Therapeutic effects of mesenchymal stem cells administered at later phase of recurrent experimental autoimmune uveitis

AIM：To test the therapeutic effects of delayed treatment of mesenchymal stem cells（MSCs） in recurrent experimental autoimmune uveitis（r EAU）.METHODS： The efficacy of different regimens of MSC administration in r EAU were tested by evaluation of clinical and pathological intraocular inflammation,as well as retinal structural and functional integrity using optical coherence tomography（OCT） and electroretinogram（ERG）.The retinal sections were also immunostained with antibodies to glial fibrillary acidic protein（GFAP）and rhodopsin（RHO）. RESULTS： Delayed treatment of MSCs effectively alleviated the severity of intraocular inflammation with relative intact of outer retinal structure and function.Moreover,double therapies with longer interval led to an even better clinical evaluation,as well as a trend of decrease in relapse and amelioration of retinal function.MSC therapies also effectively reduced GFAP expression and increased RHO expression in the retina.CONCLUSION： MSC administration can effectively treat developed diseases of rEAU,and multiple therapies can provide additional therapeutic benefits.

Immunity phenomena following olfactory ensheathing cell transplantation into experimental allergic encephalomyelitis rat brain

Olfactory ensheathing cells （OECs） can promote axonal regeneration and remyelination for the treatment of spinal cord injury. OECs can also treat experimental allergic encephalomyelitis （EAE）, but it remains unclear whether OECs might be rejected by the immune system in the brain including the destruction of the blood-brain barrier under inflammation, the release of inflammatory factors, the activation of local antigen-presenting cells （e.g., microglia cells） and antigen drainage. We found that OECs expressed major histocompatibility complex （MHC）-I molecules on the cell surface, barely expressed MHC-II, but MHC-II could be induced by interferon-v, suggesting that OECs have certain immunogenicity. When OECs were transplanted into normal animal brains, no OECs were phagocytosed by dendritic cells in the cervical lymph node, and OECs did not induce lymphocyte proliferation, which indicates that OECs share some immune privilege under normal conditions. However, OECs in the rat EAE brain were phagocytosed by dendritic cells in the cervical lymph node and enhanced lymphocyte proliferation. These findings suggest that OECs are rejected because of increased immunogenicity in EAE brain, and that brain inflammation, in particular activated dendritic cells, may be a prerequisite for rejecting OECs.

Effect of olfactory ensheathing cell transplantation on myelin repair and motor function in a rat model of experimental allergic encephalomyelitis

BACKGROUND： Olfactory ensheathing cell transplantation can activate axonal regeneration and enhance myelin repair, which are beneficial for treating demyelinating diseases. OBJECTIVE： To explore the effects of olfactory ensheathing cell transplantation on myelin repair, synaptophysin expression, and motor function in a rat model of experimental allergic encephalomyelitis. DESIGN, TIME AND SETTING： A randomized, controlled experiment was performed at the Laboratory of Provincial Hospital affiliated to Shandong University between August 2006 and September 2007. MATERIALS： Dibenzylamine （Hoechst 33342）, luxol fast blue, and rabbit anti-rat synaptophysin antibody were provided by Sigma, USA. METHODS： Olfactory ensheatbing cells extracted from neonatal Wistar rats were cultured for 10-14 days and labeled with dibenzylamine. Spinal cord extracted from a healthy guinea pig was homogenized and equally mixed with complete Freund＇s adjuvant; thereafter, the mixture was intracutaneously injected into two posterior voix pedis of healthy male Wistar rats to establish models of experimental allergic encephalomyelitis. Rats were randomly divided into a control encephalomyelitis group and an olfactory ensheathing cell transplantation group, 36 rats in each group. Physiological saline （2 μ L） or an olfactory ensheathing cell suspension （2 μ L） was separately injected along lateral cerebral ventricle at day 7 post-model induction. MAIN OUTCOME MEASURES： The migration and distribution of olfactory ensheathing cells were observed under fluorescence microscopy; myelin repair was detected using hematoxylin-eosin staining and luxol fast blue staining; synaptophysin expression was measured using immunohistochemical staining; motor function was evaluated using a motor function scale. RESULTS： Olfactory ensheatbing cells could survive in vivo and migrate to the distal end of the transplant focus and spinal cord, and survived 21 days. Hematoxylin-eosin staining and luxol fast blue staining indicated that myelin in the transplantation group was intact, and the inflammatory focus gradually disappeared. Transplantation increased synaptophysin expression （P 〈 0.05 versus control） and motor function （P 〈 0.05）. CONCLUSION： Olfactory ensheathing cell transplantation can promote myelin repair, increase synaptophysin protein expression, and ameliorate motor function in a rat model of experimental allergic encephalomyelitis.

