大鼠胚胎神经干细胞的纯化、诱导分化及鉴定

探讨胚胎神经干细胞 (Neural stem cell,NSCs)的体外纯化扩增、保存标记、诱导分化及其鉴定方法。将14 .5 d胎龄大鼠大脑额叶皮质 NSCs在无血清 DMEM/ F12 (含 2 0 ng/ ml b FGF,2 0 ng/ m l EGF及 B2 7辅助培养液 )培养 ,利用有限稀释法将悬浮生长的单个细胞所形成的克隆球挑选出来、通过亚克隆连续传代大量扩增而纯化 ,免疫组化鉴定 nestin抗原阳性 ;选取部分 NSCs冻存、复苏后 nestin抗原阳性 ;用 Brd U孵育 NSCs,被 Brd U标记的 NSCs及其血清诱导分化后仍均呈 Brd U阳性。用血清或饲养层诱导 NSCs分化为大量表达 Tubulin- (神经元特异性抗原微管蛋白 3)阳性的神经元和 GFAP(神经胶质纤维酸性蛋白 )阳性的神经胶质细胞。由该实验可知有限稀释单细胞克隆连续传代是分离纯化、大量扩增胚胎期大脑 NSCs的简单有效方法。饲养层细胞也能诱导 NSCs分化为神经细胞。 Brd U可标记、示踪神经系统疾病动物模型 NSCs的实验治疗。掌握 NSCs的体外纯化培养、保存标记、诱导分化及鉴定方法可为进一步研究 NSCs的生物学特性及神经系统疾病的治疗提供新方法。     

GDNF基因修饰的NSCs移植对暂时性缺血性脑卒中大鼠的神经保护作用研究

目的　探讨移植胶质细胞源性神经营养因子（glial cell line derived neurotrophic factor,GDNF）基因修饰的神经干细胞（neural stem cells,NSCs）对暂时性缺血性脑卒中大鼠的神经保护。 方法　用GDNF重组腺病毒载体转染新生大鼠NSCs（GDNF/NSCs）,分化培养7 d后,行免疫细胞化学染色检测微管相关蛋白2（MAP2）。采用改良的插线法制作暂时性脑缺血再灌注模型,3 d后经脑室分别移植生理盐水、NSCs和GDNF/NSCs。于再灌注后1、2、3、5、7周末处死大鼠,行免疫组织化学染色观察移植细胞在脑内的神经元分化及星形胶质细胞在缺血区形成胶质界膜情况,行Luxol fast blue（LFB）染色显示神经纤维损伤情况。 结果　GDNF/NSCs体外分化为MAP2+细胞的比例显著高于NSCs的分化。移植细胞在脑内分化为MAP2+细胞,于再灌注第5周分化达高峰,GDNF/NSCs组于再灌注第3~7周,其MAP2+细胞显著高于NSCs组。各组缺血区由星形胶质细胞形成的血管胶质界膜存在不同程度的破坏,其连续性中断。对照组在各个时间点,血管胶质界膜损伤严重,完整性差,两细胞移植组,其胶质界膜随时间延长逐渐完整,GDNF/NSCs组早于NSCs组完善对胶质界膜的修复。此外,GDNF/NSCs组的神经纤维损伤修复优于NSCs组。 结论　GDNF/NSCs比NSCs对暂时性缺血性脑卒中大鼠模型有更好的神经保护作用,可能是与GDNF提高了NSCs在脑内的神经元分化,增强了NSCs对胶质界膜及神经纤维修复有关。     

