Preliminary Research on Roles of Special Nuclear Matrix Proteins during Apoptosis of Human Esophageal Cancer EC9706 Cells

本论文以姜黄素（curcumin，Cur）处理人食管癌EC9706细胞，在鉴定姜黄素对人食管癌EC9706细胞凋亡诱导效果的基础上，综合应用亚细胞蛋白质组学分析、免疫细胞化学、细胞分子生物学等技术，对EC9706细胞凋亡过程中核基质蛋白的变化进行了较为系统的研究。分析鉴定和确证与EC9706细胞凋亡相关的特异核基质蛋白，并研究特异核基质蛋白与人食管癌EC9706细胞凋亡相关基因在细胞内的共定位关系与变化，探索它们在人食管癌细胞凋亡诱导过程中的调控作用，以期能够在较为整体水平上进一步认识人食管癌细胞癌变与凋亡机理问题，从而找出人食管癌细胞凋亡相关的靶向性蛋白和深入探索的研究方向。 实验结果表明...In this study, 30 μmol/L curcumin was used to induce the apoptosis of human esophageal cancer EC9706 cells. On this base, by the methods of subcellular proteomics, immunocytochemistry and cellular molecular biology, we studied systematically the changes of nuclear matrix proteins during apoptosis of EC9706 cells induced by curcumin, analysed their co-localizational relationship with the products o...学位：理学硕士院系专业：生命科学学院生物医学科学系_细胞生物学学号：2172006115218

Changes in expression and localization of heat shock protein 70 during curcumin-induced apoptisis of human esophageal cancer cell line EC9706

目的探讨姜黄素对人食管癌EC9706细胞凋亡的诱导作用,热休克蛋白70(HSP70)在肿瘤细胞凋亡过程中在核基质上的变化及其与凋亡调控相关蛋白的关系。方法用细胞计数和流式细胞仪检测姜黄素对人食管癌EC9706细胞的增殖抑制作用,以光学显微镜和透射电镜观察姜黄素诱导人食管癌EC9706细胞凋亡前后的细胞结构变化,琼脂糖凝胶电泳观察人食管癌EC9706细胞凋亡前后的dnA结构变化。双向凝胶电泳和质谱鉴定分析HSP70在核基质中的存在与变化;并以WESTErn blOTTIng进行确证;激光扫描共焦显微镜观察HSP70在EC9706细胞凋亡过程中的定位及其与bAX、bCl-2等基因产物的共定位关系。结果姜黄素能显著抑制人食管癌EC9706细胞增殖并诱导人食管癌EC9706细胞凋亡,双向凝胶电泳、质谱鉴定和结果发现并证实,HSP70在姜黄素处理前后的EC9706细胞核基质蛋白中的存在及其表达下调变化。激光扫描共焦显微镜观察结果显示,HSP70在EC9706细胞凋亡过程中与bAX、bCl-2等基因产物具有共定位关系,且其共定位区域发生了变化。结论姜黄素对人食管癌EC9706细胞具有显著的凋亡诱导作用;HSP70作为一种新发现的核基质蛋白,在姜黄素诱导人食管癌EC9706凋亡过程中的表达与分布发生了显著变化。HSP70与凋亡相关基因的关系对EC9706细胞凋亡具有重要影响。Objective To investigate the effect of curcumin on human esophageal cancer EC9706 cells and explore the role of heat shock protein(HSP70) in cell apoptosis by examining changes in the nuclear matrix and its relationship with apoptosis-related proteins.Methods Cell counting and flow cytometry were performed to probe the inhibitory effect of curcumin on cellular proliferation.Transmission electron microscopy and optical microscopy were used to observe the structural changes in EC9706 cells before and after apoptosis.Agarose gel electrophoresis was conducted to investigate the DNA structure of EC9706 cells before and after apoptosis.Two-dimensional polyacrylamide gel electrophoresis(2-D PAGE) and mass spectrometry(MS) analysis were performed to investigate the presence and changes of HSP70 in the nuclear matrix of EC9706 cells before and after curcumin treatment,which was further corroborated by Western blotting assay.Laser confocal scanning microscopy was used to observe the colocalization of HSP70 with Bax and Bcl-2 during apoptosis.Results The results indicated that curcumin could markedly inhibited EC9706 cell proliferation and finally induced apoptosis.Data from 2-D PAGE,MS,and Western blotting showed that HSP70 was involved in the nuclear matrix proteins and expression of HSP70 was downregulated after curcumin treatment.Laser confocal microscopy showed that HSP70 colocalized with Bax and Bcl-2,and the colocalized regions were altered by the curcumin treatment.Conclusion Our work proves that curcumin could definitely induce EC9706 cells into apoptosis.As a new found nuclear matrix protein,the expression and distribution of HSP70 are altered during the apoptosis of EC9706 cells.The colocalization of HSP70 with apoptosis-related genes evidently affects the apoptosis of EC9706 cells.福建省自然科学基金资助项目(2011J01256); 中央高校基本科研业务费专项资金(2011121061