Calculated and Experimental Research of Sheet Resistances of Laser-Doped Silicon Solar Cells

The calculated and experimental research of sheet resistances of crystalline silicon solar cells by dry laser doping is investigated. The nonlinear numerical model on laser melting of crystalline silicon and liquid-phase diffusion of phosphorus atoms by dry laser doping is analyzed by the finite difference method implemented in MATLAB. The melting period and melting depth of crystalline silicon as a function of laser energy density is achieved. The effective liquid-phase diffusion of phosphorus atoms in melting silicon by dry laser doping is confirmed by the rapid decrease of sheet resistances in experimental measurement. The plateau of sheet resistances is reached at around 15 Ω/. The calculated sheet resistances as a function of laser energy density is obtained and the calculated results are in good agreement with the corresponding experimental measurement. Due to the successful verification by comparison between experimental measurement and calculated results, the simulation results could be used to optimize the virtual laser doping parameters.

Kupffer cell and apoptosis in experimental HCC

INTRODUCTION Our previous study has proved that Kupffer cellsmay have an inhibitory effect on the process ofhepatocarcinogenesis,however,their inhibitorymechanism needs exploring deeply.We performed acomparative study on the expression of PCNA,Bax,P53 and apoptosis of liver cancer cells usingimmunohistochemical technology and terminaldeoxynucleotidyl transferase (TdT)-mediateddUTP-digoxigenin nick end labeling(TUNEL)

DYNAMIC CHANGES OF ITO CELLS IN EXPERIMENTAL LIVER CIRRHOSIS OF RAT

That Ho cells in rat liver express desmin was confirmed by immunohistochemical technique. Consequently, changes of desmin-positive cells, lysozyme-positive cells and fibronectin were further studied in experimental cirrhosis of rat. It was found that desmln- positive cells, with the transitional feature between Ito cells and myofibroblasts or fibrobiasts under electron microscope, increased in number and expression of desmin in the necrotic areas as well as in the cellular fibrous septa, but decreased in number in the fibrous septa except those areas closed to the edges of the septa. These results suggested that Ito cells, myofibroblasts and fibroblasts might belong to the same cellular system and play an important role in the pathogenesis of cirrhosis. Meanwhile, it was also noted that changes of both fibronectin and lysozymepositive cells were correlated with those of desmin-positive cells. These provide evidence in vivo that flbronectin and Kupffer cells might exert certain effects on the migration and proliferation of Ito cells in liver cirrhosis.

Experimental study on dendritic cells pulsed with brain tumor stem cells for immunotherapy of glioma

Objective To investigate the effect of dendritic cells pulsed with brain tumor stem cells which are used to treat on intracranial glioma. Methods We obtained murine brain tumor stem cells by grow ing C6 cells in epidermal grow th factor/basic fibroblast grow th factor w ithout serum.Dendritic cells isolated from rat bone marrow w ere pulsed w ith BTSCs. Rat brain

Rapamycin ameliorates experimental autoimmune uveoretinitis by inhibiting Th1/Th2/Th17 cells and upregulating CD4+CD25+ Foxp3 regulatory T cells