直流电场诱导神经干细胞桥接横断脊髓的实验研究

目的:探讨直流电场(direct current field,DC)诱导神经干细胞(neural stem cells,NSCs)桥接横断脊髓的作用。方法:将54只雌性成年SD大鼠随机分为脊髓损伤(spinal cord injury,SCI)组、NSCs移植组、NSCs移植联合直流电场诱导组(DC+NSCs组),各组分7,14,28d三个时间点取材,HE染色观察神经纤维再生情况,透射电镜下观察脊髓白质内髓鞘结构变化,免疫荧光染色检测髓磷脂碱性蛋白(myelin basic protein,MBP)表达变化。结果:在光镜下,DC+NSCs组神经纤维较其他两组都多,排布均匀且走行较规则,部分纤维穿越损伤脊髓断端两侧。在电镜下,DC+NSCs组髓鞘松散程度明显减轻,部分髓鞘结构规整,排列有序,线粒体肿胀减轻,微管空泡样变较轻,可发现大量突触,薄髓的再生髓鞘明显增多。DC+NSCs组大鼠脊髓组织中可见直径大小不等的MBP阳性的圆形结构,MBP阳性表达最多,较其他两组有显著性差异(P0.01)。结论:直流电场诱导NSCs治疗SCI可以促进损伤处脱髓的轴突再髓鞘化和MBP的表达,促进神经纤维再生,从而起到桥接横断脊髓的作用。     

星形胶质细胞条件培养液促进新生大鼠海马神经干细胞分化

目的探讨星形胶质细胞条件培养液(ACM)对神经干细胞(NSCs)体外分化的影响。方法行新生大鼠大脑皮质星形胶质细胞的纯化培养后收集ACM,将新生大鼠海马NSCs单克隆培养后行nestin免疫细胞荧光染色,诱导分化5d后行神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)、半乳糖脑苷脂(GALC)免疫细胞荧光染色;将单克隆培养的NSCs分为对照组和实验组,对照组为单纯NSCs培养液,实验组根据NSCs培养液与ACM比的不同分3组:A组(2:1),B组(1:1),C组(1:2)。各组培养1周,采用NSE免疫细胞荧光检测方法标记神经元并计数和统计学分析。结果纯化的星形胶质细胞胶质纤维酸性蛋白抗体标记阳性率为96.5%;单克隆培养的海马NSCs呈nestin阳性,诱导后呈NSE、GFAP和GALC阳性表达。ACM培养的海马NSCs各组分化为神经元的比例明显高于对照组(<0.01),其中A组的比例最高。结论ACM可以促进新生大鼠海马NSCs向神经元分化。     

移植GDNF基因修饰的神经干细胞对大鼠脑卒中的神经保护作用

目的:探讨移植胶质细胞源性神经营养因子(GDNF)基因修饰的神经干细胞(NSCs)是否比单纯NSCs移植对脑卒中有更好的神经保护.方法:用GDNF重组质粒转染NSCs,构建过表达GDNF的NSCs(GDNF/NSCs).插线法制备大鼠脑缺血卒中模型,再灌注3d,脑立体定位分别移植NSCs、 GDNF/NSCs以及生理盐水到同侧侧脑室,实验分为GDNF/NSCs组、NSCs组和对照组.再灌注第1、 2、 3、 5和7周处死大鼠,用改良的神经功能缺失评分和H-E染色分别评估大鼠神经功能和脑梗死体积;免疫组织化学显色观察移植的NSCs数量与分布和突触蛋白Syn、 PSD-95在脑组织的表达.结果:再灌注2、 3周,GDNF/NSCs组较NSCs组神经功能恢复更好.在各时间点GDNF/NSCs和NSCs组大鼠脑缺血体积较对照组显著减小;GDNF/NSCs组的NSCs细胞数量较NSCs组显著增多.再灌注2和3周,GDNF/NSCs组Syn免疫阳性产物比NSCs组显著增加;各时间点GDNF/NSCs组的PSD-95免疫阳性产物比NSCs组显著增加.结论:GDNF基因修饰的NSCs比NSCs对大鼠脑卒中有更好的神经保护作用.     

