Study on the Changes of Nuclear Matrix Proteins during the Apoptosis of Esophageal Cancer EC9706 cells by Curcumin

本文在鉴定姜黄素（curcumin，Cur）诱导人食管癌EC9706细胞凋亡的基础上，应用选择性抽提整装透射电镜和蛋白质组学分析技术，对EC9706细胞凋亡诱导过程中核基质-中间纤维系统的构型变化和核基质蛋白组成变化进行系统研究。分析鉴定与食管癌细胞凋亡相关的特异核基质蛋白，并探索它们在食管癌细胞凋亡过程中的调控作用，以期能够从较为整体的水平上进一步认识食管癌细胞凋亡及其机理，从而找出食管癌细胞凋亡研究的新方向。 实验结果显示，经8μg/mL姜黄素处理之后，EC9706细胞增殖受到明显抑制，细胞生长抑制率达69.9％；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23.0％，并发...In this study, the curcumin(Cur) was used to induce the human esophageal cancer EC9706 cells into apoptosis, and its effects were investigated by techniques of selective extraction-whole mount optic and transmission electron microscopy and proteomics. The differentially expressed nuclear matrix proteins were analyzed in order to explore the molecular mechanisms of a whole level. The results sh...学位：理学硕士院系专业：生命科学学院生物医学科学系_细胞生物学专业学号：2012005130205

Expression and Localization of hnRNP A2/B1 during Differentiation of Human Osteosarcoma MG-63 Cells Induced by HMBA

背景与目的:核基质蛋白的差异表达与细胞癌变和增殖分化调控关系密切。本研究观察了hnRNP A2/B1在诱导分化处理前后人成骨肉瘤MG-63细胞核基质中的存在和分布,及其与Actin、Prohibitin的共定位关系。方法:选择性抽提经环六亚甲基双乙酰胺(hexamethylene bisacetamide,HMBA)诱导处理前后的MG-63细胞核基质,并运用双向电泳、质谱分析、蛋白质印记杂交、免疫荧光、激光共聚焦等技术检测hnRNP A2/B1在核基质中的表达与定位变化,及其与相关蛋白的共定位关系。结果:双向电泳及蛋白质印迹杂交结果证实了hnRNP A2/B1存在于MG-63细胞核基质蛋白组分中,并在HMBA处理后细胞核基质中表达下调;免疫荧光显微镜观察显示hnRNP A2/B1定位在核基质上,经HMBA处理后hnRNP A2/B1表达减弱。激光共聚焦显微镜观察结果显示,hnRNP A2/B1与细胞核基质蛋白组分Actin、细胞增殖相关调控因子Prohibitin具有共定位关系,但在诱导处理后细胞内的共定位关系减弱。结论:hnRNP A2/B1在MG-63细胞诱导分化过程中的表达分布,及其与Actin、Prohibitin的共定位关系的改变对MG-63细胞分化具有重要影响,值得进一步探索和研究。BACKGROUND & OBJECTIVE: The differentially expressed nuclear matrix proteins have great effects on canceration and regulation of cell differentiation. This study was to explore the existence and distribution of ribonucleoprotein hnRNP A2/B1 in nuclear matrix and its co-localization with Actin and Prohibitin in human osteosarcoma MG-63 cells before and after hexamethylene bisacetamide (HMBA) treatment. METHODS: The nuclear matrix of MG-63 cells before and after treatment of HMBA were selectively extracted. The expression and localization of hnRNP A2/B1 in nuclear matrix were detected by 2-D PAGE, MALDI-TOF-MS, Western blot, and immunofluorescent staining. The co-localization of hnRNP A2/B1 with Actin and Prohibitin was observed under laser scanning confocal microscope (LSCM). RESULTS: hnRNP A2/B1 was detected in the component of nuclear matrix proteins of MG-63 cells by Western blot and immunogold staining and its expression was decreased after treatment of HMBA. hnRNP A2/B1 was located in the nuclear matrix, and its expression was weakened after HMBA treatment. hnRNP A2/B1 was co-localized with Actin or Prohibitin in MG-63 cells, while the co-localization relationship was weakened during differentiation of MG-63 cells. CONCLUSIONS: hnRNP A2/B1 is a kind of nuclear matrix protein, and localizes in the nuclear matrix. The distribution and expression of hnRNP A2/B1 and its co-localization with Actin and Prohibitin play important roles during the differentiation of MG-63 cells.国家自然科学基金项目(No.30470877)~