The Effects of Neonatal Bovine Brain Bioactive Peptide NBBP-1 on Proliferation and Associated Gene Expression of Human Neuroblastoma SK-N-SH Cells

目的:应用新生牛牛脑活性肽nbbP-1处理人神经母细胞瘤Sk-n-SH细胞,观察nbbP-1对人神经母细胞瘤Sk-n-SH细胞增殖和相关基因表达的影响。方法:通过细胞计数、流式细胞仪、光学显微镜、免疫细胞化学方法检测细胞的变化。结果:经60μg/MlnbbP-1处理后,Sk-n-SH细胞生长抑制率高达90.09%,细胞周期被阻滞在g0/g1,细胞核裂解成多个,免疫细胞化学染色结果显示,经处理后bCl-2抗凋亡基因蛋白表达减弱,而P53、fAS、bAX等促凋亡基因蛋白表达增强。结论:新生牛牛脑活性肽nbbP-1对人神经母细胞瘤Sk-n-SH细胞凋亡具有显著的诱导作用,其诱导癌细胞凋亡的机理与其调节和干预癌基因bCl-2和P53、fAS、bAX等抑癌基因的表达有关。Objective:To study the effects of neonatal bovine bioactive brain peptides in peakⅠ(NBBP-1),with which we treated SK-N-SH cells,on the proliferation and gene expression of human neuroblastoma SK-N-SH cells.Methods:The changes of SK-N-SH cells induced by NBBP-1 were detected by means of cell count,flow cytometry analysis,light microscope and immunocytochemistry.Results:Treated with 60 μg/mL NBBP-1,the proliferation of SK-N-SH cells was inhibited effectively,and the rate was up to 90.09 %.Cell cycle was arrested in G1/S phase.Nucleus broke into several parts.The results of immunocytochemistry showed that expression of anti-apoptotic gene bcl-2 was down-regulated and expression of apoptotic genes p53,fas,bax were up-regulated.Conclusions:This study suggested that NBBP-1 could regulate the proliferation of human neuroblastoma SK-N-SH cells effectively,and the effect was related with the changes of oncogene and tumor-suppressor gene.福建省科技计划重点项目(2003N0052)资

Effects of curcumin on anti-proliferation and expression of apoptosis interrelated gene of Human Osteosarcoma MG-63 Cells

目的:探讨姜黄素对人成骨肉瘤Mg-63细胞增殖抑制及凋亡相关基因表达的影响。方法:用姜黄素处理Mg-63细胞,细胞计数法检测细胞增殖抑制的效果;荧光染色观察细胞的凋亡;流式细胞仪(fCM)进行细胞周期时相分析;免疫细胞化学法和WESTErn blOTTIng免疫法检测细胞凋亡相关基因的表达水平。结果:随着姜黄素浓度的增加及其作用时间延长,对细胞的增殖抑制作用增强,最高抑制率可达89.07%;光镜下可观察到细胞发生染色质浓缩,细胞核凝聚和碎裂等典型的凋亡形态学改变;fCM检测结果显示细胞经姜黄素处理后出现明显的凋亡峰;免疫反应结果显示,凋亡相关基因bCl-2和P53表达水平降低,而bAX和fAS表达水平升高。结论:姜黄素能显著抑制Mg-63细胞增殖并可有效诱导其凋亡,其作用机制可能与bCl-2和bAX二者的比值发生变化从而接受了凋亡刺激信号有关。Objective: To investigate the regulatory effect of curcumin on proliferation inhibition and apoptosis in human osterosarcoma MG-63 cells.Methods: MG-63 cells were treated by curcumin, and the inhibitory effect on cell proliferation was assessed by cell count.The apoptosis was inspected by fluorescent staining.The cell cycle analysis was detected by FCM.The expression level of genes related with apoptosis was observed by immunohistochemistry and western blotting immunoreaction.Results: The results showed that the cells growth was inhibited with the increasing of the concentration of curcumin and the elongation of the treated time,and the highest inhibition rate was 89.07%.Parts of cells presented the typical morphological changes of apoptosis: chromatin concerntrated and agglutinatied, cell nucleus shrinked and cataclased under the microscope.The results of FCM analysis showed that curcumin could in- duce cells into apoptosis, which was proved by the emergence of apoptotic peak.The results of immunoreaction suggested that the expression level of bcl-2 and P53 were down-regulated and the expression level of bax and Fas were upregulated.Conclusions: Curcumin not only inhibited cell proliferation but also induced apoptosis of MG-63 cells, which may be related to the ratio of the expression level of bcl-2 and bax as well as reception of the signal of stimulating apoptosis.国家自然科学基金(30871241