脑源性神经营养因子基因修饰的胚胎大鼠皮质神经干细胞及其在体外的分化

目的 探讨脑源性神经营养因子（BDNF）基因修饰大鼠脑皮质神经干细胞（NSCs）后BDNF的表达,并观察其对NSCs体外诱导分化的影响。 方法 体外分离、培养大鼠胚胎脑皮质神经干细胞并鉴定;构建BDNF基因重组质粒,非脂质体法转染NSCs,实验分为pcDNA3-1- BDNF组、pcDNA3-1组和未转染NSCs组。免疫细胞化学技术和RT-PCR检测转染后BDNF蛋白和mRNA的表达;在含5％胎牛血清的分化培养基中诱导分化,βIII微管蛋白、神经胶质纤维酸性蛋白（GFAP） 和突触素( SYP)免疫细胞化学染色对分化细胞进行鉴定,并观察pcDNA3-1-BDNF转染NSCs分化过程中SYP的表达变化。 结果 体外培养获得巢蛋白（nestin）阳性的NSCs。成功构建BDNF基因重组质粒,免疫细胞化学和RT-PCR检测均显示,与pcDNA3-1组和NSCs组比较,pcDNA3-1-BDNF组NSCs的BDNF表达明显增强（P<0.05）。pcDNA3-1组和NSCs组比较,无显著性差异（P＞0.05）。分化后第4天,各组细胞均可分化为Βiii-微管蛋白阳性和GFAP阳性细胞,pcDNA3-1-BDNF组可见少量SYP阳性表达细胞。分化后第7天,与pcDNA3-1组和NSCs组比较,pcDNA3-1-BDNF组NSCs分化为βIII微管蛋白阳性神经元的比例明显增高,有显著性差异（P<0.05）,SYP阳性细胞数增多,表达增强。 结论 BDNF转染NSCs具有体外分泌BDNF的能力,能够促进NSCs定向分化为神经元,SYP表达增强,可能在促进神经元之间的突触发生中发挥作用。     

氯倍他索对大鼠神经干细胞自发分化的影响

目的:研究氯倍他索(Clo)对神经干细胞自发分化的影响,为Clo治疗中枢性神经损伤提供医学理论基础。方法:利用无血清悬浮法分离培养及鉴定大鼠NSCs;选取第三代NSCs,以含10%胎牛血清完全培养基贴壁诱导分化7 d,培养基中各加入同体积二甲基亚砜、2. 5、5μmol/L的Clo;免疫荧光染色和Western Blot方法检测NSCs分化后轴突膜蛋白(GAP-43)、酸性胶质蛋白(GFAP)、髓磷脂碱性蛋白(MBP)表达情况,进一步探讨Clo的作用机制。结果:分离培养的NSCs高表达Nestin; Clo作用组表达GAP-43和MBP较对照组明显提高(P 0. 05),GFAP表达水平低于对照组(P 0. 05),Clo作用组表达音猬因子(SHH)和锌指蛋白(Gli 1)均高于对照组(P 0.05)。结论:Clo可在体外通过SHH信号通路促进NSCs向神经元细胞和少突胶质细胞分化,与此同时Clo可抑制NSCs向星形胶质细胞分化。     

补阳还五汤联合神经干细胞移植促进脊髓损伤髓鞘再生

目的:探讨补阳还五汤(BYHWD)灌胃联合神经干细胞(NSCs)移植对脊髓损伤(SCI)后髓鞘再生的影响.方法:将48只成年雌性SD大鼠随机分为脊髓损伤(SCI)组、BYHWD灌胃组、NSCs移植组、NSCs移植联合BYHWD灌胃(BYHWD+ NSCs)组.SCI组不做治疗;NSCs组于脊髓横断后局部移植NSCs; BYHWD+ NSCs组移植NSCs,并每天BYHWD灌胃;各组在损伤后14、28 d后分别留取损伤脊髓标本,用免疫荧光双标法检测髓磷脂碱性蛋白(MBP)表达情况,透射电镜观察脊髓白质内髓鞘超微结构变化.结果:BYHWD+ NSCs组MBP阳性表达最多,较其他3组差异有统计学意义.电镜下BYHWD+NSCs组髓鞘结构较为规整,轴突微丝、微管排列有序,线粒体结构完整,可见薄髓的再生髓鞘.结论:SCI后BYHWD灌胃联合NSCs移植能够促进损伤脊髓MBP的表达及脱髓的轴突再髓鞘化.     

