沉默Stathmin基因对轴突横断的视网膜节细胞的神经保护作用研究

发布时间：2018-05-08 09:52

本文选题：视神经横断 + 视网膜节细胞　；参考：《第四军医大学》2015年博士论文

【摘要】：成年哺乳动物中枢神经系统(Central Nervous System,CNS)的损伤导致神经元不可逆的死亡,由此引起的神经功能丧失及损伤后修复一直是学术界研究的重点和难点。视神经(Optic neve,ON)是CNS的一部分,缺乏自发性再生能力,损伤后可导致视网膜节细胞(Retinal ganglion cells,RGCs)死亡及视觉功能不可逆损害。由于RGCs具有中枢神经细胞的特点,因此视神经横断成为研究中枢神经系统损伤的重要动物模型之一。微管是神经元细胞骨架最重要的组成部分,不仅可作为运输细胞内物质(如神经递质)和细胞器的轨道及维持细胞形态,而其对于细胞骨架动力学的正常调控在维持神经突起延伸和突触可塑性中起着至关重要的作用。如果调控机制紊乱,将会导致神经精神性疾病及神经退行性的疾病的发生。Stathmin蛋白作为一个强有力的微管解聚相关蛋白,参与调节微管动态平衡,在中枢神经发育、神经突起延伸和突触可塑性中发挥着重要的作用。研究报道,Stathmin m RNA表达水平改变可能与大鼠视网膜发育具有一定关联,然Stathmin蛋白是否参与视神经损伤后修复及再生至今尚无报道,其机制尚未阐明。目的:应用视神经横断大鼠模型,首先观察视神经横断后不同时间点Stathmin及其家族基因在m RNA及蛋白水平的变化情况;为了明确Stathmin蛋白是否对轴突横断的视网膜节细胞具有保护作用,制备了野生型大鼠Stathmin基因过表达及RNAi干涉重组腺病毒,在视神经横断后即刻给予玻璃体腔内分别注射Stathmin基因过表达及RNAi干涉重组腺病毒,观察视网膜节细胞的存活及小胶质细胞(Retinal microglia cells,RMGs)的活化和增殖,探讨Stathmin蛋白水平改变对损伤的视网膜节细胞的神经保护作用。方法:(1)首先应用RT-PCR、Western blot及免疫组织化学方法检测Stathmin及其家族基因在正常SD大鼠视网膜中的表达情况;其次,在SD大鼠视神经球后段1.5mm处切断视神经,构建大鼠视神经横断动物模型;应用RT-PCR、Western blot方法,分别在大鼠视神经横断后0天、3天、5天、7天、14天、21天检测Stathmin及其家族基因m RNA及蛋白水平的表达情况;应用免疫组织化学方法检测Stathmin蛋白在视神经横断后不同时间点视网膜中的表达、分布和细胞定位情况;(2)首先设计合成全长野生型大鼠Stathmin基因引物,以SD大鼠视网膜基因组DNA为模板,通过PCR的方法,扩增大鼠Stathmin全长基因并进行测序验证;同时依据si RNA的设计原则,设计合成针对Stathmin基因的特异性RNAi片段,退火形成双链结构;然后,根据设计的限制性酶切位点Eco R I、Sal I,将Stathmin全长基因连接入p DC315载体,据设计的限制性酶切位点Bam H I、HandⅢ,将针对Stathmin基因的特异性RNAi片段连接入p Silence4.1-CMV载体;重组腺病毒载体与腺病毒骨架质粒p BHGlox△E1,3Cre在脂质体介导下共转染HEK293E细胞进行同源重组,空斑技术获取病毒,并进行病毒扩增、纯化、滴度测定,将获得的病毒命分别名为Ad-Stathmin、Ad-RNAi-Sta-563、Ad-RNAi-Sta-424;同时制备不含治疗基因的野生型腺病毒Ad-wt和无关干涉腺病毒Ad-NC;最后,通过感染PC12细胞鉴定重组腺病毒对Stathmin基因的过表达和干涉效果;(3)构建大鼠视神经横断模型,应用荧光金经视神经断端逆行标记视网膜节细胞;在视神经横断后即刻给予玻璃体腔内分别注射Ad-Stathmin/Ad-wt和Ad-RNAi-Sta-563/Ad-NC重组腺病毒,首先在视神经横断后第5天应用Western blot方法检测Stathmin蛋白水平的表达情况,明确重组腺病毒在动物体内对Stathmin基因的过表达和干涉效果;其次,应用视网膜平铺方法,分别在视神经横断后3天、9天、16天观察视网膜节细胞的形态及数量变化,以期为Stathmin蛋白的视网膜节细胞保护作用提供量化的实验依据;同时也观察视网膜小胶质细胞形态及数量变化,初步明确Stathmin蛋白表达变化对视网膜小胶质细胞的影响,进一步阐明Stathmin蛋白在视神经损伤后对视网膜节细胞保护作用的机制。结果:(1)Stathmin及其家族基因在正常大鼠视网膜中均有表达,SCG-10表达水平最高,其次是SCLIP和Stathmin,RB3表达水平最低;视神经横断后3天,Stathmin及其家族基因在m RNA水平表达均显著增高(P0.01),并可持续到视神经切断后7天;14天时,SCG-10,SCLIP,RB3 m RNA表达水平明显下降,与对照组比较没有显著差异,然而,Stathmin的表达在视神经横断后14天仍高于对照组(P0.05);Western blot检测结果显示SCG-10,SCLIP,RB3蛋白水平变化基本趋势与m RNA水平变化一致,14天时与对照组之间比较没有统计学差异,而Stathmin在视神经切断后14天蛋白水平仍高于对照组(P0.05);免疫组织化学结果显示,正常大鼠视网膜中Stathmin及SCG-10蛋白阳性染色在整个视网膜可见,视网膜节细胞层和内核层阳性染色强于内网状层,SCLIP及RB3蛋白在视网膜表达较低,然而主要分布于视网膜节细胞层;Stathmin家族蛋白阳性染色定位于大鼠视网膜节细胞的胞浆;视神经切断后3天,Stathmin阳性表达呈现明显增加,可持续到视神经切断后14天;(2)限制性酶切及测序方法鉴定结果证实,成功构建了大鼠Stathmin基因过表达及RNAi干涉腺病毒载体p DC315-Stathmin、p Silence4.1-RNAi-Sta-563和p Silence4.1-RNAi-Sta-424;同源重组获得高滴度重组腺病毒,Ad-Stathmin滴度为5.9×1010TCID50/ml,Ad-RNAi-Sta-563滴度为6.5×1010TCID50/ml,Ad-RNAi-Sta-424滴度为3.4×1010TCID50/ml;重组腺病毒感染PC12细胞显示,Ad-Stathmin重组腺病毒在m RNA和蛋白水平均可增加Stathmin蛋白表达,Ad-RNAi-Sta-563及Ad-RNAi-Sta-424重组腺病毒在m RNA和蛋白水平均可抑制Stathmin蛋白表达,且Ad-RNAi-Sta-563重组腺病毒的干涉效果优于Ad-RNAi-Sta-424重组腺病毒。