發(fā)布時間：2018-05-08 09:52

  本文選題：視神經(jīng)橫斷 + 視網(wǎng)膜節(jié)細胞　； 參考：《第四軍醫(yī)大學》2015年博士論文

【摘要】：成年哺乳動物中樞神經(jīng)系統(tǒng)(Central Nervous System,CNS)的損傷導致神經(jīng)元不可逆的死亡,由此引起的神經(jīng)功能喪失及損傷后修復一直是學術界研究的重點和難點。視神經(jīng)(Optic neve,ON)是CNS的一部分,缺乏自發(fā)性再生能力,損傷后可導致視網(wǎng)膜節(jié)細胞(Retinal ganglion cells,RGCs)死亡及視覺功能不可逆損害。由于RGCs具有中樞神經(jīng)細胞的特點,因此視神經(jīng)橫斷成為研究中樞神經(jīng)系統(tǒng)損傷的重要動物模型之一。微管是神經(jīng)元細胞骨架最重要的組成部分,不僅可作為運輸細胞內物質(如神經(jīng)遞質)和細胞器的軌道及維持細胞形態(tài),而其對于細胞骨架動力學的正常調控在維持神經(jīng)突起延伸和突觸可塑性中起著至關重要的作用。如果調控機制紊亂,將會導致神經(jīng)精神性疾病及神經(jīng)退行性的疾病的發(fā)生。Stathmin蛋白作為一個強有力的微管解聚相關蛋白,參與調節(jié)微管動態(tài)平衡,在中樞神經(jīng)發(fā)育、神經(jīng)突起延伸和突觸可塑性中發(fā)揮著重要的作用。研究報道,Stathmin m RNA表達水平改變可能與大鼠視網(wǎng)膜發(fā)育具有一定關聯(lián),然Stathmin蛋白是否參與視神經(jīng)損傷后修復及再生至今尚無報道,其機制尚未闡明。目的:應用視神經(jīng)橫斷大鼠模型,首先觀察視神經(jīng)橫斷后不同時間點Stathmin及其家族基因在m RNA及蛋白水平的變化情況;為了明確Stathmin蛋白是否對軸突橫斷的視網(wǎng)膜節(jié)細胞具有保護作用,制備了野生型大鼠Stathmin基因過表達及RNAi干涉重組腺病毒,在視神經(jīng)橫斷后即刻給予玻璃體腔內分別注射Stathmin基因過表達及RNAi干涉重組腺病毒,觀察視網(wǎng)膜節(jié)細胞的存活及小膠質細胞(Retinal microglia cells,RMGs)的活化和增殖,探討Stathmin蛋白水平改變對損傷的視網(wǎng)膜節(jié)細胞的神經(jīng)保護作用。方法:(1)首先應用RT-PCR、Western blot及免疫組織化學方法檢測Stathmin及其家族基因在正常SD大鼠視網(wǎng)膜中的表達情況;其次,在SD大鼠視神經(jīng)球后段1.5mm處切斷視神經(jīng),構建大鼠視神經(jīng)橫斷動物模型;應用RT-PCR、Western blot方法,分別在大鼠視神經(jīng)橫斷后0天、3天、5天、7天、14天、21天檢測Stathmin及其家族基因m RNA及蛋白水平的表達情況;應用免疫組織化學方法檢測Stathmin蛋白在視神經(jīng)橫斷后不同時間點視網(wǎng)膜中的表達、分布和細胞定位情況;(2)首先設計合成全長野生型大鼠Stathmin基因引物,以SD大鼠視網(wǎng)膜基因組DNA為模板,通過PCR的方法,擴增大鼠Stathmin全長基因并進行測序驗證;同時依據(jù)si RNA的設計原則,設計合成針對Stathmin基因的特異性RNAi片段,退火形成雙鏈結構;然后,根據(jù)設計的限制性酶切位點Eco R I、Sal I,將Stathmin全長基因連接入p DC315載體,據(jù)設計的限制性酶切位點Bam H I、HandⅢ,將針對Stathmin基因的特異性RNAi片段連接入p Silence4.1-CMV載體;重組腺病毒載體與腺病毒骨架質粒p