穹窿海马伞切割侧海马对植入神经干细胞分化为神经元的影响

为观察成鼠神经干细胞移植入切割穹窿海马伞侧海马和正常侧海马后存活和分化为神经元的状况 ,用无血清培养和单细胞克隆技术获取成年 SD大鼠前脑室下带神经干细胞 ,进行 Brd U标记和扩增。切割 SD大鼠右侧穹窿海马伞 ,术后 2周将标记有 Brd U的神经干细胞植入双侧海马齿状回。 2月后 ,行 Nissl染色、Brd U免疫荧光、NF -2 0 0 / Brd U免疫荧光、β-tubulin- / Brd U免疫组织化学染色和 ACh E组织化学染色。结果发现 ,移植的神经干细胞在海马齿状回中存活并沿颗粒下层迁移 ,切割侧海马齿状回中有较多的 Nissl深染的大胞体神经元样细胞 ,而正常侧多为小胞体胶质样细胞。切割侧海马齿状回颗粒下层中见有数个NF-2 0 0 / Brd U、β-tubulin- / Brd U双标神经元和 ACh E阳性神经元 ,而正常侧海马中则未能见到。上述结果提示 ,移植到海马中的神经干细胞能存活、迁移 ,穹窿海马伞切割侧海马中某些物质的表达增强 ,可诱导植入其内的神经干细胞向神经元或 ACh E阳性神经元分化。     

嗅鞘细胞移植实验性自身免疫性脑脊髓炎后髓鞘碱性蛋白的表达和小胶质细胞的捕获

目的:研究嗅鞘细胞(OECs)移植实验性自身免疫性脑脊髓炎(EAE)后髓鞘碱性蛋白(Myelin basic protein,MBP)的表达及小胶质细胞捕获OECs情况。方法:用新生近交系Wistar大鼠嗅球培养出OECs,经荧光染料羧基荧光素二乙酸盐琥珀酰亚胺酯(CFSE)标记后注入同系EAE Wistar大鼠侧脑室,免疫组化检测OECs髓鞘碱性蛋白(MBP)、单核巨噬细胞诱导分子1(ED1)和CFSE共表达情况。结果:培养OECs不表达MBP,经侧脑室移植后在EAE大鼠脑内出现多量MBP+CFSE+细胞,同时在大脑广泛区域及血管周围间隙出现多量ED1+CFSE+细胞,与对照组相比差异显著。结论:OECs在EAE环境下表达MBP分子,其抗原能被脑内小胶质细胞或巨噬细胞捕获。     

18α-GA激活Nrf2对成体神经干细胞的影响

目的:明确18α-甘草次酸(18α-GA)对核因子E2相关因子2(Nrf2)的激活作用,观察上调Nrf2对成体神经干细胞(a NSCs)增殖和分化能力的影响,探讨维持a NSCs活力的有效途径。方法:体外培养新生、成年和老年小鼠的侧脑室室膜下区神经干细胞(NSCs),观察随年龄增长NSCs中Nrf2的表达变化。以18α-GA作用a NSCs,用real-time PCR和Western blot实验比较18α-GA组与DMSO对照组a NSCs的Nrf2表达变化。构建携带绿色荧光蛋白(GFP)基因的Nrf2-shRNA慢病毒载体(LV)感染a NSCs,用real-time PCR和Western blot实验检测shRNA对Nrf2的沉默效率。将a NSCs按照干预条件不同分为DMSO组、18α-GA组、LV-GFP组和LV-Nrf2-shRNA组,运用Brd U掺入实验、Tuj1免疫荧光染色、CCK-8实验、Hoechst 33342/PI双染色和活性氧簇(ROS)水平测定等方法评价18α-GA是否通过激活Nrf2对a NSCs的增殖、分化、细胞活力、细胞凋亡以及氧化应激水平产生影响。结果:随年龄增长,成年和老年小鼠a NSCs的Nrf2 mRNA表达水平较新生小鼠NSCs显著降低(P0.01),但a NSCs的ROS水平显著高于新生小鼠NSCs(P0.05)。应用18α-GA后,a NSCs的Nrf2 mRNA与蛋白表达水平较DMSO组明显升高(P0.01),并且a NSCs的Brd U阳性率也显著增加(P0.01)。2 mg/L 18α-GA作用后,a NSCs的Tuj1阳性率和细胞活力较DMSO组显著升高(P0.05),而凋亡率和ROS水平显著下降(P0.05和P0.01)。但是,敲减a NSCs的Nrf2并应用18α-GA干预后,LV-Nrf2-shRNA组Brd U阳性率、Tuj1阳性率以及细胞活力均较LV-GFP组显著下降(P0.05),ROS水平却显著上升(P0.05)。结论:18α-GA可能通过激活Nrf2提高a NSCs的抗氧化能力,促进a NSCs增殖和分化,维持a NSCs潜能。     

