人肾小球系膜细胞原代培养方法的改进

背景：肾小球系膜细胞原代培养成功率低,生存时间短,传代次数少。其中分离提取肾小球一直是培养高纯肾小球系膜细胞的最大瓶颈。 目的：建立一种操作简单、成功率高、重复性好的人肾小球系膜细胞原代培养方法。 方法：取自愿水囊引产的胎儿肾,采用肾皮质组织块法合优生选择法培养人肾小球系膜细胞。相差显微镜、透射电镜观察系膜细胞形态,免疫荧光技术鉴定细胞表型,激光共聚焦显微镜观察波形蛋白的表达。 结果与结论：培养的肾小球系膜细胞呈梭形/不规则星形和细长形,胞质中各种细胞器丰富,细胞突起明显,胞膜上有很多微绒毛,细胞表达α-肌动蛋白、肌球蛋白、波形蛋白、结蛋白,而不表达细胞角蛋白、Ⅷ因子,激光共聚焦显微镜下发现系膜细胞波形蛋白表达阳性并具有特征性纤维束。说明培养的系膜细胞符合肾小球系膜细胞的生物学特征。肾皮质组织块法合优生选择法是一种简单、高效的培养人肾小球系膜细胞的方法。     

A comparison of two factors affecting the proliferation of non-neuronal (glial) cells in vitro

Earlier studies have shown that the proliferation of sympathetic non-neuronal cells in vitro can be stimulated either by direct contact with growing neurons or by addition of sonicated neurons of the same type to the culture medium. Several lines of evidence presented herein suggest that intact neurons and neuronal sonicate probably stimulate [³H] thymidine incorporation by disticctly different mechanisms. First, mitogenic factors are present in sonicates of cell types (fibroblasts and non-neuronal cells) which do not stimulate non-neuronal cell proliferation when added asintact cells. Second, neuronal sonicate and intact neurons differ in the types of cells which are responsive no their mitogenic influence. Third, intact neurons do not appear to stimulate non-neuronal cell proliferation by the same mechanism as that of neuronal sonicate.Further similarities between stimulation of non-neuronal cell proliferation in vitro and reactive gliosis in vivo are discussed.     

cAMP类似物对人恶性胶质瘤细胞BT_(325)增殖与分化的影响

目的：观察cAMP类似物8－Br－cAMP对人恶性胶质细胞HT325增殖与分化的影响。方法：在人恶性胶质瘤细胞BT325中加终浓度为10－5mol／L的8－Br－cAMP体外培育24h后,应用酸解DNA及甲绿－派洛宁染色技术在同一标本上鉴别增殖型细胞和分化型细胞。结果：8－Br－cAMP处理组增殖型细胞占67％,分化型细胞占33％;而对照组增殖型细胞占85％,分化型细胞占15％。结论：8－Br－cAMP对BT325细胞有抑制增殖和促进分化效应。     

远志皂苷元促进人神经前体细胞系的体外增殖

Senegenin,an effective component of Polygala tenuifolia root extract,promotes proliferation and differentiation of neural progenitor cells in the hippocampus.However,the effects of senegenin on mesencephalon-derived neural progenitor cells remain poorly understood.Cells from a ventral mesencephalon neural progenitor cell line(ReNcell VM) were utilized as models for pharmaceutical screening.The effects of various senegenin concentrations on cell proliferation were analyzed,demonstrating that high senegenin concentrations(5,10,50,and 100 μmo/L),particularly 50 μmol/L,significantly promoted proliferation of ReNcell VM cells.In the mitogen-activated protein kinase signal transduction pathway,senegenin significantly increased phosphorylation levels of extracellular signal-regulated kinases.Moreover,cell proliferation was suppressed by extracellular signal-regulated kinase inhibitors.Results suggested that senegenin contributed to in vitro proliferation of human neural progenitor cells by upregulating phosphorylation of extracellular signal-regulated kinase.     