· AIM: To determine the effects of rapamycin on experimental autoimmune uveoretinitis(EAU) and investigate of role of rapamycin on T cell subsets in the disease.·METHODS: EAU was induced in rats using peptides1169 to 1191 of the interphotoreceptor binding protein(IRBP). Rapamycin(0.2 mg/kg/d) was administrated by intraperitoneal injection for a consecutive 7d after immunization. Th1/Th2/Th17 cytokines, TGF-β1, and IL-6produced by lymphocyteswere measured by ELISA, while Th17 cells and CD4 +CD25 + regulatory T cells(Tregs)from rat spleen were detected by flow cytometry.·RESULTS: Intraperitoneal treatment immediately after immunization dramatically ameliorated the clinical course of EAU. Clinical responses were associated with reduced retinal inflammatory cell infiltration and tissue destruction. Rapamycin induced suppression of Th1/Th2/Th17 cytokines, including IFN-γ, IL-2, IL-17, IL-4, and IL-10 release from T lymphocytes of EAU rats, in vitro.Rapamycin also significantly increased TGF-β1production but had no effect on IL-6 productionof T lymphocytes from EAU rats in vitro. Furthermore,rapamycin decreased the ratio of Th17 cells/CD4 +T cells and upregulated Tregs in EAU, as detected by flow cytometry.·CONCLUSION: Rapamycin effectively interferes with T cell mediated autoimmune uveitis by inhibiting antigen-specific T cell functions and enhancing Tregs in EAU.Rapamycin is a promising new alternative as an adjunct corticosteroid-sparing agent for treating uveitis.

Matrine ameliorates experimental autoimmune encephalomyelitis by regulating the differentiation of encephalitogenic Th1/Th17 and Th2/regulatory T cells

OBJECTIVE Experimental autoimmune encephalomyelitis(EAE),the classical animal model for multiple sclerosis(MS)is triggered by an impaired balance of T helper(Th)cells and regulatory T(Tregs)cells.Matrine(MAT),a quinolizidine alkaloid derived from the herb Radix Sophorae Flave,has been shown to ameliorate the clinical signs,inflammatory infiltration,demyelination in acute EAE rats.However,whether MAT protect from EAE by adjusting Th and Treg cells response in specific-cellular and molecular level is unknown.METHODS Herein,MAT was tested for its effects on Th1,Th2,Th17 and Treg cells in the spinal cord of EAE mice and splenocyte-extracted from EAE mice with MOG35-55-restimulated,respectively.RESULTS Our findings revealed that MAT significantly inhibit the proliferation of splenocyte,and remarkably down-regulate the differentiation of Th1/Th17 cells with decreased expressions of CD4+IFN-γ+cells and CD4+IL-17+cells in vivo and IL-17,IFN-γ,ROR-γt,T-bet in vitro,meanwhile it dramatically up-regulate the Th2/Treg cells response associated with increased levels of CD4+TGF-β+1cells and CD4+IL-10+cells in vivo and IL-4,IL-10,TGF-β1,Foxp3 and GATA3in vitro.CONCLUSION Considering the effective therapeutic effects of MAT on EAE,it′s worth to find its new values on other autoimmune diseases.