Discrimination between different types of neuroglial cells in rat central nervous system using combined immuno- and enzyme-histochemical methods

The central nervous system (CNS) contains several types of neuroglial cells. In the present study, we characterized different types of glial cells in rat CNS by using single and combined immuno- and enzyme-histochemical methods, and immunofluorescence techniques. Two recently developed monoclonal antibodies (mAbs) against rat macrophages-associated antigens appeared to recognize a subpopulation of glial cells in the CNS of normal adult rats. These ED4- and ED8-positive glial cells were predominantly located in the white matter of adult rat CNS and shared morphological features with microglia. ED4 and ED8 were applied in a double staining combined with mAbs and an antiserum raised against galactocerebroside (GalC) to identify oligodendrocytes, or with anti-glial fibrillary acidic protein antiserum (GFA) to identify astrocytes. We also used a mAb against myelin basic protein (MBP) to identify oligodendrocytes. It appeared that ED4 and ED8 recognized a subpopulation of oligodendrocytes. MAbs against GalC and MBP recognized cells in an immunoperoxidase staining with a morphology identical to that of the ED8-positive cells and part of the ED4-positive cells. Frozen sections of Lewis rats CNS with acute experimental allergic encephalomyelitis (EAE) were investigated, where infiltrating brain macrophages could be found which stained positively with ED4 and ED8 as well as with the monocyte/macrophage mAbs ED1 and ED2. These brain macrophages did not stain when GalC, MBP and GFA markers were applied. Furthermore, ED4+GalC+ and ED8+GalC+ oligodendrocytes were present in the CNS white matter of EAE animals with similar appearance as in normal adult rats. With the currently used markers, we could not detect a third type of neuroglial cell, besides the astrocytes and oligodendrocytes. Thus, none of our anti-macrophage monoclonals recognized the presumptive microglia. Only under pathological conditions, e.g., in inflammatory infiltrates in the course of EAE, could brain macrophages be detected in the CNS parenchyma and only in the direct vicinity of blood vessels, indicating their hematogenous origin.     

银杏提取物对实验性自身免疫性脑脊髓炎小鼠的影响

目的:探讨银杏提取物(GBE)对实验性自身免疫性脑脊髓炎(EAE)小鼠炎症脱髓鞘病变的影响。方法:应用髓鞘少突胶质细胞糖蛋白33-55(MOG33-55)配以完全弗氏佐剂(CFA)免疫小鼠,诱发EAE模型。将小鼠分为CFA对照组、EAE模型组和GBE治疗组(每日腹腔注射GBE70mg/kg)。通过神经功能评分、行为学实验以及免疫荧光染色,观察GBE对EAE小鼠的影响。结果:GBE组小鼠各时间段神经功能评分均低于EAE组(P0.05),行为学检测显示发病高峰期falling latency时间较EAE组延长10s;GBE组较EAE组视神经髓鞘碱性蛋白(MBP)表达水平增高,可见MBP阳性髓鞘结构包绕轴突;海马伞矢状切片免疫荧光染色证实GBE组CD11b阳性小胶质细胞较EAE组明显减少,但是GFAP阳性星形胶质细胞数量与EAE组无明显差别。结论:GBE可能通过抑制小胶质细胞激活从而延缓EAE小鼠脱髓鞘进程,提示GBE对多发性硬化具有一定的治疗作用。     

