损伤大鼠模型和离体RGCs培养,研究α晶体蛋白对RhoA、Rock的表达和活化情况,其下游物质磷酸化情况,RGCs轴突再生及生长锥萎缩情况的影响。从而探讨RhoA/Rock信号通路在α晶体蛋白促进视神经再生中的作用。为视神经损伤和再生的基础研究提供理论依据,为临床治疗视神经损伤探索可行方法。
方法:1、从大鼠晶状体中提取混合晶体蛋白,用分子排阻凝胶色谱法纯化分离α晶体蛋白,肽指纹质谱法鉴定其纯度;从大鼠脑组织中提取髓鞘抑制物质,并对其成份及抑制神经元轴突生长的功能进行鉴定。2、在体实验:以成年Long Evans大鼠为研究对象,分为视神经损伤后玻璃体腔注射α晶体蛋白组、RhoA/Rock抑制剂组(阳性对照组),和牛血清白蛋白组(阴性对照组)。通过western blot分析统计视网膜中RhoA/Rock表达变化,亲和沉淀法研究RhoA活性变化,并进行统计学分析。同时视神经顺行示踪检测各组视神经再生长度。3、离体实验:在髓鞘抑制物包被的培养板上进行RGCs离体培养,分析比较培养1、3、5d,α晶体蛋白组与阳性和阴性对照组有突起的细胞数、最长突起长度及cofilin和MLC磷酸化程度的统计学差异。同时观察各组生长锥的萎缩情况。
结果:1、从大鼠晶状体中提取并纯化的α晶体蛋白经肽质纹质谱分析为alphaA-crystalline和alphaB-crystalline的混合物。western blot检测提取的髓鞘抑制物中含有髓鞘抑制物NogoA和MAG;在RGCs培养液中加入髓鞘抑制物1小时后,细胞生长状态差,出现细胞碎片,生长锥的膨大明显缩小,板足回退;2h后生长锥萎缩消失,RGCs突起断裂。在髓鞘抑制物质包被的培养板上培养RGCs,培养1d、2d、3d、5d,α晶体蛋白组有突起的细胞数均较牛血清白蛋白组增多,突起长度也明显长于牛血清白蛋白组(P<0.01)。
2、正常成年大鼠视网膜中,仅RGCs层可见少量RhoA和Rock表达;视神经损伤后1天,RhoA和Rock主要分布于RGCs层;损伤3天达内丛状层;损伤7天后,分布于RGCs、内丛状层、内核层及外丛状层。
3、视神经损伤后1、3、7天,RhoA和Rock的表达均高于正常组(P<0.01～0.05),并且活化的RhoA均较正常组明显增多(P<0.01)。
4、视神经损伤后,α晶体蛋白组、fasudil组及C3转移酶组,活化的RhoA均较牛血清白蛋白组明显减少(P<0.01～0.05)。但各组RhoA和Rock在视网膜中的表达量均无显著统计学差异。
5、视神经损伤后2周,牛血清白蛋白组仅可见极少量的神经纤维长入损伤区。而α晶体蛋白组及fasudil组,不仅损伤区可见较多的神经纤维长入,而且,神经纤维越过损伤区长入损伤区的远端。
6、在蛋白上样量基本一致的情况下,α晶体蛋白组和fasudil组磷酸化-cofilin和磷酸化-MLC占总cofilin和总MLC的比率,均明显少于牛血清白蛋白组(P<0.01),α晶体蛋白组和fasudil组比较无显著统计学差异。
7、在髓鞘抑制物质包被的培养板上培养RGCs,培养1、3、5d,α晶体蛋白组和fasudil组有突起的RGCs数目均明显多于牛血清白蛋白组(P<0.01),细胞最长突起长度也显著增长(P<0.01)。与α晶体蛋白组比较,培养1d时,有突起的细胞数fasudil组明显增多(P<0.01);培养3d和5d时,两组无显著统计学差异。但fasudil组细胞最长突起长度,于培养1、3、5d均较α晶体蛋白组明显增长(P<0.01)。
8、未加髓鞘的正常生长锥末端可见明显的膨大,并且可见较多的细小指状突起。加入髓鞘后,牛血清白蛋白组细胞突起末端膨大的生长锥消失,呈断裂状且未见细小分支;α晶体蛋白组和fasudil组细胞突起末端仍可见较小的膨大,并且可见少量的指状突起。
结论:1、视神经损伤可显著增加RhoA、Rock在视网膜中的表达量,扩大其在视网膜中的分布范围,并显著增强RhoA活性。RhoA/Rock在视神经损伤后再生过程中发挥重要的作用。
2、α晶体蛋白能拮抗髓鞘抑制物质的抑制作用促进RGCs轴突再生。
3、抑制RhoA的激活,阻碍髓鞘抑制物质的信号传导,防止生长锥的萎缩是α晶体蛋白促进RGCs轴突再生的机制之一,RhoA/Rock信号通路在α晶体蛋白促进RGCs轴突再生中发挥着重要的作用。
As one of lentogenic factors, alpha-crystallin can promote axon regeneration after optic nerve injury. So far, the mechanism is still poorly defined. Subsequent death of retinal ganglion cells(RGCs) was one reason of the failure in axon regeneration. However, promoting RGCs survival only can ont result in long axon regeneration. Recent research on molecular inhibition in central nervous microenviroment have indicated the inhibitory microenviroment plays an important role in the failure of regeneration. There are many inhibitors of regeneration, such as Nogo, myelin associated glycoprotein,oligodendrocyte myelin glcoprotein, chondroitin sulfate proteoglycans, ephexin and so on. Rencent studies on molecular inhibition in central nervous microenviroment have indicated a common crucial signaling event for all axonal inhibitors and repellents is the activation of RhoA/Rock signaling pathway. Alpha-crystallin can not only promote RGCs survival, but also stimulate axon regeneration. Then, can alpha-crystallin stimulate axon regeneration through RhoA/Rock signaling pathway?
