钙敏感受体通过PI3K信号通路介导缺氧诱导的大鼠肺动脉平滑肌细胞增殖

目的:探讨钙敏感受体(CaSR)在缺氧诱导的大鼠肺动脉平滑肌细胞(PASMCs)增殖中的信号转导途径。方法:Ⅱ型胶原酶消化法提取、培养大鼠PASMCs。通过缺氧培养箱内(93%N₂、2%O₂、5%CO₂)培养24h的方法复制细胞缺氧模型。应用Western blotting分析不同处理情况下细胞周期素D1(cyclin D1)及磷酸化的蛋白激酶B(p-Akt)在PASMCs中的表达;采用流式细胞术检测不同处理因素对细胞增殖周期及增殖指数的影响,应用BrdU掺入法分析不同处理因素对细胞DNA合成的影响。结果:缺氧引起PASMCs的cyclin D1及p-Akt表达水平上调,增加BrdU掺入量、S期细胞数量和细胞增殖指数,GdCl₃能够放大缺氧的作用,但上述效应可以被LY294002抑制。结论:CaSR通过PI3K/Akt信号通路参与缺氧诱导的大鼠PASMCs增殖。     

自然衰老小鼠室管膜下区神经干细胞增龄性改变

目的 探讨自然衰老小鼠室管膜下区（SVZ）神经干细胞（NSCs）在衰老过程中生物学特点的改变。 方法 2月龄、28月龄小鼠,各10只,体外分离、培养、鉴定NSCs,5-溴-2-脱氧尿苷（BrdU）染色比较两组细胞的增殖水平;β-半乳糖苷酶（SA-β-Gal）染色检测细胞衰老水平;细胞内活性氧（ROS）试剂盒检测细胞ROS表达水平;Western blotting 检测细胞周期蛋白D1（cyclin D1）、p16及p19蛋白表达水平;体内用巢蛋白（Nestin）/性别决定基因高迁移率组蛋白-2（Sox2）检测两组小鼠SVZ区厚度的差异。 结果 年轻、年老小鼠的神经干细胞能表达Nestin及Sox2,符合神经干细胞的表达特性。与年轻小鼠相比,年老小鼠神经干细胞的BrdU阳性细胞比例显著下降,SA-β-Gal阳性细胞百分比显著升高,细胞活性氧水平显著升高,SVZ区厚度明显变薄,差异具有统计学意义（P<0.05）。在分子水平上表现为cyclin D1蛋白表达水平明显下降,p16、p19蛋白表达水平显著升高。 结论 年老小鼠NSCs出现增龄性变化,这些变化可能在大脑衰老中起重要作用。     

rhG-CSF促进体外培养神经干细胞的增殖

目的 探讨重组人粒细胞集落刺激因子(rhG-CSF)对小鼠胚胎神经干细胞(NSCs)增殖的作用.方法 分离培养小鼠NSCs,通过向培养基中添加G-CSF(10、30、60、100和200μg/L),四甲基偶氮唑蓝(MTT)比色法以及5-溴-2′-脱氧尿苷(BrdU) 免疫荧光染色标记检测NSCs的增殖.免疫印迹法检测NSCs增殖时信号转导与转录激活因子3(STAT3)和磷酸化信号转导和转录激活因子3(p-STAT3)的表达.结果 神经干细胞表达G-CSF受体,细胞活性随G-CSF的浓度不同表现出一定的剂量依赖性,当G-CSF的浓度为100μg/L时细胞的活性最高.G-CSF处理组的细胞增殖活性明显高于对照组.进一步证实,在G-CSF处理后,p-STAT3的表达则在5 min开始升高,30 min 到达峰值,之后开始下降,至75 min 接近正常水平.加入G-CSF受体的抗体后,p-STAT3的活化现象消失,并且细胞的增殖也明显低于G-CSF作用组,和对照组接近.结论 rhG-CSF可促进小鼠NSCs的增殖,其作用机制可能是通过细胞表面的G-CSF-R促进STAT3的活化.G-CSF可能作用于内源性NSCs的活化和增殖.     

