体外培养Schwann细胞缺氧模型的建立

目的:建立体外培养Schwann细胞的缺氧模型。方法:取体外培养Schwann细胞,置于通入体积分数为5%CO2和95%N2的有机玻璃盒中继续缺氧培养10、15和20min,用H-E染色观察细胞形态,MTT比色试验检测细胞的活力。结果:缺氧10min组细胞形态及活力与正常组无明显差异;而缺氧15min组的细胞突起回缩,活力为缺氧前的66·3%;缺氧20min组的细胞多数死亡,活力仅为缺氧前的20·6%。结论:本方法可以成功建立有效、简便、易行的Schwann细胞缺氧模型。     

The effect of synthetic oxygen carrier-enriched fibrin hydrogel on Schwann cells under hypoxia condition in vitro

Schwann cell (SC), which plays a key role in peripheral nerve regeneration, is one of the most classic supportive cells in neural tissue engineering. However, the biological activity of SCs seeded in nerve scaffolds decays subsequently due to local hypoxia induced by ischemia. Thus, we aimed to investigate whether a synthetic oxygen carrier-enriched fibrin gel would provide a sustained oxygen release to cultured SCs in vitro for overcoming a temporary (48 h) oxygen deprivation. In this study, perfluorotributylamine (PFTBA)-based oxygen carrying fibrin gel was prepared to provide oxygen for SCs under normoxic or hypoxic conditions. The dissolved oxygen within the culture media was measured by a blood-gas analyzer to quantify the time course of oxygen release from the PFTBA-enriched fibrin gel. SCs were cultured in the presence or absence of PFTBA-enriched fibrin gel under normoxic or hypoxic conditions. The tolerance of SCs to hypoxia was examined by a cell apoptosis assay. The growth of cells was characterized using S-100 staining and a CCK-8 assay. The migration of cells was examined using a Transwell chamber. The mRNA of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial cell derived neurotrophic factor (GDNF), neural cell adhesion molecule (N-CAM) and vascular endothelial growth factor (VEGF) in SCs were assayed by RT-PCR. In addition, SCs cultured in 3D PFTBA-enriched hydrogel were characterized by Live/Dead staining and the mRNA levels of BDNF, NGF, GDNF, N-CAM and VEGF were assayed by RT-PCR. The results showed that the PFTBA-enriched fibrin hydrogel was able to promote cell adhesion, migration, and proliferation under hypoxic conditions. Interestingly, PFTBA applied through the fibrin hydrogel dramatically enhanced the mRNA of BDNF, NGF, GDNF, N-CAM and VEGF under hypoxic condition. These findings highlight the possibility of enhancing nerve regeneration in cellular nerve grafts through PFTBA increased neurotropic secretion in SCs.     

二甲双胍联合紫杉醇对人乳腺癌细胞凋亡及AMPK信号通路的影响

目的:探索二甲双胍联合紫杉醇对乳腺癌MCF-7细胞活力和凋亡的影响及其可能的机制。方法:采用不同浓度(2、5、10、20、40和80 mmol/L)二甲双胍作用于体外培养的MCF-7细胞,MTT法检测细胞的活力。采用2 mmol/L二甲双胍和2. 4 mg/L紫杉醇单独或联合处理细胞,并加入一磷酸腺苷活化的蛋白激酶(AMPK)信号转导通路抑制剂compound C。实验分为对照组、二甲双胍组、紫杉醇组、联合组和联合+compound C组;流式细胞术检测细胞的凋亡率,采用RT-qPCR和Western blot法检测Bax、Bcl-2和caspase-3的mRNA和蛋白表达量,Western blot检测AMPK和P21蛋白的表达量。结果:不同浓度二甲双胍(2、5、10、20、40和80 mmol/L)显著抑制乳腺癌细胞的活力(P 0. 05),且具有一定浓度依赖性。与对照组相比,2 mmol/L二甲双胍和2. 4 mg/L紫杉醇单独或联合均可显著抑制细胞活力并诱导其凋亡(P 0. 05),显著下调Bcl-2水平(P 0. 05),上调Bax和caspase-3水平(P 0. 05),促进AMPK和P21蛋白表达。联合使用的效果优于单独使用。加入AMPK抑制剂可削弱此作用。结论:二甲双胍联合紫杉醇可抑制乳腺癌MCF-7细胞活力,诱导其凋亡。此作用与激活AMPK信号转导通路并调节细胞凋亡信号通路有关。     

