大鼠Olig2基因慢病毒表达载体的构建及其转染后对少突胶质前体细胞分化的影响

目的 构建大鼠少突胶质细胞转录因子2(Olig2)的慢病毒表达载体,并转染少突胶质前体细胞(OPCs),观察Olig2过表达对OPCs向少突胶质细胞(OLs)分化的影响.方法 设计合成PCR引物,从大鼠pEGFP-N3-Olig2表达质粒中扩增并克隆大鼠Olig2-EGFP片段,将其插入到pLenti6.3慢病毒表达载体中,形成pLenti6.3-Olig2-EGFP慢病毒表达载体.将其与包装质粒混合后共转染293T细胞,包装产生慢病毒后感染培养的OPCs,观察Olig2在感染细胞中的表达并鉴定其表达对OPCs向OLs分化的影响.结果 成功将大鼠Olig2-EGFP片段克隆入pLenti6.3慢病毒表达载体,构建的pLenti6.3-Olig2-EGFP慢病毒表达载体能够高效表达.与空质粒转染的OPCs相比,重组质粒转染的OPCs体外诱导分化后产生更多的OLs (n =5,P＜0.01).结论 成功建立大鼠pLenti6.3-Olig2-EGFP慢病毒表达系统,慢病毒介导的Olig2过表达促进大鼠OPCs向OLs分化.     

过表达Olig2的少突胶质前体细胞在缺血缺氧脑白质损伤新生大鼠脑内的分化

目的:观察过表达特异性转录因子Olig2的少突胶质前体细胞(oligodendrocyte precursor cells,OPCs)移植在缺血缺氧(hypoxia-ischemia,HI)脑白质损伤新生大鼠脑内的分化情况。方法:用绿色荧光蛋白(green fluorescent protein,GFP)标记的过表达Olig2的慢病毒感染原代分离纯化的大鼠大脑皮层OPCs,GFP阳性细胞计数测其感染率。将过表达Olig2的OPCs(Olig2组)或阴性对照病毒感染的OPCs(Vector组)脑立体定位注射到造模后7 d的HI模型大鼠胼胝体膝部,移植后2周,冰冻切片行免疫荧光染色观察OPCs的存活和分化情况。结果:Olig2-GV218病毒感染OPCs细胞48 h,荧光显微镜检测显示85%左右的OPCs细胞表达GFP;移植后2周,caspase-3荧光染色表明移植后绝大部分细胞存活,Vector组和Olig2组之间无统计学差异(P0.05);GFAP/GFP双阳性细胞在两组之间也无显著差异(P0.05);而Olig2组MBP/GFP双阳性细胞的荧光密度显著高于Vector组(P0.05)。结论:过表达Olig2可促进移植OPCs向少突胶质细胞分化。     

Olig对少突胶质细胞介导脱髓鞘大鼠髓鞘再生的影响

目的 探讨Olig在少突胶质细胞介导的脱髓鞘大鼠髓鞘再生中的作用。方法 40只健康Wistar大鼠,随机分成正常组、模型对照组、模型组、实验组,用形态学观察和免疫组织化学检测Olig1和Olig2的表达。结果 正常组Olig1位于少突胶质细胞的胞质,模型组胞核表达明显增多,实验组Oligl又重新转移至胞质;正常组Olig2表达位于胞核,模型组中有少量表达于胞质。 结论 在Olig1和Olig2同时缺失时,导致全脑不能形成少突胶质细胞,严重影响髓鞘的再生。     

血清-糖皮质激素诱导激酶1基因对少突胶质细胞分化的影响

目的:探讨血清-糖皮质激素诱导激酶1(SGK1)对少突胶质细胞分化的影响。方法:利用免疫荧光染色检测糖皮质激素对体外培养少突胶质前体细胞(OPCs)分化的影响;原位杂交检测SGK1在少突胶质细胞中的表达情况;通过构建SGK1真核表达载体和鸡胚神经管电穿孔转化,研究SGK1过表达对少突胶质细胞发生发展的影响。结果:在体外,SGK1的激活物氢化可的松能促进OPCs分化;抑制物GSK650394则会阻断CPCs分化,鸡胚神经管过表达SGK1会促进少突胶质细胞转录因子2和Sox10的提前表达。结论:SGK1能促进少突胶质前体细胞的发生及分化。     