The promotion of neural progenitor cells proliferation by aligned and randomly oriented collagen nanofibers through β1 integrin/MAPK signaling pathway

In regenerative medicine, accumulating evidence demonstrates that the property of substrates monitors neural stem cells behavior. However, how stem cells sense and interpret biochemical and topographical cues remains elusive. This study aimed to explore the mechanism how nanofibrous scaffold modulated stem cells behavior. Spinal cord derived neural progenitor cells (NPCs) were cultured on electrospun aligned and randomly oriented collagen nanofibrous scaffolds. A 30% increase in proliferation and an elevation of BrdU incorporation were observed in NPCs on collagen nanofibers, compared to that on collagen-coated surface. In particular, NPCs expanded faster on aligned nanofibers in comparison with that on randomly oriented nanofibers. Moreover, an alteration in cell cycle progression with a reduced percentage of cells in G0/G1 phase and increased cell proliferation index (S phase plus G2/M phase) was also detected in NPCs cultured on collagen nanofibers. Incubating NPCs with anti-β1 integrin antibody or U1026 (an inhibitor of mitogen-activated protein kinase kinase, MEK) eliminated the altered cell cycle dynamics and BrdU incorporation induced by collagen nanofibers. In addition, cyclin D1 and cyclin dependent kinase 2 (CDK2), downstream genes of β1 integrin/mitogen-activated protein kinase (MAPK) pathway that control G1/S phase transition, were correspondingly regulated by nanofibers. Collectively, these data suggested that the property of substrate modulated NPCs proliferation by promoting cell cycle through β1 integrin/MAPK pathway. Our findings provide a better understanding of the interaction between NPCs and the substrate and therefore will pave way for regenerative medicine.     

不同低氧浓度对神经干细胞增殖与凋亡的影响

目的 探讨不同的低氧浓度对离体培养胎鼠脑皮质神经干细胞( NSCs)的增殖与凋亡的影响.方法 体外不同低氧条件下(3％ O2、5％ O2、10％ O2)培养胎鼠皮质来源的细胞,实验组分别分为低氧1、3、5d组,常氧组为对照.免疫细胞化学染色鉴定NSCs,BrdU掺入法检测实验组NSCs的增殖:流式细胞仪检测实验组NSC...     

大鼠穹隆海马伞切割对海马伞内神经干细胞再生的影响

目的 检测损伤的穹隆海马伞内自体神经干细胞的再生情况.方法 SD大鼠7只,切割左侧穹隆海马伞,术后腹腔注射5-溴脱氧尿嘧啶核苷(BrdU).术后7d取脑组织制备冷冻切片,行Nestin、BrdU免疫荧光检测;取穹隆海马伞组织进行神经干细胞的体外分离培养,对获得的细胞球进行增殖性、胚胎性以及多分化潜能的检测.结果 切割...     

Notch和Wnt信号通路对神经干细胞增殖分化的影响

Notch和Wnt信号通路是调节神经干细胞(neural stem cells,NSCs)增殖、分化的重要通路,Notch信号通路的靶基因Hes1、Hes5及HES相关蛋白等分化抑制信号,通过旁侧抑制机制阻止NSCs的分化,并促进其自我更新;通过NICD与CSL DNA结合蛋白的直接结合,形成GFAP的转录激活复合物,上调GFAP的表达,从而促进NSCs向星形胶质细胞的分化。Wnt信号通过Wnt/β-catenin信号通路对细胞周期素D1和D2的转录调节,调控NSCs细胞周期的进程,使其量增殖;然而,过表达的Wnt3a和Wnt7a蛋白能够抑制NSCs的增殖,促进NSCs向神经元方向分化。     

JNK信号转导通路对神经干细胞增殖的影响

目的 探讨JNK信号转导通路对新生大鼠海马神经干细胞增殖的影响.方法 采用无血清培养技术体外培养新生大鼠海马神经干细胞,传至第4代,大多数克隆球直径约为200 μm时,进行单细胞克隆培养,单细胞克隆形成的克隆球进行传代.将培养细胞随机分为2组,对照组采用神经干细胞培养液进行培养,实验组中在神经干细胞培养液的基础上添加不...     