Experimental autoimmune encephalomyelitis in the rat: lessons in T-cell immunology and autoreactivity

Summary: Experimental autoimmune encephalomyelitis (EAE) in the rat is an acute paralytic disease from which most animals spontaneously recover. The disease can be induced in susceptible inbred Lewis and DA rats with myelin basic protein (MBP), or encephalitogenic MBP peptides administered in complete Freund's adjuvant (CFA). The disease can be adoptively transferred to syngeneic recipients with primed T cells that have been reactivated in vitro with antigen. EAE is mediated by CD4⁺ Th1 cells that secrete proinflammatory cytokines, and spontaneous remission is associated with CD4⁺ T cells that secrete transforming growth factor-beta (TGF-β). Studies of EAE in susceptible rats have provided many important insights into the interactions of T cells and accessory cells that culminate in the induction of the autoimmune response.     

Regulatory T cells in spontaneous autoimmune encephalomyelitis

Summary: Spontaneous experimental autoimmune encephalomyelitis (EAE) develops in 100% of mice harboring a monoclonal myelin basic protein (MBP)-specific CD4⁺αβ T-cell repertoire. Monoclonality of the αβ T-cell repertoire can be achieved by crossing MBP-specific T-cell receptor (TCR) transgenic mice with either RAG^−/− mice or TCR α^−/−/TCR β^−/− double knockout mice. Spontaneous EAE can be prevented by a single administration of purified CD4⁺ splenocytes or thymocytes obtained from wild-type syngeneic mice. The regulatory T cells (T-reg) that protect from spontaneous EAE need not express the CD25 marker, as effective protection can be attained with populations depleted of CD25⁺ T cells. Although the specificity of the regulatory T cells is important for their generation or regulatory function, T cells that protect from spontaneous EAE can have a diverse TCR α and β chain composition. T-reg cells expand poorly in vivo , and appear to be long lived. Finally, precursors for T-reg are present in fetal liver as well as in the bone marrow of aging mice. We propose that protection of healthy individuals from autoimmune diseases involves several layers of regulation, which consist of CD4⁺CD25⁺ regulatory T cells, CD4⁺CD25⁻ T-reg cells, and anti-TCR T cells, with each layer potentially operating at different stages of T-helper cell-mediated immune responses.     

小鼠背根神经节中痒觉神经元的分布特性

目的观察小鼠背根神经节(DRG)中痒觉特异性Mrgpr A3+神经元的分布特征。方法采用遗传学方法将Mrgpr A3+神经元特异性标记强化绿色荧光蛋白(EGFP)和td Tomato;选取3只纯合Mrgpra3EGFP-Cre;ROSA26td Tomato成年转基因小鼠,分离皮肤和背根神经节组织;采用激光扫描共焦成像技术观察Mrgpr A3+神经元的外周神经纤维在小鼠躯体皮肤的投射分布特征;采用双光子成像技术观察Mrgpr A3+神经元在整体背根神经节中的三维空间分布情况。结果脸颊、背部和脚掌皮肤的Mrgpr A3+神经纤维分布密集,粗且长,分布广泛;颈部和腹部皮肤的Mrgpr A3+神经纤维分布稀疏,短且小,呈散点状分布;Mrgpr A3+神经纤维在皮肤的有毛和无毛区域都有投射分布,且不同部位分布特点不同;几乎所有的Mrgpr A3+神经元都为小直径感觉神经元,且在颈、胸、腰、尾段背根神经节中均有分布;颈段、胸段、腰段和尾段的Z轴成像深度分别为350μm、250μm、400μm和200μm;躯体不同部位背根神经节中的Mrgpr A3+神经元的三维空间分布在不同节段存在明显差异。结论小鼠躯体不同部位皮肤的Mrgpr A3+神经纤维的分布特征和整体背根神经节中Mrgpr A3+神经元的三维空间分布特征都存在较大差异,痒觉神经元和末梢分布的差异可能是不同区域存在痒觉生理反应差异的结构基础。     