Identification of growth factors that promote long-term proliferation of olfactory ensheathing cells and modulate their antigenic phenotype

Olfactory ensheathing cells can develop into distinct subtypes in culture after incubation in serum-free medium conditioned by astrocytes, which have Schwann cell-like and astrocyte-like properties. It has not been possible so far to modulate and grow large numbers of these olfactory ensheathing cell subtypes. In this study, we have shown that astrocyte-conditioned medium, although promoting differentiation of the two olfactory ensheathing cell types, is growth-restrictive after 14 days, probably due to the upregulation of p16 and p27. Growth arrest can be overridden and cells maintained for a further 11 weeks, by a mitogen mix of fibroblast growth factor 2, forskolin, and heregulin (olfactory mitogen medium) combined with astrocyte-conditioned medium. In the absence of astrocyte-conditioned medium, combinations of the same factors can also override growth arrest but to a lesser extent. Olfactory mitogen medium combined with astrocyte-conditioned medium upregulates O4 and low-affinity nerve growth factor receptor expression on olfactory ensheathing cells, leading to a 100% Schwann cell--like phenotype. If cells are maintained in olfactory mitogen medium alone, or if they are treated with forskolin or fibroblast growth factor 2 diluted in serum-free medium, O4 and low-affinity nerve growth factor receptor expression remains at 100%, but there is also an increase in expression of E-NCAM, the astrocyte-like marker. Medium containing serum also overrides growth arrest, but for only 4 weeks, during which time most differentiation-specific markers disappear. These studies have allowed us to define conditions to modulate the olfactory ensheathing cell phenotype.     

CNTF对神经干细胞体外增殖的影响

目的 探讨睫状神经营养因子(ciliary neurotrophic factor,CNTF)对神经干细胞体外增殖的影响.方法 原代培养新生SD大鼠的室下区神经干细胞并鉴定,经传代纯化后,用不同浓度CNTF (0.1、0.5、1、5、10、20、30 ng/ml)处理5～7 d,MTT实验检测神经干细胞增殖活性.不完全...     

EGF infusion stimulates the proliferation and migration of embryonic progenitor cells transplanted in the adult rat striatum

Immature progenitor cells (generated by in vitro propagation) may provide a useful alternative to primary cells (from dissected embryonic tissue) for transplantation if their migratory and proliferative and differentiation properties can be controlled and directed in vivo. In this study E15 murine EGF-responsive progenitor cells were transplanted to the striatum of adult rats. Simultaneously, these animals received continuous infusion of either epidermal growth factor (EGF) or vehicle, to the lateral ventricle, for 8 days. In animals that received EGF, the transplanted progenitors migrated toward the lateral ventricle and proliferated, as evidenced by bromodeoxyuridine incorporation. Progenitor cells transplanted to rats that received vehicle infusions showed neither of these responses. In all animals, transplanted progenitors expressed an immature astrocyte or oligodendrocyte phenotype, the majority of cells being astrocytes. We conclude that EGF stimulates the migration and proliferation of murine progenitor cells in vivo, either directly or indirectly, but does not influence their phenotypic differentiation.     

晚期糖基化终末产物对大鼠雪旺细胞增殖和凋亡影响的体外研究

目的 探讨晚期糖基化终末产物(AGEs)对大鼠雪旺细胞(RSC-96)增殖和凋亡的影响及其机制.方法 (1)体外培养RSC-96细胞(大鼠源性雪旺细胞系),细胞培养24 h后用AGEs处理RSC-96细胞造模;(2)细胞增殖实验:细胞培养24 h后,用0、100、250、500、1 000 μg/mL浓度的AGEs处理...     

Influence of the visual cortex on responses of retinal ganglion cells in the rat

The objective of the present investigation was to answer the following question: Does the visual cortex affect the neuronal firing of retinal ganglion cells in the rat? To test this hypothesis, the visual cortex was inactivated by a reversible cryoblockade. Action potentials of a ganglion cell were recorded from its axon at the optic tract level prior to, during, and following cortical blockade. The results indicated that indeed the visual cortex influenced the retinal output since its inactivation led to a modification of the firing pattern evoked in response to a flash of light. In most cases the modification was an increase of the bursting pattern of the evoked discharges. By contrast cooling nonvisual areas failed to modify ganglion cells' discharge. A comparison between cortico-geniculate and cortico-retinal feedback loops seems to suggest that the first path is involved mostly with the spatial organization of center-surround receptive fields, whereas the second path is associated with temporal aspects of the retinal responses in the rat.     