(3)视神经横断后即刻玻璃体腔内分别注射Ad-Stathmin、Ad-RNAi-Sta-563重组腺病毒,在视神经横断后5天Western blot结果显示,Ad-Stathmin重组腺病毒可明显上调视网膜中Stathmin蛋白表达,Ad-RNAi-Sta-563重组腺病毒可显著下调视网膜中Stathmin蛋白表达;(4)大鼠视神经横断后即刻玻璃体内注射Ad-Stathmin重组腺病毒,在视神经横断后第9天存活的RGCs密度为539±41个/mm2,显著低于Ad-wt注射组1039±67个/mm2及溶剂注射组1130±74个/mm2(p0.05);Ad-Stathmin重组腺病毒注射组在视神经横断后第3天,视网膜中可见少量活化的RMGs,视神经横断后第9天,Ad-Stathmin重组腺病毒注射组视网膜中活化增殖的RMGs明显增多,密度为585±24个/mm2,明显高于溶剂注射组402±19个/mm2、Ad-wt注射组398±38个/mm2,具有统计学意义(p0.05),且其活化程度进一步增加。(5)视神经横断后即刻玻璃体腔内注射Ad-RNAi-Sta-563重组腺病毒,在视神经横断后3天存活的RGCs密度与Ad-NC及溶剂组间无统计学差异;视神经横断后9天,Ad-RNAi-Sta-563注射组存活的RGCs密度为2183±125个/mm2,与溶剂组1209±132个/mm2、Ad-NC组1236±157个/mm2有显著差异(p0.01);Ad-RNAi-Sta-563注射组几乎没有显影的RMGs,密度仅为21±1个/mm2,显著低于溶剂注射组435±21个/mm2、Ad-NC注射组425±13个/mm2(p0.001),而在Ad-NC注射组,显影的RMGs数量显著增多,多为分枝状形态,并可见杆状及双极状的RMGs;视神经横断后16天,注射Ad-RNAi-Sta-563实验组RGCs密度为1465±82个/mm2,与溶剂组341±66个/mm2、Ad-NC组303±47个/mm2比较显著增高(p0.001);Ad-RNAi-Sta-563注射组RMGs密度为246±29个/mm2,与溶剂组665±47个/mm2、Ad-NC组613±32个/mm2比较显著降低(p0.01),可见典型的阿米巴样RMGs。结论:(1)Stathmin及其家族基因在正常大鼠视网膜中均有表达,SCG-10表达水平最高,其次是Stathmin和SCLIP,RB3表达水平最低;Stathmin及SCG-10蛋白阳性染色在整个视网膜可见,视网膜节细胞层和内核层阳性染色强于内网状层,SCLIP及RB3蛋白在视网膜表达较低,然而主要分布于视网膜节细胞层;Stathmin家族蛋白阳性染色定位于大鼠视网膜节细胞的胞浆。(2)视神经横断后3天,Stathmin及其家族基因m RNA和蛋白水平表达均显著增高,并可持续到7天;Stathmin蛋白在视神经切断后14天蛋白水平仍高于对照组。(3)成功构建了大鼠Stathmin基因过表达及针对Stathmin基因的RNAi干涉重组腺病毒,体内外实验显示Ad-Stathmin重组腺病毒具有显著的Stathmin过表达作用,Ad-RNAi-Sta-563重组腺病毒具有显著的Stathmin基因表达干涉作用。(4)大鼠视神经横断后即刻玻璃体腔内注射Ad-Stathmin重组腺病毒,在视神经横断后第3天、9天,可促进视网膜小胶质细胞活化,在视神经横断后第9天,存活的视网膜节细胞显著降低,提示视神经损伤后过表达Stathmin基因不利于损伤视网膜节细胞的存活。(5)大鼠视神经横断后即刻玻璃体腔内注射Ad-RNAi-Sta-563重组腺病毒,在视神经横断后第9天及16天,可显著促进损伤的视网膜节细胞的存活,同时可显著抑制视网膜小胶质细胞的活化,提示视神经损伤后抑制Stathmin基因表达对损伤的视网膜节细胞的具有一定的保护作用。
[Abstract]:The damage of Central Nervous System (CNS) in adult mammals leads to the irreversible death of neurons. The loss of nerve function and repair after injury have been the key and difficult point in academic research. The optic nerve (Optic neve, ON) is a part of CNS, which lacks the ability to spontaneously regenerate and can cause the retina after injury. Retinal ganglion cells (RGCs) death and irreversible impairment of visual function. As RGCs has the characteristics of central nervous cells, the transection of the optic nerve becomes one of the most important animal models for the study of the damage of the central nervous system. Microtubules are the most important part of the neuron cytoskeleton, which can not only be used as the substances in the transport cells (such as the cells of the cells (such as) Neurotransmitters and organelles orbit and maintain cell morphology, and the normal regulation of cytoskeleton dynamics plays a vital role in maintaining neurite protuberance and synaptic plasticity. If the regulatory mechanism is disturbed, the.Stathmin protein of neuropsychic diseases and neurodegenerative diseases will be used as one. A strong microtubule depolymerization associated protein, involved in regulating the dynamic balance of microtubules, plays an important role in central nervous development, neurite protuberance and synaptic plasticity. It is reported that the changes in the expression level of