两类抗精神病药对脱髓鞘小鼠模型髓鞘修复的影响

目的:探讨抗精神病药物对双环己酮草酰二腙(CPZ)诱导的慢性脱髓鞘动物模型中髓鞘修复的作用。方法:持续给予CPZ喂养12周诱导小鼠慢性脱髓鞘模型,采用快蓝染色、髓鞘碱性蛋白(MBP)免疫组织化学显色等方法检测各组髓鞘变化,观察典型性抗精神病药氟哌啶醇和非典型性抗精神病药喹硫平对髓鞘修复的作用。通过免疫组织化学标记少突胶质前体细胞(OPCs)标志物Olig2,比较药物对OPCs的影响。结果:氟哌啶醇可以阻止髓鞘的自发性修复;喹硫平能促进慢性髓鞘再生的进程。在髓鞘修复过程中氟哌啶醇使OPCs数目减少;喹硫平可以促进OPCs增生。结论:在脱髓鞘模型中,氟哌啶醇抑制髓鞘自发修复,喹硫平可以促进髓鞘的慢性修复,其机制可能与两种药物对OPCs的不同作用有关。OPCs可能是抗精神病药治疗的靶点。     

Olig1过表达对大鼠神经干细胞向少突胶质细胞分化的影响

目的:Olig1基因修饰的神经干细胞(NSCs)移植治疗中枢神经脱髓鞘疾病具有重要的应用前景。本研究目的是构建大鼠olig1真核表达载体,并观察其过表达对大鼠NSCs向少突胶质细胞分化的影响。方法:采用RT-PCR,以新生大鼠脊髓RNA为模板,扩增olig1基因,定向克隆到pEGFP-N3载体中;用电穿孔方法转染pEGFP-N3-olig1表达载体至NSCs中,然后用RT-PCR鉴定olig1的表达,免疫荧光染色鉴定NSCs向少突胶质细胞的分化情况。结果:成功构建了pEGFP-N3-olig1真核表达载体,olig1在重组质粒转染的NSCs中能够高效表达。重组质粒转染的NSCs在体外诱导分化后,能够较空质粒转染的NSCs产生更多的少突胶质细胞(P0.01)。结论:Olig1过表达能够显著促进大鼠NSCs向少突胶质细胞方向分化。     

少突胶质前体细胞与神经元突触联系的特点及功能

<正>少突胶质前体细胞(oligodendrocyte precursor cells,OPCs)广泛分布于中枢神经系统(central neural system,CNS)中,约占成年大鼠脑内总细胞数的5%~10%,占人脑内白质区细胞总数的20%,且约80%的OPCs处于活化状态,能够不断增殖或分化~([1])。因OPCs表面表达神经/胶质抗原2(nerve/glial antigen 2,NG2)蛋白多糖,故又被称为NG2     

少突胶质前体细胞分离纯化方法的比较

目的 对少突胶质前体细胞(OPCs)的分离、培养及传代方法进行改良,并建立少突胶质细胞谱系的体外分化模型,以模拟体内OPCs的生长和发育过程.方法 将12只雄性balb/c乳鼠随机分成2组,每组各6只,分别用于神经干细胞诱导分化法和传统的混合胶质细胞分离法,前者将海马及皮层中的神经干细胞分离并在条件性分化培养基中定向培养,通过差速消化法和差速贴壁法结合纯化OPCs;后者是将全脑的胶质细胞共同培养,细胞生长出现明显分层后通过差速离心法分离中间层的OPCs.不同分化阶段免疫标记物(O2、CC1和MBP)荧光染色鉴定以确定细胞分化情况,Ki-67染色用以以判断OPCs的增殖能力.结果 两种方法均可培养出具有分化能力的OPCs,但经神经干细胞诱导方法获得的OPCs具有更好的增殖能力(P<0.05),经过成熟分化培养后获得的成熟的少突胶质细胞数量也多于传统的混合胶质细胞分离法(P<0.05).该方法的整个培养周期短,获得的细胞数量多,细胞纯度高.结论 神经干细胞诱导法获得的OPCs具有更好的增殖和分化能力,可为少突胶质前体细胞研究提供稳定的细胞来源.     