LncRNA NORAD通过miR-513b-5p/GREM1轴调节颅内动脉瘤血管平滑肌细胞增殖、迁移、侵袭和凋亡的作用机制

目的:探讨长链非编码RNA DNA损伤诱导的非编码RNA(LncRNA NORAD)通过miR-513b-5p/GREM1轴调节颅内动脉瘤血管平滑肌细胞(VSMC)增殖、迁移、侵袭和凋亡的机制。方法:采用实时荧光定量聚合酶链式反应(PCR)法检测人颅内动脉瘤组织和正常组织中LncRNA NORAD、miR-513b-5p及GREM1表达。体外分离培养人VSMC,随机分为对照组、LncRNA NORAD siRNA组、miR-513b-5p mimics组、共转染(LncRNA NORAD siRNA+miR-513b-5p inhibitor)组、共转染阴性对照(LncRNA NORAD siRNA阴性对照+miR-513b-5p inhibitor阴性对照)组,分组转染后,采用实时荧光定量PCR法检测各组细胞LncRNA NORAD、miR-513b-5p及GREM1 mRNA表达;采用细胞计数试剂盒(CCK-8)和免疫荧光染色检测各组细胞增殖情况;采用Hoechst 33342染色和免疫荧光染色检测各组细胞凋亡情况;采用细胞划痕实验和Transwell实验检测各组细胞迁移、侵袭情况;采用免疫印记实验检测各组细胞上皮间充质转化(EMT)标志蛋白神经钙黏素(N-cadherin)、E-钙黏素(E-cadherin)、波形蛋白(Vimentin)表达;采用双荧光素酶报告实验分析VSMC中LncRNA NORAD对miR-513b-5p、miR-513b-5p对GREM1的靶向调控。结果:与正常组织比较,颅内动脉瘤组织LncRNA NORAD、GREM1 mRNA表达明显升高(P<0.05),miR-513b-5p表达明显降低(P<0.05)。与对照组比较,LncRNA NORAD siRNA组、miR-513b-5p mimics组细胞GREM1 mRNA表达、增殖率、Ki67阳性率、迁移率、侵袭数及N-cadherin、Vimentin蛋白表达降低(P<0.05),miR-513b-5p表达、凋亡率及Bax/Bcl-2、E-cadherin蛋白表达升高(P<0.05);共转染阴性对照组各指标差异无统计学意义(P>0.05)。与LncRNA NORAD siRNA组比较,共转染组细胞GREM1 mRNA表达、增殖率、Ki67阳性率、迁移率、侵袭数及N-cadherin、Vimentin蛋白表达升高(P<0.05),miR-513b-5p表达、凋亡率及Bax/Bcl-2、E-cadherin蛋白表达降低(P<0.05)。结论:敲低LncRNA NORAD可通过上调miR-513b-5p表达而降低GREM1表达,从而抑制VSMC增殖与侵袭迁移,并促使其凋亡。

苦瓜皂苷L调控PI3K/AKT通路促进内皮祖细胞增殖能力和内皮功能的实验研究

目的:探讨苦瓜皂苷L对内皮祖细胞(EPC)增殖能力和内皮功能的作用及机制。方法:体外分离、培养、鉴定外周血EPC。使用不同浓度苦瓜皂苷L处理EPC 24 h后,应用细胞计数试剂盒(CCK-8)法检测各组细胞活力。使用AutoDock Tools 5.6软件,分别以磷脂酰肌醇3-激酶(PI3K)和蛋白激酶B(AKT)为受体,以药物分子苦瓜皂苷L为配体采用盲对接法进行分子对接。使用PI3K/AKT抑制剂LY294002和最佳使用浓度的苦瓜皂苷L单独或同时处理EPC 24 h后检测各组细胞活力和磷酸化PI3K(p-PI3K)、磷酸化AKT(p-AKT)及磷酸化内皮型一氧化氮合酶(p-eNOS)表达水平。结果:与对照组比较,苦瓜皂苷L呈剂量依赖性地促进EPC增殖,且40μmol/L的苦瓜皂苷L是最佳药物浓度。与对照组比较,LY294002可下调EPC细胞活力,苦瓜皂苷L可上调细胞活力。与苦瓜皂苷L组比较,共同使用苦瓜皂苷L和LY294002可降低细胞活力。分子对接结果显示,苦瓜皂苷L与PI3K和AKT均存在直接作用,形成稳定的锁钥结构。与对照组比较,LY294002可显著下调p-PI3K、p-AKT和p-eNOS蛋白水平;苦瓜皂苷L可上调p-PI3K、p-AKT和p-eNOS蛋白水平。与苦瓜皂苷L组比较,使用苦瓜皂苷L和LY294002可降低p-PI3K、p-AKT和p-eNOS蛋白水平。结论:苦瓜皂苷L通过PI3K/AKT信号通路靶向调节EPC的增殖能力,进而上调内皮型一氧化氮合酶(eNOS)活化水平以促进EPC内皮功能。