Apoptosis of Human Esophageal Carcinoma Cell Line EC9706 Induced by Curcumin

目的:应用姜黄素处理人食管癌EC9706细胞,研究姜黄素对人食管癌EC9706细胞凋亡的诱导作用。方法:应用细胞计数、流式细胞仪、琼脂糖凝胶电泳、Hoechst染色、H.E染色和透射电镜检测经姜黄素诱导处理后人食管癌EC9706细胞的凋亡。结果:经姜黄素诱导处理后,人食管癌EC9706细胞生长抑制率达69.9%;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23%;琼脂糖凝胶电泳显示出细胞凋亡典型的180-200 bp及其倍体的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;光镜和电镜下可见典型的细胞凋亡特征:细胞体积缩小,染色体凝集,可见有成群或单独存在的凋亡细胞,电镜下可见凋亡小体存在。结论:姜黄素能够有效诱导人食管癌EC9706细胞的凋亡,从而进一步为食管癌等恶性肿瘤疾病的治疗和凋亡机理的研究提供重要基础和科学依据.Objective: To study the apoptosis effects of the human esophageal carcinoma cell line EC9706 cells induced by curcumin(Cur). Methods: The apoptosis effects of EC9706 cells induced by curcumin were detected by cell count, flow cytometry analysis, light microscope and electron microscope. Results: After treated with curcumin, the proliferation of EC9706 cells was inhibited, and the inhibitory rate was 69.9%. The results of flow cytometry analysis showed that curcumin could induce the emergence of the phase of apoptosis, and the rate was 23%. 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 concerntrated, chromatin agglutinated, mitochondria swelled, and apoptosis body formed. Conclusion: This study suggested that curcumin could induce apoptosis of the human esophageal carcinoma cell line EC9706 effectively, and provided important foundation and research proofs to study more about the therapy of esophageal carcinoma, the malignant tumor and apoptosis mechanisms.国家自然科学基金项目(30470877

Effects of Retinoic Acid on Proliferation and Associated Gene Expression of Human Osteosarcoma MG-63 Cells

目的:研究维甲酸对人成骨肉瘤Mg-63细胞增殖和相关基因表达的影响,以探索其对成骨肉瘤细胞的生物学效应。方法:以1μMOl/l维甲酸处理人成骨肉瘤Mg-63细胞,生长曲线测定,流式细胞仪分析、光镜观察和免疫细胞化学检测等研究维甲酸对Mg-63细胞的生长曲线、细胞周期和相关癌基因、抑癌基因表达的影响,并对其作用机理进行初步分析。结果:维甲酸处理7天后,Mg-63细胞生长抑制率达到42.2%,g0/g1期比例达到61.8%,细胞形态铺展,排列趋于规则,癌基因C-MyC、C-fOS的表达降低,而抑癌基因rb、P27表达上调。结论:1μMOl/l维甲酸可以有效抑制细胞的增殖活动,改变细胞恶性形态特征,下调癌基因C-MyC、C-fOS和上调抑癌基因rb、P27的表达,从而对人成骨肉瘤细胞分化具有诱导作用。Objective:To explore effect of Retinoic Acid(RA) on proliferation of human osteosarcoma MG-63 cells and its mech-anism of antitumor.Methods:The MG-63 cells treated by 1μmol/L RA were subjected to growth curve counts,flow sytometry,im-muncocytochemical assay and light microscopy.We investigated the influence of RA on proliferation,cell cycle,expression of associated oncogene and tumor suppressor gene of MG-63 cells.Then we analyzed the mechanism of RA in inducing differentiation primarily.Re-sults:Seven days after treated by 1 μmol/L RA,the inhibition rate of proliferation of MG-63 cells amounted to 42.2 % and RA reduced the rate of progression from G1 to S phase.The cells tend to be flat and spread.Meanwhile,the expression level of oncogene was down-regulated and the expression level of tumor suppressor gene was upregulated.Conclusions:1μmol/L RA could inhibit proliferation of MG-63 cells,reverse the malignant phenotype characters,downregulate expression of Oncogene c-Myc,c-fos and upregulate expression of Tumor Suppressor Gene Rb,p27,as a result,induce MG-63 cells into differentiation effectively.国家自然科学基金资助项目(30470877

Expression and Localization of Nucleophosmin During HMBA-induced Differentiation in Human Hepatocarcinoma SMMC-7721 Cells

To explore the existence and distribution of nucleophosmin in the nuclear matrix and its co-localization with the other related gene products following HMBA treatment in the human hepatocarcinoma SMMC-7721 cells,the nuclear matrix of SMMC-7721 cells was extracted pre/post HMBA induced differentiation.2D PAGE proteomics analyses showed that nucleophosmin existed in the fractions of nuclear matrix proteins and was down-regulated after HMBA treatment with further confirmation by Western blot analysis.The immunofluorescence observation revealed that nucleophosmin located in the nuclear matrix,HMBA treatment altered its expression level and distribution profile.The co-localization of nucleophosmin with cancer-related genes and the products of oncogenes or tumor repression genes,including c-fos,c-myc,p53 and Rb,using laser scanning confocal microscopy,were evaluated,and substantial differences were observed following HMBA treatment.The results implies that nucleophosmin,as a nuclear matrix protein,the level of its expression and the colocalization with cancer-related gene products may play an important role during the differentiation of SMMC-7721 cell.国家自然科学基金资助项目(No.30470877)~

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

The Effect of Retinoic Acid on Proliferation and Associated Gene Expression of SK-N-SH Cell