槲皮素对大鼠缺氧损伤体外培养少突胶质前体细胞增殖的作用

目的:探讨槲皮素(Quercetin,Qu)对大鼠缺氧损伤后少突胶质前体细胞(oligodendrocyte precursor cells,OPCs)增殖的作用。方法:取新生1～2 d Sprague-Dawley(SD)大鼠大脑皮质细胞进行原代培养,振荡分离后纯化3 d行Olig2和A2B5免疫荧光染色鉴定。纯化的细胞分为正常对照组、模型组(缺氧9 h)和Qu处理组(3,9,27,81μmol/L)。缺氧后3 d光镜下观察细胞形态学变化,CCK-8法检测细胞存活率,Olig2免疫荧光染色计数阳性细胞数。结果:纯化培养3 d的细胞胞体呈圆形,有双极或三极突起。免疫荧光染色显示,绝大部分细胞为A2B5和Olig2阳性,A2B5/DAPI阳性细胞率为98.54%。缺氧9 h后,模型组很多细胞出现突起皱缩或崩解,细胞密度较Qu处理组明显降低;培养至3 d,光镜下计数和CCK-8检测结果均表明9μmol/L和27μmol/L Qu处理组细胞数较模型组显著增加(P<0.01,P<0.05);Olig2免疫细胞化学染色结果进一步表明9μmol/L和27μmol/L Qu处理组OPCs数较模型组显著增加(P<0.01,P<0.05)。结论:9μmol/L和27μmol/L Qu有促进缺氧损伤OPCs增殖的作用。     

弥漫性轴索损伤后胶质细胞的反应性变化

目的 探讨大鼠弥漫性轴索损伤（DAI）后胶质细胞的反应性变化规律及与轴索继发损伤的相互关系。 方法 SD 成年大鼠随机分为对照组及DAI损伤1d、2d、3d、7d组,每组8只。参照Marmarou方法制做 DAI 模型,并对大鼠的脑干部位进行胶质纤维酸性蛋白（GFAP）、离子钙结合适配器分子1（Iba1）、少突胶质细胞系转录因子2（Olig 2）、CC-1、NG2免疫组织化学染色及TUNEL染色,并且通过透射电子显微镜进一步观察脑干超微结构变化。 结果 DAI大鼠损伤后3d脑干Iba1标记的小胶质细胞细胞数目显著增多,一直持续至伤后7d,且激活的小胶质细胞呈肥大样形态学改变;DAI大鼠伤后1周内脑干GFAP标记的星形胶质细胞数目虽有所增多,但并没有统计学意义;CC-1标记的成熟少突胶质细胞在DAI后1d即开始明显降低,且随损伤时间的延长而进一步降低。DAI大鼠脑干TUNEL标记的凋亡细胞随损伤时间延长持续增多,与成熟少突胶质细胞的丢失成正相关。Olig2标记的少突胶质细胞系表达在DAI损伤后1周内各时间点均明显增高,于损伤后3d达峰值。NG2标记的少突胶质细胞前体细胞数量于损伤后3d和7d显著增多。透射电子显微镜结果示,DAI大鼠损伤呈现由髓鞘到轴索的特点,最早表现为髓鞘松解,轴索完整;且脱髓鞘会进一步促进轴索骨架崩解,最终导致轴索变性。 结论 成熟少突胶质细胞对DAI损伤具有选择易感性,髓鞘脱失是轴索继发损伤的重要因素。少突胶质细胞前体细胞增殖,并伴随小胶质细胞的激活。探索胶质细胞的动态变化将为进一步解释轴索继发损伤的病理生理学机制奠定基础。     