液晶器件制备虚拟仿真实验平台的设计与教学实践

针对液晶器件制备实验设备多、成本高、安全隐患大、实验时间长等问题,采用client/server和browser/server混合架构建设了液晶器件制备虚拟仿真实验平台,该实验平台高度仿真了液晶器件制备的完整过程.平台同时建立了实验预习、虚拟操作练习和项目考核的多维度、全过程考核评价方法.从实践教学效果来看,平台的建设能够使学生系统掌握液晶器件制备流程,提高了学生自主学习能力、操作能力和创新能力.

医学细胞分子学虚拟仿真课件的建设

基于国内医学院校基础医学本科生/研究生教学与实验中最常用的细胞分子生物学实验,首都医科大学细胞生物学系精选了其中代表性的15个实验,制备出精致可视化的仿真视频教学课件,包括实验原理、实操步骤和问题与解答,旨在从深层次探讨机能教学中某些重要疾病发生发展的细胞与分子生物学基础。该套课件适用于医学生和医学工作者从事科学研究和临床实践,为医学院校开展高层次实验课教学提供一套全新模式。

细胞增殖与凋亡的检测方法及其在医学细胞生物学实验中的应用

细胞增殖和凋亡是医学细胞生物学研究中至关重要的过程,其准确的检测方法对于解疾病机制、药物筛选及治疗方案的制定具有关键性意义。文章阐述了细胞增殖的常用检测方法,即MTT法、CCK-8法及BrdU标记法等,以及细胞凋亡的检测方法,即Annexin V-FITC/PI染色法、TUNEL法及Caspase活性检测法等,探讨了细胞增殖与凋亡的检测方法在医学细胞生物学实验和教学中的具体应用,进而为医学细胞生物学实验和相关教学实验提供精确的参考数据。

Dynamic interplay of T helpercell subsets in experimental autoimmune encephalomyelitis

AIM： To investigate the temporal onset and dynamic interplay of CD4^＋ T helper cell subsets in experimental autoimmune encephalomyelitis （EAE）.METHODS： EAE was induced in C57BL/6 mice by im-munization with myelin oligodendrocyte glycoprotein peptide p35-55. The clinical signs were scored and the tissue samples and immune cells isolated for analysis at different phases of EAE. The expression levels of inflammatory cytokines and related transcription fac-tors were detected by quantitative reverse transcription polymerase chain reaction （PCR） and enzyme linked immunosorbant assay （ELISA）. The percentages of Th1, Th17, Th2, Treg and memory T cell subsets in EAE were analyzed by immunostaining and fow cytometry. The data were analyzed by statistical techniques.RESULTS： Quantitative real-time PCR analysis showed that EAE mice express elevated levels of Th1 [interferon gamma （ IFNγ ）, interleukin （ IL ） -12p40 ], Th17 [ IL-17 , related orphan receptor gamma （RORγ ）, IL-12p40] and Treg [ Foxp3, Epstein-Barr virus induced gene 3 （EBI3）, IL-10] genes in the central nervous system at the peak of the disease. Whereas, the expression of Th1 （ IFNγ , T-bet, IL-12p35, IL-12p40 ）, Th17 （RORγ, IL-12p40 ）, Th2 （ IL-4） and Treg （ Foxp3, EBI3） response genes was reduced in the spleen during pre-disease but gradually recovered at the later phases of EAE. ELISA and fow cytometry analyses showed an increase in Th17 re-sponse in the periphery, while Th1 response remained unchanged at the peak of disease. The mRNA levels of IFNγ, IL-17 and IL-12p40 in the brain were increased by 23 （P 〈 0.001）, 9 （P 〈 0.05） and 14 （P 〈 0.01） fold, respectively, on day 21 of EAE. Conversely, the mRNA expression of IL-10 was increased by 2 fold （P 〈 0.05） in the spleen on day 21. CD4^＋CD25^＋Foxp3＋Treg response was reduced at pre-disease but recovered to na？ve levels by disease onset. The percentage of CD25 Foxp3 regulatory T cells decreased from 7.7% in the na？ve to 3.2% （P 〈 0.05） on day 7 of EAE, which then increased to 8.4% by day 28. Moreover, the CD4＋CD127＋CD44high memory T cell response was increased during the onset and recovery phases of EAE. The memory and effector cells showed an in-verse relationship in EAE, where the memory T cells increased from 12.3% in nave to 20% by day 21, and the effector cells decreased from 32% in na？ve to 21% （P 〈 0.01） by day 21. The wild type C57BL/6 mice with EAE showed elevated levels of effector-memory T cells （TEM） with concomitant reduction in central-memory T cells （TCM）, but the EAE-resistant IL-7R defcient mice showed elevated TCM with no effect on TEM cells in EAE.CONCLUSION： Our fndings highlight the temporal on-set and dynamic interplay of effector, memory and regu-latory CD4^＋ T cell subsets and its signifcance to clinical outcome in EAE and other autoimmune diseases.