Comparative analysis of the frequency and distribution of stem and progenitor cells in the adult mouse brain

The neurosphere assay can detect and expand neural stem cells (NSCs) and progenitor cells, but it cannot discriminate between these two populations. Given two assays have purported to overcome this shortfall, we performed a comparative analysis of the distribution and frequency of NSCs and progenitor cells detected in 400 mum coronal segments along the ventricular neuraxis of the adult mouse brain using the neurosphere assay, the neural colony forming cell assay (N-CFCA), and label-retaining cell (LRC) approach. We observed a large variation in the number of progenitor/stem cells detected in serial sections along the neuraxis, with the number of neurosphere-forming cells detected in individual 400 mum sections varying from a minimum of eight to a maximum of 891 depending upon the rostral-caudal coordinate assayed. Moreover, the greatest variability occurred in the rostral portion of the lateral ventricles, thereby explaining the large variation in neurosphere frequency previously reported. Whereas the overall number of neurospheres (3730 +/- 276) or colonies (4275 +/- 124) we detected along the neuraxis did not differ significantly, LRC numbers were significantly reduced (1186 +/- 188, 7 month chase) in comparison to both total colonies and neurospheres. Moreover, approximately two orders of magnitude fewer NSC-derived colonies (50 +/- 10) were detected using the N-CFCA as compared to LRCs. Given only 5% of the LRCs are cycling (BrdU+/Ki-67+) or competent to divide (BrdU+/Mcm-2+), and proliferate upon transfer to culture, it is unclear whether this technique selectively detects endogenous NSCs. Overall, caution should be taken with the interpretation and employment of all these techniques.     

Ndrg2对培养大鼠神经干细胞增殖和分化的影响

目的:检测Ndrg2在培养大鼠神经干细胞(NSCs)中的表达情况,探索改变Ndrg2表达对NSCs增殖和分化的影响。方法:用免疫细胞化学染色和Western Blot法检测Ndrg2在培养NSCs中的表达;利用AAV-Ndrg2过表达病毒及LV-Ndrg2-RNAi干扰病毒感染大鼠NSCs后,通过BrdU掺入实验观察干预Ndrg2表达后NSCs增殖的变化,利用神经元标记物Tuj1和星型胶质细胞标记物GFAP的免疫细胞化学染色分析NSCs的分化情况。结果:Ndrg2高表达于大鼠NSCs中;与对照组(感染病毒空载体)相比,上调Ndrg2表达可显著增加BrdU~+和Tuj1~+细胞数(P0.05),但GFAP~+细胞数目无明显变化(P0.05);相反,下调Ndrg2表达可减少BrdU~+细胞数(P0.05),但不影响Tuj1~+和GFAP~+细胞数(P0.05)。结论:Ndrg2表达于大鼠NSCs,它可正性调控NSCs的增殖并促进NSCs向神经元分化。     

雌二醇对大鼠海马神经干细胞增殖的影响

目的观察雌二醇对大鼠海马神经干细胞增殖的影响及其作用机制。方法取20只孕17d的SD大鼠海马组织,进行神经干细胞的培养和增殖,添加不同浓度的雌二醇(E2)。通过5-溴脱氧尿嘧啶核苷(BrdU)免疫荧光、MTT方法检测神经干细胞的增殖;通过免疫荧光技术和巢蛋白(Nestin)双标观察神经干细胞球中雌激素受体ERα和ERβ的表达情况。结果 BrdU与MTT检测结果显示,随着雌二醇浓度从10-10mol/L增加至10-8mol/L,神经干细胞数量逐渐增加。雌二醇在10-8mol/L时,细胞增殖数目最多而且细胞活力最好。随着雌二醇浓度进一步增加至10-6mol/L,神经干细胞增殖能力逐渐下降。免疫荧光技术检测显示神经干细胞均表达ERα和ERβ两种受体。结论在一定浓度范围内,雌二醇能促进海马神经干细胞的增殖,并可能是通过ERα和ERβ介导促进神经干细胞增殖。     