目的:研究维甲酸对SK-N-SH细胞的增殖和相关基因表达的影响。方法:通过苔盼蓝排除法绘制细胞生长曲线、流式细胞技术测定细胞周期、HE染色光镜观察细胞形态改变以及SABC免疫细胞化学染色法观察维甲酸对SK-N-SH相关癌基因、抑癌基因表达的影响。结果:1μmol/L RA处理后,SK-N-SH细胞的增殖活动受到明显的抑制,处理第7天抑制率达36.16%;周期测定显示处理后出现明显的G0/G1期阻滞,由对照组的49.7%增加至62.7%,增加了26.2%;免疫细胞化学染色结果显示,处理后细胞癌基因c-myc、c-fos的表达较对照组明显降低,而抑癌基因p53、p27的表达则有所加强。结论:维甲酸能有效抑制SK-N-SH细胞的增殖活动,其对细胞的增殖抑制作用与RA下调c-myc、c-fos等癌基因以及上调p53、p27等抑癌基因的表达有关。Objective:To study the effect of retinoic acid on proliferation and associated gene expression of SK-N-SH cell.Meth- ods:Effects of retinoic acid on proliferation of SK-N-SH cells were determined with Taipan-blue dye exclusion test,effects on cell cycle were detected by flow cytometry,effects on morphology were detected by HE staining.And expression levels of oncogene and tumor suppressor gene were examined by immunocytochemistry.Results:The experiment results showed that after treaed with 1μmol/L RA,the proliferation rate of SK-N-SH cells was inhibited,the inhitbion rate amounts to 36.16%,and the cell cycle was arrested at G0/G1 phase, it was increased from 49.7% to 62.7%.The immunocytochemistry assay also revealed that the expression level of oncogene including c-myc c-fos was downregulated,and the expression level of tumor suppressor gene including p53 and p27 was upregulated.Conclusions: The results showed that the RA could effectively inhibit the proliferation activity of the SK-N-SH cell,and the mechanism of RA was re- lated to its downregulating the expression levels of c-myc c-fos and upregulating the expression levels of p53 and p27.国家自然科学基金(No.30470877

The Localizational and Expressive Alteration of Nucleophosmin during the Differentiation of Human Osteosarcoma MG-63 cells Induced by the Combination of Ginsenoside Rg1

选择性抽提经中药有效成分人参皂甙Rg1组合(简称RCT)诱导处理前后的人成骨肉瘤MG-63细胞核基质,对nucleophosmin(NPM)在核基质中的存在、分布及其与相关基因产物在MG-63细胞中的共定位关系进行了观察研究。双向凝胶电泳和质谱鉴定结果显示NPM存在于MG-63细胞核基质蛋白组分中,在RCT处理后细胞核基质蛋白中表达下调。蛋白质印迹杂交结果证实了NPM在RCT处理前后的MG-63细胞核基质蛋白中的存在及其表达下调变化。免疫荧光显微镜观察显示NPM定位于MG-63细胞核基质上,经RCT处理后出现分布位置与表达水平的变化。免疫荧光-激光扫描共聚焦显微镜的观察结果显示NPM在MG-63细胞中与c-fos、c-myc、RB、p53等基因产物具有共定位关系,并在RCT处理后细胞核内其共定位区域发生了变化。研究结果证实了NPM存在于核基质上,是一种核基质蛋白,其在RCT诱导人成骨肉瘤MG-63分化过程中的表达与分布变化及其与相关癌基因、抑癌基因的关系显然对MG-63细胞分化具有重要影响,这为深入揭示中药有效成分诱导肿瘤细胞分化的机制提供了重要科学依据和深入探索的新方向。To explore the existence, distribution of nucleophosmin (NPM) in the nuclear matrix of the osteosarcoma MG-63 cells and the co-localizational relationship between NPM and the products of some interrelated genes, the MG-63 cells treated with or without the combination of ginsenoside Rg1, cinnamic acid and tanshinone II A (RCT) were selectively extracted and subjected to proteomic methodologies and laser scanning confocal microscopy (LSCM). 2-D PAGE, MS and Western blotting methods confirmed the existence of NPM and its decreased expression in the nuclear matrix proteins. LSCM observation showed that there were altered co-localization after RCT treatment between NPM and the product of genes c-fos, c-myc, RB and p53, respectively. Our study confirmed the existence of NPM in the nuclear matrix and the important role its expression and distribution played during the differentiation of MG-63 cells induced by effective ingredients from Chinese materia medica.国家自然科学基金资助项目(No.30470877)~

一种共掺杂的纳米氧化锌粉体及制备方法

本发明公开了一种共掺杂的纳米氧化锌粉体及其制备方法，共掺杂的元素包括第一金属元素和第二金属元素，第一金属元素是钛元素，第二金属元素是铝、镓、铟或锡元素；按物质的量计，第一金属元素、第二金属元素和氧化锌中锌元素的总和为100份，其中第一金属元素和第二金属元素的总和占0.005～30份，并且第一金属元素与第二金属元素的份数比范围为1∶100至100∶1。与现有技术相比，本发明方法反应条件精确可控，所用设备简单、成本低廉，便于污染控制，易于大规模工业化生产；得到的共掺杂氧化锌粉体的掺杂均匀度高，粉体颗粒均匀呈近球形，具有较高的烧结活性，在高含量掺杂元素掺杂的前提下实现了纳米粉体的单相性