Stathmin m RNA may be associated with the development of the retina of rats, but whether Stathmin protein is involved in the optic nerve injury The mechanism of repair and regeneration has not yet been reported, and its mechanism has not been elucidated. Objective: To observe the changes of Stathmin and its family genes at m RNA and protein levels at different time points after optic nerve transection, and to determine whether Stathmin protein has protective effect on the retinal ganglion cells of axonaxal transection. The overexpression of Stathmin gene and RNAi interference recombinant adenovirus were prepared. The overexpression of Stathmin gene and RNAi interference recombinant adenovirus were injected into the vitreous cavity immediately after the optic nerve transection, and the survival of the retinal ganglion cells and the activation and proliferation of the microglia (Retinal microglia cells, RMGs) were observed and the Stathmin was discussed. The neuroprotective effect of protein level on damaged retinal ganglion cells. Methods: (1) first, RT-PCR, Western blot and immunohistochemistry were used to detect the expression of Stathmin and its family genes in the retina of normal SD rats. Secondly, the optic nerve was cut off in the posterior segment of the optic nerve of SD rats, and the transverse optic nerve was constructed in the rat. The expression of M RNA and protein levels of Stathmin and its family genes were detected by RT-PCR and Western blot methods at 0 days, 3 days, 5 days, 7 days, 14 days and 21 days after the transection of the optic nerve in rats. The expression of Stathmin protein in the retina at different time points after optic nerve transection was detected by immunohistochemistry. Cell location; (2) first, a full-length wild type Stathmin gene primer was designed, and the SD rat retina genome DNA was used as a template. The whole Stathmin length gene of the rat was amplified by PCR method and was sequenced. At the same time, according to the design principle of Si RNA, the specific RNAi fragment for Stathmin gene was designed and synthesized, and the annealing formation was formed. Then, according to the designed restriction enzyme site Eco R I, Sal I, the Stathmin full length gene is connected to the P DC315 vector, and the designed restrictive enzyme cutting site Bam H I, Hand III is connected to the specific fragment of the gene. 1,3Cre was co transfected with HEK293E cells mediated by liposome, and the virus was obtained by plaque technique, and the virus was amplified, purified, and the titer was measured. The acquired virus was named Ad-Stathmin, Ad-RNAi-Sta-563, Ad-RNAi-Sta-424, and the wild type of adenovirus Ad-wt and unrelated interference adenovirus Ad-NC without the therapeutic base were prepared. Finally, the overexpression and interference effect of recombinant adenovirus to the Stathmin gene was identified by infected PC12 cells. (3) the rat optic nerve transection model was constructed and