基于蒙特卡罗法的斜齿轮随机啮合效率可靠性分析

基于蒙特卡罗法，建立了斜齿轮随机啮合效率的可靠性模型，选择对啮合效率影响较大的5个参数作为随机参数，通过对随机参数的正态分布抽样，分别得到各个随机参数对啮合效率的可靠性影响。结果表明，法向模数和螺旋角对斜齿轮啮合效率的影响最小，两者相近；其次是齿宽、法向压力角；影响最大的是齿顶高系数h。所以，在考虑啮合效率的斜齿轮传动系统的设计和制造中，可以通过优化法向模数、螺旋角β和齿宽来提高斜齿轮的接触强度和弯曲强度，以减少对啮合效率的影响

Expression and localization changes of nucleophosmin during apoptosis of human esophageal cancer EC9706 cells induced by curcumin

目的探讨核磷蛋白nPM在癌细胞诱导凋亡过程中在细胞内、细胞核基质上的定位与表达变化,以及nPM与凋亡调控相关蛋白的关系,探索其在凋亡调控中的作用。方法在姜黄素诱导人食管癌EC9706细胞凋亡的基础上,以亚细胞蛋白质组学方法分析nPM在核基质中的存在与变化,并以免疫印迹法杂交实验进行确证;激光扫描共焦显微镜观察nPM在EC9706细胞凋亡过程中的定位与变化,以及nPM与bAX、bCl-2等基因产物的共定位关系。结果 nPM存在于EC9706细胞核基质蛋白组分中,并在姜黄素处理后表达下调。nPM在EC9706细胞凋亡过程中发生显著的胞质-核之间的穿梭定位变化,并与bAX、bCl-2等蛋白具有共定位关系,且共定位区域发生了变化。结论 nPM是一种核基质结合蛋白,在EC9706细胞凋亡中的表达与定位变化,及其与凋亡调控蛋白的共定位关系提示,它在EC9706细胞凋亡调控中具有重要作用。Objective To explore the expression and localization change of nucleophosmin( NPM) in the nuclear matrix of cancer cells and its relationships with apoptosis regulating proteins during induced apoptosis,and to discuss its regulating functions in apoptosis.Methods Curcumin was used to induce apoptosis of human esophageal cancer EC9706 cells.By using subcellular proteomics technology we analyzed the presence and expression change of NPM in the nuclear matrix.We performed a Western blotting analysis to confirm the change.Subsequently,we examined the localization change of NPM and its co-localization with apoptotic gene products such as Bax and Bcl-2 through laser scanning confocal microscopy.Results The results of this study showed that NPM existed in the nuclear matrix of EC9706 cells with a remarkable down-regulation after curcumin treatment.We demonstrated that NPM exhibited a distinct nucleocytoplasmic shuttling during induced apoptosis.NPM was co-localized with Bax,Bcl-2 and their co-localization regions changed during the process of apoptosis.Conclusion NPM is a nuclear matrix associated protein.Its altered expression and localization, as well as its co-localization with apoptosis regulating proteins suggest the important regulative functions in apoptosis of EC9706 cells.国家自然科学基金资助项目(81071669;81272921;81272245