人参皂苷Rg1在造血干/祖细胞连续移植中对延缓细胞衰老的作用与去乙酰化酶6/核因子-κB信号轴的关系

目的探讨人参皂苷Rg1在造血干/祖细胞(HSC/HPC)连续移植中对抗细胞衰老的作用与去乙酰化酶6/核因子-κB(SIRT6/NF-κB)信号轴的关系。方法免疫磁性分选法分离纯化雄性供体小鼠干细胞抗原阳性(Sca-1+)HSC/HPC,连续移植3代构建HSC/HPC衰老体内模型。60Coγ射线致死剂量辐射雌性受体鼠后分4组,照射对照组;衰老模型组;Rg1治疗衰老组;Rg1预防衰老组。造血祖细胞混合集落(CFU-Mix)培养,细胞周期分析和衰老相关β-半乳糖苷酶(SA-β-Gal)染色分析Rg1体内调控Sca-1+HSC/HPC衰老的作用。实时定量PCR及Western blotting检测衰老调控分子SIRT6、NF-κB mRNA及蛋白的表达。结果连续移植后受体鼠Sca-1+HSC/HPC出现细胞衰老特征,随移植代数的增加,Sca-1+HSC/HPC G0/G1期细胞比例及SA-β-Gal染色阳性率增高,CFU-Mix数量下降。与同代衰老模型组相比,Rg1治疗衰老组及Rg1预防衰老组受体鼠Sca-1+HSC/HPC G0/G1期细胞比例、SA-β-Gal染色阳性率下降,CFU-Mix数量升高;SIRT6 mRNA及蛋白表达上调,NF-κB mRNA及蛋白表达下调;Rg1预防衰老组各指标变化均较Rg1治疗衰老组明显。结论 Rg1可能通过调控SIRT6/NF-κB信号轴发挥其对抗连续移植过程中Sca-1+HSC/HPC衰老的作用。     

Molsidomine ameliorates experimental allergic encephalomyelitis in Lewis rats

Experimental allergic encephalomyelitis (EAE) is an autoimmune CD4⁺ T cell-mediated disease of the central nervous system (CNS). Nitric oxide (NO) plays an important role in preventing the development of EAE. Molsidomine (Mol) is a drug used for the treatment of coronary artery disease. Its therapeutic effects are the consequences of NO formation. In this study, we investigated the effects of Mol on EAE development in myelin basic protein (MBP)-immunized Lewis rats. All rats immunized with MBP developed typical clinical signs of acute EAE. In the EAE rats receiving Mol, the severity of clinical signs and the infiltration of inflammatory cells in CNS were clearly reduced. Furthermore, Mol administration significantly reduced the production of interferon-γ, a Th1 inflammatory cytokine, but increased the production of interleukin-10, a Th2 anti-inflammatory cytokine. Our findings suggest that the administration of the exogenous NO donor Mol is of considerable benefit in limiting the development of EAE and other Th1 cell-mediated inflammatory diseases.     

IL-17 Eliminates the Therapeutic Effects of Myelin Basic Protein-Induced Nasal Tolerance in Experimental Autoimmune Encephalomyelitis by Activating IL-6

Interleukin (IL)-17 is a proinflammatory cytokine primarily secreted by Th17 cells, which are a CD4⁺ T-cell subset. Th17 cells and IL-17 are important in the pathogenesis of multiple sclerosis and in its established animal model, experimental autoimmune encephalomyelitis (EAE). However, it is unclear whether IL-17 contributes to EAE immune tolerance. We used the myelin basic protein (MBP) peptide MBP 68–86 to induce nasal tolerance to EAE, and simultaneously interfered with the tolerance by treatment with different doses of IL-17. We found that IL-17 dramatically interfered with MBP 68–86-induced immune tolerance. IL-17 administration increased IL-6 release, skewing T cell differentiation towards Th17 cells and decreasing the number of Treg cells. This led to an imbalance between Treg cells and Th17 cells and spurred the development of EAE.