the retinal ganglion cells were retrograde labelled with the broken end of the fluorescent optic optic nerve, and Ad-Stathmin/Ad-wt and Ad-RNAi-Sta-563/Ad-NC were injected into the vitreous cavity immediately after the optic nerve transection. Recombinant adenovirus, first fifth days after the optic nerve transection, the Western blot method was used to detect the expression of Stathmin protein level, and the overexpression and interference effect of the recombinant adenovirus on the Stathmin gene in the animal body was determined. Secondly, the retinal ganglion cells were observed at 3 days, 9 days, and 16 days after the optic nerve transection. In order to provide quantitative experimental basis for the protection of retinal ganglion cells of Stathmin protein, the morphological and quantitative changes of retinal microglia are also observed, and the effect of the changes of Stathmin protein expression on retinal microglia is preliminarily clarified, and the Stathmin protein is further clarified after the optic nerve injury. The mechanism of retinal ganglion cell protection. Results: (1) Stathmin and its family genes are expressed in the retina of normal rats, the expression of SCG-10 is the highest, followed by SCLIP and Stathmin, the expression level of RB3 is the lowest, and at the 3 day after the optic nerve transection, the expression of Stathmin and its family genes are significantly increased (P0.01) at the level of M RNA (P0.01), and sustainable to the view. The expression level of SCG-10, SCLIP, RB3 m RNA decreased significantly at the 14 day, and there was no significant difference between the control group and the control group. However, the expression of Stathmin was still higher than that of the control group at 14 days after the optic nerve transection (P0.05); Western blot detection results showed SCG-10, SCLIP, the basic trend of the change of RB3 protein level was consistent with the level of 14 days. There was no statistical difference between the control group and the control group, but the protein level of Stathmin was still higher than that of the control group at 14 days after the optic nerve cut off (P0.05). The immunohistochemical results showed that the positive staining of Stathmin and SCG-10 protein in the retina of the normal rats was visible in the whole retina, and the positive staining of the retinal ganglion cell layer and the core layer was stronger than the inner net shape in the retina of the normal rats. The expression of SCLIP and RB3 protein in the retina was low, but mainly distributed in the retinal ganglion cell layer; Stathmin family protein positive staining was located in the cytoplasm of rat retinal ganglion cells. The positive expression of Stathmin increased obviously at 3 days after the optic nerve cut off, and was sustainable to the optic nerve after the severing of the optic nerve; (2) restriction enzyme digestion and sequencing method. The results confirmed that the overexpression