缺氧性神经干细胞钙离子浓度变化和葡萄糖保护作用的研究

为了从钙离子角度探讨葡萄糖对神经干细胞缺氧性损伤保护作用机制,本实验将三气培养箱的氧气浓度调至5%制备缺氧环境。分别在缺氧前后于无血清培养基中加入不同剂量的葡萄糖,同时设常氧常糖正常对照。通过MTT法检测干细胞的存活和增殖情况,用激光扫描共聚焦显微镜和Fluo-3荧光探针标记技术检测神经干细胞内钙离子浓度。结果显示,缺氧前加入30mmol/L葡萄糖,神经干细胞的存活率和增殖率较常糖缺氧组明显增高,其胞内钙离子浓度显著低于常糖缺氧组。缺氧后再加入葡萄糖时,其保护作用不明显。本实验提示:缺氧前给予足够浓度的葡萄糖可通过抑制胞内钙超载机制对神经干细胞损伤起到一定的保护作用。     

嗅鞘细胞对神经干细胞增殖与分化的影响

观察嗅鞘细胞(OECs)对神经干细胞(NSCs)增殖与分化的影响。取孕14d的SD胚鼠嗅球和腹侧中脑组织,分为OECs+NSCs共培养组和NSCs单独培养组进行培养。用p75免疫组化法,p75、BrdU/nestin、GFAP、NF(神经原纤维)和p75/nestin免疫荧光法分别鉴定OECs和NSCs并观察其增殖与分化。在培养7d时,绝大多数OECs呈梭形且发出2~3个突起,少量呈扁平椭圆形,两者均呈p75阳性。在7d时,单独培养的NSCs表现为典型的神经球悬浮生长,呈BrdU/nestin阳性;14d后偶见神经球贴壁分化,球中的少数细胞呈绿色荧光标记的NF阳性,大部分呈GFAP阳性。在5d时,OECs+NSCs共培养组形成神经球;10d时球体积不断增大,球心透亮度良好;12d后神经球的体积不再增大,开始贴附在OECs上生长,可见神经球向四周伸出突起并开始分化;14d时NSCs紧贴OECs生长并同OECs广泛交织在一起,可见NSCs和OECs分别呈nestin和p75阳性,NSCs中的大部分呈NF阳性,小部分为GFAP阳性。NSCs单独培养组和OECs+NSCs共培养组中NF阳性细胞率分别为47.2%和69.5%,前者明显少于后者(P<0.05)。以上研究结果提示OECs有促进NSCs增殖和诱导其分化的作用。     

沉默HIF-1α基因表达对大鼠肝癌细胞增殖的影响

 目的： 探讨沉默缺氧诱导因子　1α（HIF-1α）基因表达对缺氧状态下肝癌细胞增殖的影响。方法：选用大鼠CBRH-7919肝癌细胞株作为研究对象,利用氯化钴（CoCl2）建立缺氧模型。制备3组特异性较强的HIF-1α siRNA-脂质体复合物,转染于肝癌细胞。利用real-time RT-PCR、Western blotting等方法分别检测肝癌细胞HIF-1α、血管内皮生长因子(VEGF)、p21和cyclin D1在mRNA和（或）蛋白水平的表达。MTT及BrdU 掺入实验检测细胞增殖的变化。结果：缺氧条件下,肝癌细胞的HIF-1α和VEGF mRNA及蛋白表达显著增多（P<0.05）。HIF-1α基因表达沉默后,HIF-1α、VEGF及cyclin D1 mRNA和（或）蛋白表达明显减少（P<0.05）,p21蛋白表达明显增加（P<0.05）。HIF-1α siRNA转染组的BrdU阳性细胞比例明显少于对照组(P<0.05)。结论：沉默HIF-1α基因表达对缺氧状态下肝癌细胞的增殖有显著抑制作用。     