SchWann细胞在壳聚糖纤维上的立体培养

目的：探讨壳聚糖纤维与体外培养Schwann细胞的相容性和亲和力。方法：新生SD鼠的臂丛和坐骨神经组织块种植于壳聚糖纤维支架上培养Schwann细胞。结果：壳聚糖纤维支架上的Schwann细胞为橄榄形或椭圆形,细胞在纤维上分裂、迁移,形成链状结构,细胞突起的末端呈扁平状膨大,伸出爪形伪足贴附于纤维。结论：壳聚糖纤维不仅与Schwann细胞的相容性良好,而且对在其表面培养的Schwann细胞形成髓鞘可能有诱导作用。     

不同浓度人羊膜匀浆上清液培养大鼠许旺细胞的增殖

背景：许旺细胞是周围神经修复过程的重要细胞,而研究发现人羊膜细胞分泌的多种细胞因子能够促进许旺细胞增殖。 目的：观察不同浓度人羊膜匀浆上清液对鼠许旺细胞(RSC96)生长的影响。 方法：使用含体积分数20%胎牛血清的高糖DMEM培养基原代培养RSC96细胞株,传代至第2代用于实验研究。根据人羊膜匀浆上清液在培养基中的不同体积分数(0,10,15,20,25%)分组。 结果与结论：人羊膜匀浆上清液的总蛋白浓度为675 mg/L,表皮生长因子、碱性成纤维生长因子和血管内皮生长因子浓度分别为(470.625±2.546),(4.121±0.026)和(0.172±0.002) ng/L。在培养第1-7天,10%和15%人羊膜匀浆上清液组的增殖率大于20%和25%人羊膜匀浆上清液组(P < 0.05);10%、15%人羊膜匀浆上清液组显示出促进细胞增殖的作用,而20%、25%人羊膜匀浆上清液组显示出抑制细胞增殖的作用;各实验组的细胞活力与对照组接近(P > 0.05)。提示人羊膜匀浆上清液低浓度时(10%和15%)具有促进RSC96增殖作用,高浓度时(20%和25%)抑制RSC96增殖。 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：     

Effect of Hypoxia/Reoxygenation on Cell Viability and Expression and Secretion of Neurotrophic Factors (NTFs) in Primary Cultured Schwann Cells

As the primary myelin‐forming cells of the peripheral nervous system, Schwann cells (SCs) play a key role in the regeneration of injured peripheral nerves. However, hypoxia causes injury of SCs, as observed in peripheral neuropathies, including those caused by diabetes. So we investigated the effect of hypoxia/reoxygenation (H/R) on SCs in this study. To do so, SCs were cultured in hypoxic condition in vitro and then in normal condition for 24 hr; The effects H/R on SCs were evaluated by MTT (3(4,5‐dimethylthiazol‐2‐yl)2,5‐diphenyltetrazolium bromide) assay, Hoechst staining, immunocytochemistry, western blotting, ELISA, and RT‐PCR. H/R resulted in a significant decrease in SCs survival and an increase in caspase‐3 activity. H/R also reduced the mRNA level of BDNF (brain derived neurotrophic factor) and its secretion, but NGF mRNA level was elevated in these cells. These observations showed that H/R induces death of primary cultured SCs, and different mechanisms responsible for regulating NGF and BDNF expression. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

利用贴壁特点提取和纯化乳鼠雪旺细胞

目的主要利用乳鼠的雪旺细胞比成纤维细胞贴壁快的特点,介绍简单而快速提取和纯化乳鼠雪旺细胞的方法。方法取3天SD大鼠双侧坐骨神经,在解剖镜下剥离去除神经外膜,剪碎后用Ⅰ型胶原酶消化,差速法去除成纤维细胞,分小皿培养进一步纯化细胞;光镜下计算细胞纯度;S-100免疫荧光鉴定细胞性质。结果获取的雪旺细胞纯度大于96%;S-100免疫荧光鉴定细胞为阳性。结论本方法可以简单而快速地获得高纯度雪旺细胞。     