of Stathmin gene and the RNAi interference adenovirus vector p DC315-Stathmin, P Silence4.1-RNAi-Sta-563 and P Silence4.1-RNAi-Sta-424 were successfully constructed, and the recombinant adenovirus with high titer was obtained by homologous recombination, the Ad-Stathmin titer was 5.9 x 1010TCID50/ml, and the Ad-RNAi-Sta-563 titer was 6.5 * 1010TCID50/ml. The titer was 3.4 x 1010TCID50/ml, and the recombinant adenovirus infected PC12 cells showed that the Ad-Stathmin recombinant adenovirus could increase the expression of Stathmin protein at the level of M RNA and protein, and the Ad-RNAi-Sta-563 and Ad-RNAi-Sta-424 recombinant adenovirus could inhibit the Stathmin protein expression at m RNA and protein levels, and the interference effect of the recombinant adenovirus of Ad-RNAi-Sta-563 recombinant adenovirus was effective. The fruit was superior to Ad-RNAi-Sta-424 recombinant adenovirus. (3) Ad-Stathmin and Ad-RNAi-Sta-563 recombinant adenovirus were injected into the immediate vitreous cavity of the optic nerve after transection of the optic nerve. The result of Western blot in the 5 day after the optic nerve transection showed that the recombinant adenovirus of Ad-Stathmin could obviously increase the expression of Stathmin protein in the retina, and the recombinant adenovirus of Ad-RNAi-Sta-563 could be shown to be obvious. The expression of Stathmin protein in the retina was downregulated; (4) the Ad-Stathmin recombinant adenovirus was injected into the vitreous body immediately after the optic nerve transection in rats. The RGCs density of the surviving ninth days after the optic nerve transection was 539 + 41 /mm2, significantly lower than the 1039 + 67 /mm2 in the Ad-wt injection group and 1130 + 74 /mm2 (P0.05) in the solvent injection group; and the Ad-Stathmin recombinant adenovirus was injected. In group third days after optic nerve transection, a small amount of activated RMGs was seen in the retina, and ninth days after the optic nerve transection, the proliferation of RMGs in the retina of the Ad-Stathmin recombinant adenovirus injection group increased significantly, with a density of 585 + 24 /mm2, significantly higher than that in the solvent injection group of 402 + 19 /mm2, and 398 + 38 /mm2 in the Ad-wt injection group, with statistical significance (P0.05). And the activation degree was further increased. (5) the Ad-RNAi-Sta-563 recombinant adenovirus was injected into the immediate vitreous cavity after the optic nerve transection, and there was no statistical difference between the RGCs density and the Ad-NC and the solvent group for the 3 day after the optic nerve transection. The RGCs density of the Ad-RNAi-Sta-563 injection group was 2183 + 125 /mm2, and 12 was in the solvent group at 9 days after the optic nerve transection. 