外伤性视神经损伤后视神经及视网膜形态的动态观察

目的:了解外伤性视神经损伤后的病理变化、溃变特点与时相间的关系。方法:参照Allen脊髓损伤法,造成视神经眶尖段间接600gcm力冲击、挤压伤。伤后对视神经和视网膜行形态学动态观察。结果:①伤后48h,视神经轻度肿胀和空泡反应;1周时损伤处视神经出现溃变,神经胶质细胞增生,视网膜神经节细胞(retinalganglioncells,RGCs)形态改变不明显;2周时神经纤维轴束间空泡样改变,局灶性坏死,RGCs核固缩和细胞数量减少。术后3月,视神经损伤部位直径缩小,形成胶质疤痕,RGCs数量明显减少,核固缩细胞增多。②RGCs数量于术后48h、1周、2周、1月和3月分别比正常对照组低3.35%、13.23%、19.74%、23.20%、29.28%。③视网膜细胞在48h内出现凋亡。结论:本实验模型可造成明确的视神经和视网膜损伤,神经元的损伤程度从节细胞、中间神经元、感光细胞的次序依次递减。视网膜和视神经损伤的严重程度与时间呈相关性。RGCs数量在48h至1周时下降速率最快。     

The value of neurophysiological and MRI assessment in demyelinating optic neuritis (DON).

PURPOSE: To study, by neurophysiological means, the possible involvement of the retina, in demyelinating optic neuritis (DON). MATERIAL AND METHODS: Thirty-five patients fulfilling strict criteria of unilateral DON were investigated with a battery of neurophysiological tests and MRI within 3 weeks of the onset of their symptoms. Flash-ERG (F-ERG) in photopic conditions, Flash-VEPs and PR-VEPs were recorded. MRI of the brain and the optic nerve were performed. RESULTS: The amplitude of b-wave of F-ERG in photopic conditions was statistically significantly lower in the affected eye (p < 0.001) compared to normal controls, whereas in the unaffected eye, it was also statistically significantly lower than normal controls (p < 0.01). All patients had statistically significant prolongation of P100 latency in PR-VEPs of the affected eye (p < 0.001) in comparison to normal controls. The P100 wave of the unaffected eye was also delayed (p < 0.01). In MRI, Gd-DTPA enhancement was observed in 7 symptomatic nerves with only minimal enhancement of the optic nerve between optic chiasm and optic canal, whereas 11 patients were presented with intracranial associated plaques. Five of the above patients had optic nerve enhancement and diffused demyelinating findings simultaneously. CONCLUSION: These results are a neurophysiological indication of involvement of the retina in DON, probably of vascular origin.     

Astrocyte invasion and vasculogenesis in the developing ferret retina

We studied the time course of astrocyte invasion and blood vessel formation in the developing ferret retina using glial fibrillary acidic protein (GFAP)-immunohistochemistry for astrocytes and isolectin B4 histochemistry for blood vessels. As in other mammals, strongly GFAP positive astrocytes invade the ferret retina from the optic nerve. At birth, strongly GFAP positive astrocytes have reached about 22% of the distance between optic disc and outer retinal edge whereas weakly GFAP positive processes already extend to the edge of the retina. At postnatal days P30–P37 about 82% of the distance between optic disc and outer retinal edge and in the adult 88% of this distance is covered with strongly labelled astrocytes. Superficial blood vessels form from the optic disc. They reach up to about 24% of the retinal radius at birth and grow radially across the retina during further development. At P30–P37, the whole retina is covered with superficial blood vessels. The deep vascular layer forms later (around P30) through sprouting from superficial vessels. The radial pattern of astrocyte and vessel growth from the optic disc is not affected by the formation of the area centralis and visual streak.     

Optic nerve invasion of uveal melanoma

The aim of the study was to identify the histopathological characteristics associated with the invasion of the optic nerve of uveal melanoma and to evaluate the association between invasion of the optic nerve and survival. In order to achieve this, all uveal melanomas with optic nerve invasion in Denmark between 1942 and 2001 were reviewed (n=157). Histopathological characteristics and depth of optic nerve invasion were recorded. The material was compared with a control material from the same period consisting of 85 cases randomly drawn from all choroidal/ciliary body melanomas without optic nerve invasion. Prelaminar/laminar optic nerve invasion was in multivariate analysis associated with focal retinal invasion, neovascularization of the chamber angle, and scleral invasion. Postlaminar invasion was further associated with non-spindle cell type and rupture of the inner limiting membrane of the retina. The optic nerve was invaded in four different ways: 1) by tumor extension from the neuroretina through the lamina cribrosa; 2) by direct extension into the optic nerve head between Bruch's membrane and the border tissue of Elschnig; 3) by direct invasion through the border tissue of Elschnig; and 4) in one case a tumor spread along the inner limiting membrane to the optic nerve through the lamina cribrosa. Invasion of the optic nerve had no impact on all-cause mortality or melanoma-related mortality in multivariate analyses. The majority of melanomas invading the optic nerve are large juxtapapillary tumors invading the optic nerve because of simple proximity to the nerve. A neurotropic subtype invades the optic nerve and retina in a diffuse fashion unrelated to tumor size or location.     

