Study on the Changes of Nuclear Matrix Proteins and Its Functions during the Apoptosis of the Human Osteosarcoma MG-63 cells

本论文在鉴定姜黄素（curcumin）诱导人成骨肉瘤MG-63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MG-63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的变化进行系统研究。分析鉴定与肿瘤细胞凋亡相关的特异核基质蛋白，并研究了特异核基质蛋白与人成骨肉瘤MG-63细胞凋亡相关基因表达产物在细胞内的共定位关系与变化，探索它们在人成骨肉瘤MG-63细胞凋亡诱导过程中的调控作用，以期能够在较为整体的水平上进一步认识人成骨肉瘤细胞凋亡及其机理，从而找出人成骨肉瘤细胞凋亡相关的靶向性蛋白和深入探索的研究方向。 实验结果显示，7.5mg/L姜黄素能有效抑制...In this study, the curcumin was used to induce the human osteosarcoma MG-63 cells into apoptosis, and its effects were investigated by cellular biolgy and immunocytochemistry. The differentially expressed nuclear matrix proteins were analyzed by subcelluar proteomics methods and laser confocal scanning microscope in order to find out a new way to explore the molecular mechanisms of carcinogenes...学位：理学博士院系专业：生命科学学院生物学系_细胞生物学学号：2162006015331

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

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

Curcumin-induced Apoptosis Affects the Expression and Localization of hnRNP A2/B1 in Human Osteosarcoma MG-63 Cells

姜黄素(CurCuMIn)诱导处理的人成骨肉瘤Mg-63细胞,在光镜和电镜观察细胞凋亡的基础上,对HnrnP A2/b1在核基质中存在、分布及其与凋亡相关基因产物在Mg-63细胞中的共定位关系进行了研究.经姜黄素处理后,细胞出现染色质凝聚、细胞核固缩、凋亡小体等典型的细胞凋亡形态特征;双向凝胶电泳和质谱鉴定结果显示,HnrnP A2/b1存在于Mg-63细胞核基质蛋白组分中,在姜黄素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HnrnP A2/b1在姜黄素处理前后的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,HnrnP A2/b1定位于Mg-63细胞核基质纤维上,经姜黄素处理后出现分布位置与表达水平变化.激光扫描共聚焦显微镜的观察结果显示,HnrnP A2/b1在Mg-63细胞凋亡过程中与bAX、bCl-2、fAS和P53等基因产物具有共定位关系,且其共定位区域发生了变化.研究结果证实了HnrnP A2/b1定位于核基质纤维上,是一种核基质蛋白,在姜黄素诱导人成骨肉瘤Mg-63凋亡过程中的表达与分布变化及其与凋亡相关基因的关系显然对Mg-63细胞凋亡具有重要影响,这为深入揭示肿瘤细胞凋亡的机制提供了重要科学依据和深入探索的新方向.The morphology of human osteosarcoma MG-63 cells under light microscopy and transmission election microscopy was altered when treated with curcumin.The distribution of heterogeneous nuclear ribonucleoprotein (hnRNP)A2 /B1 in the nuclear matrix and its colocalization with the apoptosis-related gene products were examined.The results demonstrated that curcumin induced typical apoptotic responses,such as chromatin agglutination,nuclei condensation,and apoptotic body formation.Twodimentional PAGE and mass spectrum analyses showed that hnRNP A2 /B1 was complexed with the nuclear matrix.Confirmed by Western blotting,curcumin treatment resulted in a down-regulation of hnRNP A2 /B1 expression.An altered distribution of hnRNP A2 /B1 with the nuclear matrix was observed in immunofluorescence assays.Laser confocal microscopy showed that hnRNP A2 /B1 colocalized with apoptosis-related molecules,including Bax,Bcl-2,Fas and p53,with different patterns before and after curcumin treatments.Our study showed that expression level and the distribution of hnRNP A2 /B1,a nuclear matrix protein,were altered by curcumin treatments and mought be involved in the process of apoptosis in MG-63 cells.国家自然科学基金项目(No.30871241)---

SARS的激素治疗与胸片所见

【目的】探讨严重急性呼吸综合征（SARS）的糖皮质激素治疗与胸片所见的关系。【方法】收集广州市458例SARS患者的一般临床资料及每次胸片资料。所有患者都临床确诊并经免疫学检查证实,临床资料及胸片资料同时较齐全才作为研究对象。根据应用激素与否将其分为两组。【结果】应用激素组257例,未用激素组201例。两组在首次见到肺内病变时间之差异无统计学意义（t=1.417,P=0.157）;激素用于临床表现较严重的病例,胸片上肺内病变达高峰时间较未用激素组长（t=3.242,P=0.001）,所累及的肺小区个数较多（t=3.982,P〈0.001）,住院时间较长（F=2.731,P〈0.001）。始用激素于病程10d之内及10d之后比较,后者肺内病变达高峰时所累及的肺小区个数较多。最大日剂量较大者,其病变存留较久、住院时间较多（F=3.040,7.044;P〈0.001,〈0.001）。【结论】激素用于症状较重的SARS患者的情况下,病死率不高,提示激素在SARS治疗中有重要意义

Numerical Investigation on a Transition Prediction Model

采用了新型的转捩预测模式,将间歇因子耦合到湍流脉动输运方程中去,计及流体转捩的内在因素,因而能够反映转捩的具体发展过程.以二维不可压平板边界层和二维NACA0012翼型为例,研究了该转捩模式在预测不可压转捩流动时的作用,得到与实验值符合良好的计算结果.同时也研究了该转捩模式在不同网格分辨率下的计算能力和对近壁面网格分辨率的要求

气固磁稳定流化床屈服应力的实验研究

气固磁稳定流化床是分选6～1mm细粒煤的有效手段,屈服应力对气固磁稳定流化床的稳定性和被分选颗粒的运动具有重要影响.采用拉板法,以0.074～0.045mm磁铁矿粉为加重质,对床层的屈服应力进行了研究.结果表明,磁稳定流化床的屈服应力是磁场强度、床层深度和表观气速的函数,分别随磁场强度及床层深度的增加而增大,随流化气速的增大而减小,磁场强度对屈服应力的影响最大,床层深度次之,表观气速影响最小.构建了3个因素对屈服应力的单因素作用模型及综合作用模型,为气固磁稳定流化床分选6～1mm细粒煤提供了理论基础