Accessory function of human glioma cells for the induction of CD3-mediated T cell proliferation: a potential role of glial cells in T cell activation in the central nervous system

Accessory function of human glial cells for the induction of anti-CD3 antibody-mediated proliferation of T cells was investigated by using seven glioma cell lines. Three of them were found to function as accessory cells and one of them, U118, was used for further analysis. U118 cells showed the cell-contact-mediated accessory function for T cell proliferation. It was found that protein synthesis was required to reveal this function, suggesting that some surface molecules are synthesized and expressed on U118 cells during interaction with T cells to mediate effective signals in T cells. Intercellular adhesion molecule-1 seemed to be one of such inducible molecules and was shown to contribute to effective accessory cell-T cell interaction, but necessity of other molecule(s) was also suggested.     

Influence of thyroid hormone on some electrophysiological properties of developing rat skeletal muscle cells in culture

The metabolic activity in the brains of adult spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY), and Wistar (W) rats was assessed before and after fasting using cytochrome oxidase (COX) histochemistry. Before fasting, metabolic activity in the paraventricular (PVH) and supraoptic (SON) nuclei of the hypothalamus in SHR was greater than in control rats. Fasting elicited a decrease in arterial pressure (AP) in SHR and WKY; in SHR the decrease in AP was accompanied by a decrease of metabolic activity in the PVH and SON. The findings of this study support the hypothesis that the PVH and SON are involved in the hypertension and in the increased levels of sympathetic nervous activity and vasopressin production known to occur in SHR. In addition, the PVH and possibly the SON may be involved in the suppression of sympathetic nervous system activity and the lowering of arterial pressure which are associated with fasting.     

逆转录酶抑制剂AZT对胶质瘤干细胞端粒酶活性和细胞增殖的影响

目的 探讨逆转录酶抑制剂3-叠氮-3-脱氧胸腺核苷(AZT)对脑胶质瘤干细胞增殖的影响及相关机制.方法 原代分离培养脑胶质瘤干细胞和脑胶质瘤细胞并鉴定,两种细胞同时设立为实验组(0.125 mot/L、0.250 mol/L、0.500 mol/L AZT)和对照组.MTT法检测AZT对两种细胞增殖的影响;流式细胞仪检测细胞凋亡和细胞周期的改变;端粒重复序列扩增技术-酶联免疫吸附试验(TRAP-ELISA)检测端粒酶活性的变化.结果 AZT对两组细胞的生长抑制呈浓度-时间依赖性(均P<0.05),在同一浓度和时间点,AZT对脑胶质瘤干细胞的生长抑制作用弱于脑胶质瘤细胞(均P<0.05).0.250 mol/L、0.500 mol/L AZT作用72 h后,脑胶质瘤干细胞的凋亡率分别为(4.21±1.53)%、(10.60±0.38)%,而脑胶质瘤细胞的凋亡率分别为(6.75±1.25)%,(14.30±2,59)%,明显高于胶质瘤干细胞(均P<0.05).AZT对两组细胞端粒酶活性的抑制呈浓度-时间依赖性(均P<0.05),且在同一浓度和时间点对脑胶质瘤细胞的作用明显强于脑胶质瘤干细胞(均P<0.05).结论 逆转录酶抑制剂AZT对脑胶质瘤干细胞和脑胶质瘤细胞均有明显的生长抑制作用,可能机制是通过抑制端粒酶活性、调控细胞周期和诱导细胞凋亡实现.脑胶质瘤干细胞的耐药性强于胶质瘤细胞,其机制有待进一步研究.     

稳定增殖神经干细胞的实验研究

目的探讨稳定、可靠建立神经干细胞体外增殖的方法,并对增殖培养的细胞进行鉴定。方法获取胚胎大鼠的脑组织,通过加入神经生长因子和采用保留细胞联系技术操作,使脑组织中的神经干细胞在体外克隆增殖并稳定传代。以免疫荧光方法对增殖克隆的神经干细胞球进行鉴定。结果神经干细胞不断增殖形成神经干细胞球且神经干细胞能快速稳定传代增殖。培养的细胞为神经干细胞特异性巢蛋白(nestin)染色阳性细胞。结论在神经生长因子的作用下,利用保留细胞联系技术操作,神经干细胞可以在体外快速、稳定克隆增殖并传代。     

Time-dependent influence of the somatostatin analogue octreotide on the proliferation of rat astrocytes and glioma cells

The somatostatin receptor subtype sst₂ was visualized by immunostaining on cultivated rat astrocytes and C6 rat glioma cells. Octreotide, a metabolically stable sst₂ agonist reduced [³H]thymidine incorporation into DNA of both cell types dose-dependently only after short-time application (2–5 h), after prolonged incubation (>12 h) no antiproliferative effect was measurable. We conclude that sst₂ receptors may be desensitized. Thus, desensitization might hinder application of octreotide to reduce glial tumour growth.     