Apoptosis of Human Osteosarcoma MG-63 Cell Induced by Curcumin

通讯作者。Tel: 0592-2185363, E-mail: [email protected][中文文摘]应用细胞培养、细胞计数、流式细胞仪、琼脂糖凝胶电泳、Hoechst33258染色、苏木精-伊红(HE)染色、透射电镜观察等技术研究姜黄素对人成骨肉瘤MG-63细胞凋亡的诱导作用。实验结果显示,姜黄素可明显诱导MG-63细胞的凋亡,经7.5mg/L姜黄素诱导处理后,人成骨肉瘤MG-63细胞的增殖活动受到显著的抑制,细胞生长抑制率达63.14%;细胞周期检测出现亚二倍体(亚G1期)细胞峰值,细胞凋亡率达21.8%;琼脂糖凝胶电泳出现细胞凋亡典型的DNA"梯状"条带;细胞核经Hoechst33258染色出现浓染致密的固缩形态和颗粒状荧光;光镜和电镜观察结果显示,姜黄素处理组细胞体积缩小,细胞核固缩,染色质凝聚、线粒体肿胀、有凋亡小体形成等显著的凋亡特征。研究结果表明,姜黄素对人成骨肉瘤MG-63凋亡的诱导具有显著作用,从而为进一步研究细胞凋亡机制和多种骨骼疾病的研究提供重要基础和研究依据。 [英文文摘]The apoptosis effects of curcumin (Cur) on human osteosarcoma MG-63 cells have been studied using the methods of cell culture, cell count, flow cytometry analysis, DNA gel electrophoresis, Hoechst33258 staining, HE staining, electron microscope. The results showed that proliferation of MG-63 cells could be inhibited by 7.5 mg/L Cur, and the inhibitory rate is 63.14%. The results of flow cytometry analysis showed that curcumin could induced the emergence of the phase of apoptosis, and the rate is 21.8%. Agarose gel electrophoresis revealed that cell DNA fragment exhibited characteristic “DNA ladder”. Cell nucleus concentrated and appeared granular fluorescence by Hoechst33258 staining. Light microscope and electron microscope showed that the morphology of the cells treated with curcumin appeared shrinked, cell nucleus concentrated, chromatin agglutination, mitochondria swelling, and apoptosis body forming. This study suggested that curcumin had induced apoptosis of the human osteosarcoma MG-63 cells effectively, and provides important foundation and research proofs to study more about the apoptosis mechanisms of the osteosarcoma cells and the study of disease caused by osteoplast.国家自然科学基金项目资助(No.30470877

Apoptosis of Human Neuroblastoma SK-N-SH Cells Induced by Neonatal Bovine Brain Bioactive Peptides NBBP-1

应用新生牛牛脑活性肽nbbP-1处理人神经母细胞瘤Sk-n-SH细胞,通过细胞计数、流式细胞仪、琼脂糖凝胶电泳、HOECHST染色、HE染色和电子显微镜检测等方法,研究新生牛牛脑活性肽nbbP-1对人神经母细胞瘤Sk-n-SH细胞凋亡的诱导作用.实验结果显示,60μg/Ml新生牛牛脑活性肽nbbP-1能有效抑制人神经母细胞瘤Sk-n-SH细胞增殖活动,生长抑制率高达88.84%;细胞周期检测出现亚g1细胞凋亡峰,细胞凋亡率达19.20%;琼脂糖凝胶电泳显示出现细胞凋亡典型的dnA梯状条带;HOECHST染色显示细胞核内出现浓染致密的固缩形态或颗粒状荧光;光学显微镜和电子显微镜下可见典型的细胞凋亡特征:细胞核固缩、染色质凝聚与凋亡小体等典型的细胞凋亡形态和超微结构特征.因此,新生牛牛脑活性肽nbbP-1能够有效诱导人神经母细胞瘤Sk-n-SH细胞的凋亡.To investigate the effects of neonatal bovine bioactive brain peptides in peakⅠ(NBBP-1) on the apoptosis effects of human neuroblastoma SK-N-SH cells,we detected the changes of SK-N-SH cells treated with NBBP-1 by means of cell count,flow cytometry analysis,agarose gel electrophoresis,Hoechst staining,HE staining and electron microscope.After 60 μg/mL NBBP-1 treatment,the proliferation of SK-N-SH cells was inhibited effectively and the inhibitory rate of cellular growth was up to 88.84 %.We found that NBBP-1 could induce the emergence of the phase of apoptosis sub-G1,and the rate was 19.20%.Cell DNA fragment exhibited characteristic DNA ladder.Cell nucleus concentrated and appeared granular fluorescence by Hoechst33258 staining.Characters typical of apoptosis such as cell nucleus concentrated,chromatin agglutination and apoptosis body forming,were observed by the means of light microscope and electron microscope.It could be concluded that NBBP-1 could induce apoptosis of the human neuroblastoma SK-N-SH effectively.福建省重点科技项目(2003N0052)资

永磁旋转振荡电机及电动设备

本实用新型提供一种永磁旋转振荡电机，包括转子部分和定子部分；转子部分包括轴、永磁体、永磁体盖板、支撑前座，支撑后座和弹性装置；定子部分包括第一定子铁心、第一定子线圈、第一线圈骨架、第二定子铁心、第二定子线圈、第二线圈骨架和隔磁块。第一定子铁心和第二定子铁心通过隔磁块隔开。定子部分为轴对称结构。当第一定子线圈和所述第二定子线圈通入高频电流时产生磁场，从而驱动所述转子部分旋转运动，本实用新型还涉及一种电动设备。本实用新型的永磁旋转振荡电机及电动设备，结构简单，线圈置于定子上，转子部分为永磁体结构，提供更大的气隙磁密，质量更轻，动态响应快，在提供相等安匝电流情况下，输出力矩更大