流式细胞仪平台支撑本科细胞生物学实验课教学改革的实践

在流式细胞术检测细胞凋亡实验中,借助流式细胞仪平台,从教学模式、实验内容、仪器准备、教学素材等方面对细胞生物学实验课进行改革,取得了较好的教学效果。这种仪器平台与教学团队共同探索教学改革的模式,充分发挥大型仪器设备在人才培养方面的潜能,可为其他高校仪器平台建设提供参考。

中高能重离子束全径迹辐照材料和细胞的实验装置设计及效应分析

中高能重离子束注入生物材料的径迹显著深于生物靶,且不同径迹点上的剂量分布、理化效应等又大不相同,所以,同一靶点通常不可能接受所有理化因子的作用(即全径迹辐照)。本实验设计思路:基于CR-39核径迹探测手段,在纯水介质中验证全径迹辐照综合效应特征;通过沿径迹上连续逆向移动靶材料或细胞培养液,以探究全径迹的辐照效应。最后,与对照组相比较,揭示重离子束全径迹的辐照效应及其潜在的分子改性、细胞改良和发酵工程等方面的应用价值。

太阳能电池板功率影响因素仿真实验的优化设计

太阳能电池的输出功率测量实体实验存在着无法实现极端温度下的测试环境,无法实现大功率太阳能功率输出户外现场实验,实验难度大且存在采集数据的时间跨度长等问题。利用计算机建模和现代信息交互技术,开发了影响太阳能电池输出功率变化因素的探究虚拟仿真实验项目。分析了太阳光强变化的数学建模、实验报告系统中非线性数据自动评判、异常操作提示等问题,实验操作步骤中有机融合了验证性、综合性和设计性实验内容。为工科虚拟仿真实验项目的构建提供了参考,也激发了学生学习光伏技术的兴趣,提高了学生自主创新能力。

双硫死亡基因BAK1在口腔鳞癌细胞中的表达及对细胞增殖、迁移和侵袭的影响

目的:识别口腔鳞状细胞癌新标志物,探究双硫死亡在口腔鳞状细胞癌增殖、迁移和侵袭中潜在的生物学价值。方法:检索相关文献,汇总双硫死亡相关基因。基于TCGA数据库,利用生物信息学方法识别具有临床意义的双硫死亡相关基因,并通过细胞生物学实验探究该基因在肿瘤发生发展过程中具有的生物学价值。结果:细胞生物学实验显示,BAK1在口腔鳞状细胞癌中均高表达。此外,BAK1过表达或敲低对于细胞增殖、迁移和侵袭能力均产生较大影响。结论:BAK1可作为口腔鳞状细胞癌新标志物,具有临床意义。同时,双硫死亡在口腔鳞状细胞癌增殖、迁移和侵袭中具有一定的生物学价值。