09 + 132 /mm2, group Ad-NC 1236 + 157 /mm2 significant difference (P0.01), Ad-RNAi-Sta-563 injection group almost no developing RMGs, the density is only 21 + 1 /mm2, significantly lower than the solvent injection group 435 + 21 /mm2, Ad-NC injection group 425 + 13 /mm2 (p0.001), but in the Ad-NC injection group, the number of development significantly increased, and more branch shape, and can be 16 days after the transection of the optic nerve, the density of RGCs was 1465 + 82 /mm2, 341 + 66 /mm2 in the solvent group and 303 + 47 /mm2 in the Ad-NC group (p0.001), and the RMGs density of the Ad-RNAi-Sta-563 injection group was 246 + 29 /mm2, compared with the solvent group of 665 + 47 /mm2, and the group 613 + 32. The typical amoeba like RMGs. conclusion is found (P0.01): (1) Stathmin and its family genes are expressed in the retina of normal rats, the expression level of SCG-10 is the highest, followed by Stathmin and SCLIP, the expression level of RB3 is the lowest, Stathmin and SCG-10 positive staining is visible throughout the retina, and the retinal ganglion cell layer and the core layer are positive. The expression of SCLIP and RB3 protein was lower in the retina, but mainly in the retinal ganglion cell layer, and the positive staining of Stathmin family protein was located in the cytoplasm of the rat retinal ganglion cells. (2) the expression of M RNA and protein levels of Stathmin and its family genes increased significantly at the 3 day after the optic nerve transection, and continued to 7 days. The protein level of Stathmin protein was still higher than that of the control group at 14 days after the optic nerve cut off. (3) the overexpression of Stathmin gene and the RNAi interference recombinant adenovirus for Stathmin gene were successfully constructed. The experiment in vitro and in vivo showed that the recombinant adenovirus of Ad-Stathmin had a significant Stathmin overexpression, and the recombinant adenovirus of Ad-RNAi-Sta-563 was significant. Stathmin gene expression interfered. (4) Ad-Stathmin recombinant adenovirus was injected into the immediate vitreous cavity after the optic nerve transection in rats. The retinal microglia activation could be promoted at the third day after the optic nerve transection, 9 days after the optic nerve transection, and the surviving retinal ganglion cells decreased significantly at the ninth day after the optic nerve transection, suggesting that the Stathm was overexpressed after the optic nerve injury. In gene is not conducive to the survival of retinal ganglion cells. (5) Ad-RNAi-Sta-563 recombinant adenovirus is injected into the immediate vitreous cavity after the optic nerve transection in rats. The survival of the damaged retinal ganglion cells can be significantly promoted in the ninth and 16 days after the optic nerve transection, and the activation of the retinal microglia can be inhibited significantly, suggesting the optic nerve loss. Inhibition of Stathmin gene expression after injury can protect the injured retinal ganglion cells.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R774.1

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