Objective: To study the antagonism effect of alpha-crystaliin on myelin and stimulate RGCs axon regeneration . And to study whether alpha-crystallin can stimulate axon regeneration through RhoA/Rock signaling pathway or not by analysing the expression and activation of RhoA and Rock, the phosphorylation of cofilin and myosin light chain(MLC), RGCs axon regeneration and cone growth collapse in vivo and in vitro.
Methods: 1. Extracted and identified the myelin and alpha-crystallin from rats. 2. Alpha-crystallin, bovine serum albumin(BSA) and the inhibitor of RhoA/Rock were injected into vitreous cavity respectively. The expression of RhoA and Rock were analysed by western blot and the activation of RhoA assayed by affinity precipitation. At the same time, the regeneration length of optic nerve were measured by anterograde tracing using cholera toxin subunit B (CTB). 3. RGCs were cultured on myelin-coated dishes with DMEM containing alpha-crystallin , BSA, and the inhibitor of RhoA/Rock. The density of RGCs with neurite and the longest neurite of the cells were measured on day 1,,3 and 5. The phosphorylation of cofilin and MLC were assayed by western blot. And the morphology of growth cone was observed by scanning electron microscope.
Results: 1. Peptide mass fingerprinting analysis showed that alpha-crystallin was composed of alphaA-crystallineand alphaB-crystalline. The results of western blot showed that myelin contained NogoA and myelin associated glycoprotein.And myelin could induced RGCs growth cone retraction.
2. In normal retina, RhoA and Rock were only distributed in RGCs layer. One day after optic nerve injury, the distribution of RhoA and Rock was the same as that in normal retina. After 3 days, RhoA and Rock existed in both the RGCs and inner plexiform layers. Its immunoreactivity was abundant in RGCs layers, inner plexiform layers, inner nuclear layers and outer plexiform layers 7 days after optic nerve injury.
3. Compared to the normal retina, the expression of RhoA and Rock and RhoA activation were enhanced significantly 1, 3 and 7 days after optic nerve injury.
4. Compared to BSA, treatment of alpha-crystallin, C3 transferase and fasudil resulted in significant decrease in GTP-RhoA after optic nerve injury. However, the expression of RhoA and Rock can not be decrease.
5. Tow weeks after optic nerve injury, a little RGCs axon regenerated into crushed region. Whereas, a lot of axons regrowed across the crushed region and entered the distal optic nerve after treated with alpha-crystallin or fasudil.
6. Compared to BSA, alpha-crystallin and fasudi resulted in significant decrease in phosphorylation of cofilin and MLC. And the phosphorylation of cofilin and MLC were similar between alpha-crystallin and fasudil.
7. Compared to the BSA control group, alpha-crystallin and fasudil could increase the density of RGCs with neurite significantly after cultured 1, 3 and 5 days. Alpha-crystallin and fasudil could also promote neurite outgrowth in comparison with the BSA.
8. In normal RGCs axon, growth cone, filopodia and lamellipodia can be seen. The growth cone disappeared after treated with BSA and myelin, and no filopodia and lamellipodia can be seen. However, alpha-crystaliin and fasudil could antagonize myelin and inhibite growth cone retraction in a certain extent. Although, the filopodia and lamellipodia were not very typical, same processes can be seen at the end of the axon after treated with alpha-crystallin and fasudil.
Conclusions: 1. The expression of RhoA and Rock was enhanced significantly, and the disstribution was extended after optic nerve injury. RhoA/Rock was involved in optic nerve degeneration.
2. Alpha-crystaliin could antagonize myelin and stimulate RGCs axon regeneration.
3. Inhibiting RhoA activation was one of the mechanisms of alpha-crystallin stimulating RGCs axon regeration. Alpha-crystallin could stimulate axon regeneration through RhoA/Rock signaling pathway.
引文
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