三维有限元模型力学分析可预测视乳头的形状变化

背景：视乳头形状变化的准确测量及预测是青光眼的早期诊断及病程预测的关键,因此高眼压下视乳头形状的测量与分析具有重要意义。 目的：建立视乳头三维有限元模型,并分析急性高眼压下视乳头形状及视网膜厚度的变化。 方法：利用前房灌注的方法建立急性高眼压动物模型,利用光相干断层扫描仪获得猫在正常眼压及5 320,    7 980,10 640,13 300,15 960 Pa眼压时的断层图片,测量视网膜特征点处厚度的变化;基于正常眼压猫眼视乳头的断层数据,利用MIMICS软件获得视网膜及脉络膜的三维结构,并进行组装获得视乳头三维模型;利用有限元软件ABAQUS分析高眼压状态下视乳头的形状变化及视网膜厚度变化,并与实验测试结果进行比较。 结果与结论：随着眼压的升高,发现视网膜厚度逐渐变薄,视乳头深度及视乳头宽度均逐渐增加,视乳头宽度与深度的比值也逐渐增加。提示急性高眼压导致视网膜变薄,视乳头宽度增大,视乳头深度增大。结果证明,利用光学相干断层扫描仪进行活体视乳头建模和分析是可行的,力学分析可预测视乳头的形状变化,对进一步确定青光眼的病理进程有一定的指导意义。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

骨髓间充质干细胞调节大鼠视神经损伤后小胶质细胞的活化

背景:外伤性视神经损伤是引起视力丧失的重要原因,治疗方法也比较局限,为探求更好的治疗方法,该实验从小胶质细胞方向入手进行探究。在神经病理条件下,激活小胶质细胞能够维持中枢神经系统的稳定,但小胶质细胞过度活化会产生大量的炎症因子,使损伤进行性加重。目的:探讨视神经损伤后小胶质细胞活化情况以及骨髓间充质干细胞对其过度表达的调节作用。方法:选取8周龄SD大鼠18只,将其随机分为移植组、模型组和假手术组,每组6只,其中模型组、移植组选取左眼进行视神经钳夹造模后分离视网膜和视神经,假手术组只分离视网膜和视神经,不进行钳夹,移植组在损伤后立即向左眼玻璃体内注入第3代骨髓间充质干细胞(注入细胞1×108,细胞量为2μL),模型组、假手术组玻璃体内注入等量的PBS,术后15 d全部处死,在灌流固定后取视网膜连带视神经用于苏木精-伊红染色和免疫组织化学检测。结果与结论:模型组视神经及视网膜小胶质细胞活化标记物Ox-42以及炎症因子TNF-α的表达量均高于假手术组(P<0.05),移植组视神经及视网膜中Ox-42和TNF-α表达量降低(P<0.05)并且接近假手术组水平。结果表明,小胶质细胞的过度活化与视神经损伤相关,骨髓间充质干细胞可以抑制小胶质细胞过度活化及炎症因子的释放,从而在一定程度上保护视网膜和视神经免受损伤。     