Normal and neoplastic Schwann cells in fish

Summary Peripheral nerve sheath (PNS) neoplasms, primarily neurofibromas, schwannomas and maliganant schwannomas, are among the most common tumors in fishes. Model systems involving PNS tumors in fishes are also valuable because mammalian models of PNS tumors are rare. Schwann cells, the primary cell type suspected of neoplastic transformation in these tumors, have been difficult to culture. We describe techniques for culturing normal and neoplastic Schwann cells from fish. We also present methods for preparing cells on culture dishes for electron microscopy which are especially useful when specific cells in a culture must be located for ultrastructural examination.Abbreviations PNS peripheral nerve sheath - SC Schwann cells     

雪旺细胞增殖的研究进展

随着周围神经组织工程学的发展,雪旺细胞的研究越来越受到重视。在周围神经损伤后,雪旺细胞对周围神经再生过程中的形态和功能的修复起着不可替代的作用,因此雪旺细胞的增殖情况对于周围神经损伤后的修复就尤为重要。其增殖速度的提高可大大改善神经桥接体移植的存活率,为临床神经移植术的成功赢得宝贵的时间。本文从雪旺细胞的功能、增殖机制及其影响因素入手,为雪旺细胞的临床应用提供理论依据。     

Effect of Xymedon on Cell Survival in the System Sensory Neuron Schwann Cell

Pyrimidine derivative xymedon inhibits neuronal death in L4-L5 spinal ganglia 30 days after ligation of rat sciatic nerve. After treatment with xymedon the number of neurons on the operated side decreased by 22.1% compared to that on the contralateral side, while in the control group this parameter decreased by 28.7%. At the same terms, the number of Schwann cells on the operated side after xymedon injection increased by 27.7% in comparison with that on the contralateral side, while in the control group this parameter decreased by 57.3%.     

bFGF缓释微球的制备及其促雪旺细胞分裂增殖的初步研究

研究碱性成纤维细胞生长因子(bFGF)缓释微球的制备方法及其对雪旺细胞的促分裂增殖作用。以聚乳酸-羟基乙酸共聚物(PLGA)为载体材料,采用复乳包囊法制备bFGF-PLGA缓释微球,并对微球的形态学、粒径分布、载药量和包封率、及体外释药进行研究。将bFGF、bFGF-PLGA微球分别加入不同组的雪旺细胞培养液中,分别测定雪旺细胞的数量、活力和细胞周期。结果显示,复乳包囊法制备的bFGF-PLGA缓释微球表面光滑圆整,球体均匀度好;微球平均粒径为1.552±0.015μm,平均径距为1.310±0.010;载药量和包封率分别为(27.18×10-3)%±(0.51×10-3)%、66.43%±1.24%;微球的体外释药过程较为稳定,11d释药率为72.47%。体外细胞试验中,培养1、2d时,bFGF组的细胞计数、吸光度明显高于bFGF缓释微球组;培养3、4d时,bFGF组和bFGF缓释微球组的细胞计数、吸光度无统计学差异;培养6、8d时,bFGF缓释微球组的细胞计数、吸光度明显高于bFGF组。流式细胞仪检测结果显示,培养2d后,bFGF组的G2/M S期百分数高于bFGF缓释微球组;培养4、8d后,bFGF缓释微球组的G2/M S期百分数高于bFGF组,差异具有统计学意义。说明采用复乳包囊法制备bFGF-PLGA缓释微球的工艺可行,微球中bFGF的生物活性保存良好,能缓慢持续释放活性bFGF,促进雪旺细胞的分裂增殖。     

The activation of AMPK in cardiomyocytes at the very early stage of hypoxia relies on an adenine nucleotide-independent mechanism