BHGlox△E1,3Cre在脂質體介導下共轉染HEK293E細胞進行同源重組,空斑技術獲取病毒,并進行病毒擴增、純化、滴度測定,將獲得的病毒命分別名為Ad-Stathmin、Ad-RNAi-Sta-563、Ad-RNAi-Sta-424;同時制備不含治療基因的野生型腺病毒Ad-wt和無關干涉腺病毒Ad-NC;最后,通過感染PC12細胞鑒定重組腺病毒對Stathmin基因的過表達和干涉效果;(3)構建大鼠視神經(jīng)橫斷模型,應用熒光金經(jīng)視神經(jīng)斷端逆行標記視網(wǎng)膜節(jié)細胞;在視神經(jīng)橫斷后即刻給予玻璃體腔內分別注射Ad-Stathmin/Ad-wt和Ad-RNAi-Sta-563/Ad-NC重組腺病毒,首先在視神經(jīng)橫斷后第5天應用Western blot方法檢測Stathmin蛋白水平的表達情況,明確重組腺病毒在動物體內對Stathmin基因的過表達和干涉效果;其次,應用視網(wǎng)膜平鋪方法,分別在視神經(jīng)橫斷后3天、9天、16天觀察視網(wǎng)膜節(jié)細胞的形態(tài)及數(shù)量變化,以期為Stathmin蛋白的視網(wǎng)膜節(jié)細胞保護作用提供量化的實驗依據(jù);同時也觀察視網(wǎng)膜小膠質細胞形態(tài)及數(shù)量變化,初步明確Stathmin蛋白表達變化對視網(wǎng)膜小膠質細胞的影響,進一步闡明Stathmin蛋白在視神經(jīng)損傷后對視網(wǎng)膜節(jié)細胞保護作用的機制。結果:(1)Stathmin及其家族基因在正常大鼠視網(wǎng)膜中均有表達,SCG-10表達水平最高,其次是SCLIP和Stathmin,RB3表達水平最低;視神經(jīng)橫斷后3天,Stathmin及其家族基因在m RNA水平表達均顯著增高(P0.01),并可持續(xù)到視神經(jīng)切斷后7天;14天時,SCG-10,SCLIP,RB3 m RNA表達水平明顯下降,與對照組比較沒有顯著差異,然而,Stathmin的表達在視神經(jīng)橫斷后14天仍高于對照組(P0.05);Western blot檢測結果顯示SCG-10,SCLIP,RB3蛋白水平變化基本趨勢與m RNA水平變化一致,14天時與對照組之間比較沒有統(tǒng)計學差異,而Stathmin在視神經(jīng)切斷后14天蛋白水平仍高于對照組(P0.05);免疫組織化學結果顯示,正常大鼠視網(wǎng)膜中Stathmin及SCG-10蛋白陽性染色在整個視網(wǎng)膜可見,視網(wǎng)膜節(jié)細胞層和內核層陽性染色強于內網(wǎng)狀層,SCLIP及RB3蛋白在視網(wǎng)膜表達較低,然而主要分布于視網(wǎng)膜節(jié)細胞層;Stathmin家族蛋白陽性染色定位于大鼠視網(wǎng)膜節(jié)細胞的胞漿;視神經(jīng)切斷后3天,Stathmin陽性表達呈現(xiàn)明顯增加,可持續(xù)到視神經(jīng)切斷后14天;(2)限制性酶切及測序方法鑒定結果證實,成功構建了大鼠Stathmin基因過表達及RNAi干涉腺病毒載體p DC315-Stathmin、p Silence4.1-RNAi-Sta-563和p Silence4.1-RNAi-Sta-424;同源重組獲得高滴度重組腺病毒,Ad-Stathmin滴度為5.9×1010TCID50/ml,Ad-RNAi-Sta-563滴度為6.5×1010TCID50/ml,Ad-RNAi-Sta-424滴度為3.4×1010TCID50/ml;重組腺病毒感染PC12細胞顯示,Ad-Stathmin重組腺病毒在m RNA和蛋白水平均可增加Stathmin蛋白表達,Ad-RNAi-Sta-563及Ad-RNAi-Sta-424重組腺病毒在m RNA和蛋白水平均可抑制Stathmin蛋白表達,且Ad-RNAi-Sta-563重組腺病毒的干涉效果優(yōu)于Ad-RNAi-Sta-424重組腺病毒。