NOV对人神经干细胞增殖和分化的影响

目的 探讨NOV蛋白对人神经干细胞(HNSCs)增殖和分化的影响。方法 NOV基因重组质粒转染COS—7细胞，收集COS-7细胞和NOV基因修饰COS—7细胞的条件培养液(COS—CM和NOV—CM)，作用于培养的HNSCs，^3H—TdR掺入液闪检测HNSCs的增殖，免疫细胞化学和流式细胞仪检测HNSCs的分化。结果 (1)NOV—CM能明显促进HNSCs对^3H—TdR的摄入，说明NOV—CM能明显促进HNSCs的增殖，另外NOV—CM的促细胞增殖作用具有一定的量效关系；(2)免疫细胞化学和流式细胞仪结果显示，NOV—CM促进HNSCs向神经元方向分化。结论 NOV蛋白可能具有促进HNSCs增殖和分化的作用。     

星形胶质细胞源性因子对神经干细胞分化的实验研究

目的探讨星形胶质细胞源性因子对神经干细胞分化的影响。方法分离和培养新生大鼠脑组织的神经干细胞;采用差速贴壁法和振荡法分离纯化星形胶质细胞,用免疫细胞化学染色法,胶质纤维酸性蛋白(GFAP)标记星形胶质细胞,进行细胞的纯度鉴定;将星形胶质细胞和神经干细胞在互不接触的情况下进行共培养,免疫荧光法观察神经干细胞分化后神经元特异性烯醇化酶(NSE)、GFAP和酪氨酸羟化酶(TH)的表达。结果纯化的星形胶质细胞GFAP抗体标记阳性,细胞纯度达98%;星形胶质细胞与神经干细胞共培养时,神经干细胞贴壁分化加快,NSE阳性细胞及TH阳性细胞明显多于对照组(P<0·05)。结论星形胶质细胞源性因子可快速诱导神经干细胞向神经元细胞、包括多巴胺神经元细胞分化,提示星形胶质细胞支持神经元发生。     

低氧条件下星形胶质细胞对神经干细胞增殖的影响

目的 探讨体外低氧条件下星形胶质细胞对海马神经干细胞( NSCs)增殖的影响.方法 将分离出的新生小鼠海马NSCs分别与低氧条件星形胶质细胞培养液(低氧组)、低氧条件星形胶质细胞培养液+磷脂酰肌醇-3激酶/蛋白激酶B( PI3K/Akt)通路拮抗剂LY294002(拮抗剂组)以及常氧条件星形胶质细胞培养液(对照组)共培养;分别于培养24 h、48 h、72 h应用四甲基偶氮唑盐(MTT)法检测各组NSCs活性;应用Western Blot法检测各组NSCs磷酸化Akt (p-Akt)和磷酸化糖原合成酶激酶(p-GSK)-3β含量.结果 (1)共培养24h、48 h及72 h,低氧组NSCs活性的光密度(OD)值分别为0.292±0.006、0.382 ±0.005、0.649±0.028,拮抗组分别为0.197±0.003、0.255±0.005、0.325±0.012,对照组分别为0.107±0.006、0.198±0.008、0.254±0.006;低氧组各时间点NSCs活性显著高于拮抗剂组和对照组(均P＜0.05);(2)共培养24h、48 h和72 h,低氧组NSCs p-Akt和p-GSK-3β的吸光度(A)值分别为0.52、0.71、0.86及0.49、0.65、0.82;拮抗剂组分别为0.39、0.42、0.61及0.31、0.35、0.40;对照组分别为0.34、0.38、0.39及0.28、0.31、0.35;低氧组各时间点p-Akt及p-GSK-3β含量显著高于拮抗剂组和对照组(均P＜0.05).结论 缺氧刺激可激活星形胶质细胞PI3K/Akt信号通路,促进NSCs增殖.