The energy status of a cell plays a key role in its survival, and the exposure of eukaryotic cells to the hypoxia that accompanies the depletion of intracellular ATP triggers specific systemic adaptive responses. AMP-activated protein kinase (AMPK) has emerged as a key regulator of energy metabolism in the heart and plays a critical role in inducing these responses. However, the specific mechanism responsible for AMPK activation in cardiomyocytes at very early stages of hypoxia remain unclear. The goals of this study were to assess the relative contribution to AMPK activation of phosphorylation by AMPK kinase (AMPKK) and of positive allosterism due to AMP:ATP ratios in the early stages of hypoxia. Our results demonstrated that, compared with normoxic controls, neither intracellular AMP concentrations nor AMP:ATP ratios significantly increased within 1h of hypoxia onset. In contrast, a SAMS peptide phosphorylation assay and an immunoblot analysis revealed significant increases in both AMPK activity and ACC phosphorylation within 5min of hypoxic treatment. Furthermore, exposure of cardiomyocytes to hypoxia significantly increased AMPK phosphorylation within 5min, by 3- to 4-fold compared with controls (P<0.01), while overall levels of AMPKα protein did not differ between aerobic and anoxic cardiomyocytes. We also observed increased AMPKK activity in anoxic cardiomyocytes, through use of an α₃₁₂ substrate. Taken together, our findings demonstrate that in the early stage of hypoxia in cardiomyocytes, increases in AMPK activity occur prior to and independently of increases in AMP concentration or in the AMP:ATP ratio. Instead, under these circumstances, AMPK is primarily activated by phosphorylation of the conserved Thr-172 residue in its activation loop by its upstream kinase AMPKK.     

二甲双胍对体外培养人卵巢颗粒细胞分泌功能的影响

目的本研究探讨不同浓度二甲双胍在不同时间点对其分泌类固醇激素的影响。方法收集因输卵管因素不孕或者男性因素不孕行IVF/ICSI-ET患者促排卵后的含有黄素化颗粒细胞的卵泡液,经密度梯度离心后分离获得颗粒细胞,行体外培养。分别加入含有二甲双胍浓度为0 ng/ml,10 ng/ml,100ng/ml,1000ng/ml,10000ng/ml的DMEM培养基对其进行体外培养,每24h收集细胞培养上清,用化学发光法检测上清液中雌二醇及孕酮的水平。结果体外培养颗粒细胞分泌类固醇激素在72h到96h达到高峰,120h后开始递减,这与二甲双胍体外生长曲线一致;二甲双胍都能够抑制类固醇激素的分泌,且二甲双胍浓度在10000ng/ml,能够极显著抑制类固醇激素的分泌,在每个时间点均呈剂量依赖性。结论二甲双胍在颗粒细胞体外发育整个过程中均抑制。     

一种简便的雪旺细胞培养方法

目的介绍一种简便的雪旺细胞培养方法。方法采用反复组织块种植法,进一步在2.5%胎牛血清条件下抑制成纤维细胞生长,进行雪旺细胞培养;以S-100抗体染色鉴定雪旺细胞。结果获得纯度在98%以上的大量高活性雪旺细胞。结论本方法可以简便的获得较高纯度的雪旺细胞,值得进一步推广。     

低氧对人脐带间充质干细胞生物学特性及增殖的影响简

目的探讨低氧培养条件对人脐带间充质干细胞（hUC-MSCs）的生长和增殖的影响。方法采用人脐带酶消化法分离培养hUC-MSCs,分别将细胞置人体积分数20％、10％、3％0,条件下培养,通过细胞成脂、成骨分化诱导及流式细胞仪检测其表面标志物.鉴定hUC-MSCs：绘制细胞生长曲线,用流式细胞仪检测细胞周期及凋亡情况。结果低氧条件培养hUC-MSCs具有成脂、成骨等多项分化的潜能：流式细胞仪检测呈CD105、CD73、CD90、CD44、HLA-ABC高表达,CDl06、CD29、CD45、CD34、HLA-DR低表达;体积分数3％O2培养组与正常体积分数20％O2培养组及体积分数10％0,培养组相比（各组倍增时间分别为17.2h、20.8h、18.9h）,细胞增殖速度加快（P〈0.05）;G0/G1期细胞减少（各组G0／G1期细胞数分别为77.11％±3.89％、83.92％±5.59％、80.19％±5.16％）,S期细胞增多（S期细胞数分别为15.73％±2.56％、10.91％±1.86％、13.31％-4-2.31％）,增殖指数升高（P〈0.05）;细胞凋亡率降低（流式细胞仪检测各组凋亡率分别为13.41％±1.39％、20.83％±1.81％、19.11％±2.44％）（P〈0.05）。结论体积分数3％O：持续培养可促进hUC-MSCs的增殖.减少细胞凋亡。     