(3)視神經(jīng)橫斷后即刻玻璃體腔內分別注射Ad-Stathmin、Ad-RNAi-Sta-563重組腺病毒,在視神經(jīng)橫斷后5天Western blot結果顯示,Ad-Stathmin重組腺病毒可明顯上調視網(wǎng)膜中Stathmin蛋白表達,Ad-RNAi-Sta-563重組腺病毒可顯著下調視網(wǎng)膜中Stathmin蛋白表達;(4)大鼠視神經(jīng)橫斷后即刻玻璃體內注射Ad-Stathmin重組腺病毒,在視神經(jīng)橫斷后第9天存活的RGCs密度為539±41個/mm2,顯著低于Ad-wt注射組1039±67個/mm2及溶劑注射組1130±74個/mm2(p0.05);Ad-Stathmin重組腺病毒注射組在視神經(jīng)橫斷后第3天,視網(wǎng)膜中可見少量活化的RMGs,視神經(jīng)橫斷后第9天,Ad-Stathmin重組腺病毒注射組視網(wǎng)膜中活化增殖的RMGs明顯增多,密度為585±24個/mm2,明顯高于溶劑注射組402±19個/mm2、Ad-wt注射組398±38個/mm2,具有統(tǒng)計學意義(p0.05),且其活化程度進一步增加。(5)視神經(jīng)橫斷后即刻玻璃體腔內注射Ad-RNAi-Sta-563重組腺病毒,在視神經(jīng)橫斷后3天存活的RGCs密度與Ad-NC及溶劑組間無統(tǒng)計學差異;視神經(jīng)橫斷后9天,Ad-RNAi-Sta-563注射組存活的RGCs密度為2183±125個/mm2,與溶劑組1209±132個/mm2、Ad-NC組1236±157個/mm2有顯著差異(p0.01);Ad-RNAi-Sta-563注射組幾乎沒有顯影的RMGs,密度僅為21±1個/mm2,顯著低于溶劑注射組435±21個/mm2、Ad-NC注射組425±13個/mm2(p0.001),而在Ad-NC注射組,顯影的RMGs數(shù)量顯著增多,多為分枝狀形態(tài),并可見桿狀及雙極狀的RMGs;視神經(jīng)橫斷后16天,注射Ad-RNAi-Sta-563實驗組RGCs密度為1465±82個/mm2,與溶劑組341±66個/mm2、Ad-NC組303±47個/mm2比較顯著增高(p0.001);Ad-RNAi-Sta-563注射組RMGs密度為246±29個/mm2,與溶劑組665±47個/mm2、Ad-NC組613±32個/mm2比較顯著降低(p0.01),可見典型的阿米巴樣RMGs。結論:(1)Stathmin及其家族基因在正常大鼠視網(wǎng)膜中均有表達,SCG-10表達水平最高,其次是Stathmin和SCLIP,RB3表達水平最低;Stathmin及SCG-10蛋白陽性染色在整個視網(wǎng)膜可見,視網(wǎng)膜節(jié)細胞層和內核層陽性染色強于內網(wǎng)狀層,SCLIP及RB3蛋白在視網(wǎng)膜表達較低,然而主要分布于視網(wǎng)膜節(jié)細胞層;Stathmin家族蛋白陽性染色定位于大鼠視網(wǎng)膜節(jié)細胞的胞漿。(2)視神經(jīng)橫斷后3天,Stathmin及其家族基因m RNA和蛋白水平表達均顯著增高,并可持續(xù)到7天;Stathmin蛋白在視神經(jīng)切斷后14天蛋白水平仍高于對照組。(3)成功構建了大鼠Stathmin基因過表達及針對Stathmin基因的RNAi干涉重組腺病毒,體內外實驗顯示Ad-Stathmin重組腺病毒具有顯著的Stathmin過表達作用,Ad-RNAi-Sta-563重組腺病毒具有顯著的Stathmin基因表達干涉作用。(4)大鼠視神經(jīng)橫斷后即刻玻璃體腔內注射Ad-Stathmin重組腺病毒,在視神經(jīng)橫斷后第3天、9天,可促進視網(wǎng)膜小膠質細胞活化,在視神經(jīng)橫斷后第9天,存活的視網(wǎng)膜節(jié)細胞顯著降低,提示視神經(jīng)損傷后過表達Stathmin基因不利于損傷視網(wǎng)膜節(jié)細胞的存活。(5)大鼠視神經(jīng)橫斷后即刻玻璃體腔內注射Ad-RNAi-Sta-563重組腺病毒,在視神經(jīng)橫斷后第9天及16天,可顯著促進損傷的視網(wǎng)膜節(jié)細胞的存活,同時可顯著抑制視網(wǎng)膜小膠質細胞的活化,提示視神經(jīng)損傷后抑制Stathmin基因表達對損傷的視網(wǎng)膜節(jié)細胞的具有一定的保護作用。
[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.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R774.1

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