H89和wortmannin对霍乱毒素促进远端视神经损伤后视网膜节细胞再生的影响

目的：探讨H89和wortmannin对霍乱毒素(CTx)促进成年金黄地鼠远端视神经受损后视网膜节细胞(RGCs)再生的影响。方法：远端切断视神经并对接一段自体坐骨神经(AG)，玻璃体内注射CTx及H89、wortmannin。动物随机分为AG；AG CTx组；AG CWx H89组；AG CTx wortmannin组，4w后粒蓝逆行标记再生的RGCs，荧光显微镜下观察计数。结果：AG CTx组再生的视网膜节细胞平均数(43．2)明显高出AG组(2.6)，AG CTx H89组平均再生数为3．2，与AG组相近，明显低于AG CTx组。AG CTx W组平均再生数为9.6，明显高于AG，低于AG CTx组。结论：H89和wortmannin可部分抑制CTx对视神经远端切断后视网膜节细胞再生的促进作用。     

Macrophages during retina and optic nerve development in the mouse embryo: relationship to cell death and optic fibres

We compared the spatial and temporal patterns of distribution of macrophages, with patterns of naturally occurring cell death and optic fibre growth during early retina and optic nerve development, in the mouse. We used embryos between day 10 of embryogenesis (E10; before the first optic fibres are generated in the retina) and E13 (when the first optic fibres have crossed the chiasmatic anlage). The macrophages and optic axons were identified by immunocytochemistry, and the apoptotic cells were detected by the TUNEL technique, which specifically labels fragmented DNA. Cell death was observed in the retina and the optic stalk long before the first optic axons appeared in either region. Subsequently, specialized F4/80-positive phagocytes were detected in chronological and topographical coincidence with cell death, which disappeared progressively. As development proceeded, the pioneer ganglion cell axons reached the regions where the macrophages were located. As the number of optic fibres increased, the macrophages disappeared. Therefore, cell death, accompanied by macrophages, preceded the growth of fibres in the retina and the optic nerve. Moreover, these macrophages synthesized NGF and the optic axons were p75 neurotrophin receptor (p75^NTR)- and TrkA-positive. These findings suggest that macrophages may be involved in optic axon guidance and fasciculation.     

Is Thy-1 expressed only by ganglion cells and their axons in the retina and optic nerve?

Summary The distribution of Thy-1 in the retina and optic nerve has been examined immunohistochemically, and compared to that of the astrocytic marker glial fibrillary acidic protein. The axons and cell bodies of ganglion cells were found to be Thy-1 positive as were processes within the inner plexiform layer. Transection of the optic nerve in the neonatal rat results in the rapid degeneration of the ganglion cells but some Thy-1 staining remains in the inner plexiform layer. We have estimated using an immunoassay of normal and optic nerve transected retinae that about 70% of the Thy-1 in the retina is on ganglion cells and their axons and the remainder is on cells which contribute processes to the inner plexiform layer, presumably amacrine, bipolar or Müller cells.In the optic nerve the Thy-1 was found to be limited to the fascicles of optic nerve fibres and the intrafascicular spaces, containing astrocytes and their processes, were not stained. Axotomy of the adult nerve, which produced axonal degeneration and astrocytic proliferation, led to a loss of over 95% of the Thy-1 from the nerve. We found no evidence that the astrocytes of the retina or optic nerve were Thy-1 positive in normal animals or during degeneration.     

Anomalous axonal outgrowth at the retina caused by injury to the optic nerve or tectal ablation in adultXenopus

Summary The retina and optic nerve head have been examined by light and electron microscopy in adultXenopus laeuis after injury to optic nerve fibres. Intraorbital resection, transection or crush of the optic nerve all resulted in the appearance at the retina of a mass of actively growing axons which formed a ring around the intraretinal and adjacent choroidal portions of the optic nerve head. Formation of this heterotopic axon population was first noted at two weeks after nerve injury and fibres persisted for at least six months. The ectopic fibres were seperated from the optic nerve head by astrocytes within the retina or by blood vessels and fibroblasts of the leptomeninges at extraretinal locations. In general, the orientation of the ectopic fibres was perpendicular to the fibres of the optic nerve. Bundles of axons were found between the ring of ectopic fibres and the pigment epithelial layer of the retina or among the blood sinuses of the choroid. Similar ectopic fibres were seen following transection of the optic nerve at the chiasm and after tectal ablation although the onset of these changes was slower than that seen after nerve resection. It is concluded that damage to visual pathways in the frog induces dramatic morphological alterations in the optic nerve and retina far proximal to the site of injury in this regenerating system.