Models and methods to study Schwann cells

Schwann cells (SCs) are fundamental components of the peripheral nervous system (PNS) of all vertebrates and play essential roles in development, maintenance, function, and regeneration of peripheral nerves. There are distinct populations of SCs including: (1) myelinating SCs that ensheath axons by a specialized plasma membrane, called myelin, which enhances the conduction of electric impulses; (2) non‐myelinating SCs, including Remak SCs, which wrap bundles of multiple axons of small caliber, and perysinaptic SCs (PSCs), associated with motor axon terminals at the neuromuscular junction (NMJ). All types of SCs contribute to PNS regeneration through striking morphological and functional changes in response to nerve injury, are affected in peripheral neuropathies and show abnormalities and a diminished plasticity during aging. Therefore, methodological approaches to study and manipulate SCs in physiological and pathophysiological conditions are crucial to expand the present knowledge on SC biology and to devise new therapeutic strategies to counteract neurodegenerative conditions and age‐derived denervation. We present here an updated overview of traditional and emerging methodologies for the study of SCs for scientists approaching this research field.     

胰岛素样生长因子-1对雪旺细胞氧化损伤的保护作用

目的:探讨胰岛素样生长因子-1(IGF-1)对雪旺细胞氧化损伤的保护作用。方法:用体外纯化培养的雪旺细胞建立氧化损伤模型,将培养细胞分成氧化损伤组、IGF-1保护组和正常对照组。四甲基偶氮唑蓝比色法(MTT)检测细胞的活性,生化技术检测细胞内超氧化物歧化酶(SOD)的含量,免疫印迹法检测凋亡蛋白Bcl-2的表达水平。结果:与对照组相比,H2O2处理组细胞胞体积缩小、空泡化,细胞活性降低,SOD含量明显减少,Bcl-2表达减弱;而IGF-1保护组细胞存活率明显升高,SOD含量较损伤组高,Bcl-2表达明显上调。结论:IGF-1对氧化损伤的雪旺细胞有保护作用。     

The serum of dysautonomia patients enhances proliferation and signaling in Schwann cells

Disorders of the autonomic nervous system, or dysautonomias, affect a large segment of the population, especially women, and represent a diagnostic challenge. Identification of biomarkers for autonomic disorders, and the subsequent development of screening methods, would benefit diagnosis and symptom management. We studied the effect of sera from fifteen well-characterized dysautonomia patients (mean age 49 ± 16 years, 10 females, 5 males) and ten control subjects (mean age 31 ± 14 years, 5 females, 5 males) on the proliferation of cultured Schwann cells and activity of mitogen-activated protein kinases (MAPKs) in these cells. We correlated characteristics of patients with the effects on cell proliferation and signaling. Overall, we observed a significant increase in proliferation when Schwann cells were incubated with sera from female dysautonomia patients when compared to control subjects and male patients. Interestingly, removal of IgGs significantly reduced the proliferative effect of patient sera. We also observed significant activation of p38 MAPK following incubation with both male and female patient sera. These results suggest that patient sera contain factors that contribute to aberrant Schwann cell proliferation and signaling and may ultimately lead to autonomic nerve dysfunction. Our observations represent a promising first step in the identification of dysautonomia biomarkers.     

电刺激对背根神经节和雪旺细胞髓鞘化的影响

目的:建立体外的背根神经节（DRG）—雪旺细胞（SCs）—电刺激模型,为研究电刺激促周围神经成髓鞘机制提供基础。方法:以函数发生器和电刺激小室构建电刺激细胞培养系统,DRG/SCs共培养24 h后分为对照（Ctrl）组和电刺激（ES）组。ES组给予矩形波电刺激,6 Vp-p,10 Hz,1 h/d,7 d。Ctrl组无电刺激。利用CCK8观测电刺激对细胞毒性的影响,利用髓磷脂碱性蛋白（MBP）免疫荧光检测电刺激对DRG/SCs髓鞘化的影响。结果:CCK8细胞增殖毒性实验显示,ES组光密度值略高于Ctrl组,但两组之间无统计学意义。免疫荧光检测结果显示ES组MBP的荧光强度明显高于Ctrl组（P<0.01）。结论:本文设计的电刺激细胞培养系统对神经细胞安全无毒,连续7 d的电刺激可提高神经髓鞘化率。