EphB2 induces proliferation and promotes a neuronal fate in adult subventricular neural precursor cells

The subventricular germinal zone (SVZ) retains an active population of stem cells and neural precursor cells throughout adulthood. EphrinB signaling mediates angiogenesis and vasculogenesis in the developing and adult brain. Recent studies indicate that molecules involved in angiogenesis often influence neurogenesis as well. However, little work has been done considering a role for EphB2/EphrinB in adult neural precursor cells. We therefore examined whether the EphB2 receptor tyrosine kinase could directly effect proliferation of SVZ neural precursors and/or direct the cell fate of SVZ cells in vitro. Here, we found that clustered EphB2 increased bromodeoxyuridine (BrdU) incorporation and proliferation of SVZ neurosphere cultures. Immunostaining and RT-PCR analysis for beta-tubulin III (Tuj1) and GFAP indicated 4-day treatment with EphB2 promoted a neuronal phenotype, suggesting that the EphB2 receptor might also direct SVZ cell fate. EphB2 transiently down-regulated SVZ cell mRNA of Notch1 and Zic1, genes that regulate neurogenesis and neuronal differentiation. Notch1 has been implicated in apoptosis of neural precursors, however, a cell viability assay revealed no statistical difference between EphB2-treated and control cultures. When SVZ neurospheres were cultured upon Matrigel, EphB2 attenuated radial migration of SVZ cells in vitro. These results demonstrate that EphB2/EphrinB signaling directly induces SVZ proliferation, decreases migration, and promotes a neuronal fate of SVZ neural precursors independent of cell survival.     

成年小鼠神经干细胞体外培养、鉴定及分化

目的:建立一种简便易行的从成年小鼠脑组织分离、培养和鉴定神经干细胞(neural stem cells,NSCs)的方法,为相关研究提供新的研究手段。方法:采用机械分离和酶消化结合方法分离成年昆明种小鼠的脑组织,用无血清培养基悬浮培养;倒置相差显微镜观察细胞形态;MTT法(四甲基偶氮唑盐微量酶反应比色法)观察NSCs的自我增殖能力;免疫荧光细胞化学技术检测NSCs标志物巢蛋白(Nestin)的表达;多聚赖氨酸铺板和撤除培养基中FGF和EGF的条件下,给予5%血清和1μm维甲酸诱导NSCs分化,免疫荧光细胞化学技术检测GFAP(标记胶质细胞),β-tubulin Ⅲ(标记神经元)蛋白的表达来测定NSCs的分化能力。结果:从成年小鼠脑组织分离的细胞,在无血清培养液中可形成神经球,并可在体外扩增和连续传代,免疫荧光细胞化学表明神经球Nestin阳性表达,在给予血清和维甲酸条件下神经球可表达GFAP和β-tubulin Ⅲ。结论:本研究成功建立了体外培养成年小鼠脑组织分离和培养NSCs的方法,培养的NSCs具有自我更新、增殖及多向分化潜能。此方法是一个稳定、简便易行的方法,可广泛用于神经干细胞相关的基础和应用研究。     

Correlative association between circadian expression of mousePer2 gene and the proliferation of the neural stem cells

We examined the circadian expression of mousePeriod (mPer) genes (mPer1 and mPer2) and the proliferation of the neural stem cells in vitro. The neural stem cells from the ganglionic eminence of embryonic mice were expanded by the neurosphere method and then treated with epidermal growth factor (EGF) to stimulate their mitotic activity. The time courses of the proliferation were examined by WST-8 assay and bromodeoxyuridine (BrdU) incorporation assay and the expression of mPer1 and mPer2 genes was examined by RT-PCR and immunocytochemistry. We have found that EGF treatment elicited the circadian change in both the increase in viable cell number and DNA synthesis activity of the neural stem cells. Also, the gene expression of mPer2, but not mPer1, changed rhythmically with a period of 24 h and correlated negatively with the DNA synthesis activity rhythm. Furthermore, the treatment with an antisense oligonucleotide against mPer2 increased the DNA synthesis activity of the neural stem cells. These results suggest that mPer2 might periodically suppress the proliferation of neural stem cells.     

Characterization of neural stem cells in the dorsal vagal complex of adult rat by in vivo proliferation labeling and in vitro neurosphere assay

The dorsal vagal complex, located in the brainstem, is the major integrative center of the autonomic nervous system. By combining in vivo bromodeoxyuridine incorporation and phenotypic immunolabeling, we have previously reported that neurogenesis occurs in the adult rat dorsal vagal complex [Bauer S, Hay M, Amilhon B, Jean A, Moyse E (2005) In vivo neurogenesis in the dorsal vagal complex of the adult rat brainstem. Neuroscience 130:75-90.]. In the present study we asked whether adult dorsal vagal complex contains proliferative and/or neural stem cells. Using Ki-67 immunolabeling and cyclin D1 Western blot, we showed intrinsic cell proliferation in the dorsal vagal complex and its stimulation by vagotomy. Detailed time-course analysis revealed that vagotomy-induced proliferation in the dorsal vagal complex peaked three days after lesion. In order to directly assess the presence of intrinsic stem cells, primary cell cultures from adult rat dorsal vagal complex were performed in the presence of epidermal growth factor and basic fibroblast growth factor (neurosphere assay). A discrete subpopulation of dorsal vagal complex cells proliferated as neurospheres, self-renewed when passaged, and differentiated into neurons, astrocytes and oligodendrocytes. Proliferation and neuron-differentiating potentials of dorsal vagal complex neurospheres were both lower than those of subventricular zone neurospheres from the same rats. The relationship between in vitro neurosphere-forming cells of dorsal vagal complex and in vivo dorsal vagal complex neurogenesis is discussed and remains to be directly addressed. The present data demonstrate the occurrence of neural stem cells in the dorsal vagal complex of adult rat brain.     

音猬因子对神经干细胞增殖与分化的影响

目的 探讨音猬因子(SHH)对神经干细胞(NSCs)增殖与分化的影响. 方法从大鼠胚胎端脑皮质及海马分离培养的NSCs分为SHH组、RA组和PBS组.分别用SHH、维甲酸(RA)及PBS条件培养液处理后,通过免疫细胞化学等方法检测NSCs的增殖及分化情况.结果体外培养的NSCs能快速增殖并形成神经球,其中的细胞均表达NSCs特异性标志物nestin.BrdU作用于NSCs 3 h后,SHH组的BrdU阳性率明显高于RA组和PBS组,而RA组和PBS组之间无显著差异.当NSCs在分化条件培养液中培养7d后,SHH组和RA组中的βⅢ-tubulin阳性率及Rip阳性率显著高于PBS组,SHH组又显著高于RA组和PBS组.结论 SHH可以促进NSCs增殖及其向神经元和少突胶质细胞的分化.     

18α-甘草次酸通过抗氧化作用促进成年小鼠室管膜下区神经干细胞增殖

目的 探讨18 α-甘草次酸(18α-GA)对成年小鼠室管膜下区(SVZ)神经干细胞(NSCs)增殖的影响及其机制.方法 100只6月龄BALB/c小鼠随机平均分为18α-GA组(腹腔注射18α-GA 40 mg/kg两个月,以DMSO为溶解介质)和DMSO对照组(腹腔注射含等体积DMSO溶解介质的PBS溶液),每组5...