CDld基因稳定转染Panc-1细胞系的建立

实验采用PCR法获得CD1d基因片段,将其插入慢病毒pReceiver-Lv201载体（带GFP荧光）中获得EX-S0249-LV201重组质粒,经转染试剂EndoFectin-Lenti转染到293T细胞中获得慢病毒LP-S0249-LV201,用包装获得的慢病毒感染胰腺癌Panc-1细胞,在不同时间段用倒置荧光显微镜观察绿色荧光,并用反转录酶聚合酶链反应（RT-PCR）和Western blot方法验证获得的表达细胞株.实验结果显示经RT-PCR和Western blotting检测证实慢病毒LP-S0249-LV201 转染至Panc-1细胞株后,该细胞株表达CD1d基因和蛋白,成功建立了稳定表达CD1d基因的Panc-1细胞系,为进一步研究CD1d基因在胰腺癌免疫基因治疗中的作用奠定基础.     

天然高分子牛血红蛋白对钾离子的吸附研究

用离子选择电极法研究了牛血红蛋白在各种条件下对钾离子的吸附.结果表明,在中性条件下K~ 几乎没有被吸附;随着溶液pH的增加和K~ 浓度的增加,牛血红蛋白对K~ 的吸附量也增加,在一定条件下达到饱和.牛血红蛋白对K~ 的吸附呈可逆性,但蛋白质本身结构,当pH由>11调至<8.5后,呈不可逆变化.如溶液中存在Na~ ,则K~ 的吸附量下降.表明存在竞争吸附.这些实验结果支持了缔合一诱导理论.     

人参皂苷Compound K对人膀胱癌T24细胞凋亡的影响

对人膀胱癌T24细胞进行体外培养,采用MTT方法观察不同浓度人参皂苷Compound K处理细胞24、48、72h后对细胞增殖的抑制作用,利用Hoechst33342对细胞进行染色,荧光显微镜观察细胞形态学变化,应用流式细胞术观察人参皂苷Compound K对T24细胞凋亡的诱导作用,并应用WesternBlotting检测不同浓度人参皂苷Compound K处理后T24细胞中caspase-3及相关蛋白的表达情况。结果显示,人参皂苷Compound K对膀胱癌T24细胞的增殖具有较强的抑制作用,呈浓度和时间依赖关系,并诱导肿瘤细胞凋亡,同时caspase-3活化型表达量增加。说明人参皂苷Compound K能够显著抑制膀胱癌T24细胞的活力,有效地诱导T24细胞凋亡。     

基于改进算法递归神经网络的研究

动态递归神经网络具有递归单元及记忆功能，使其在系统辨识和控制中有独特的作用．针对BP算法的不足，提出了一种递推预报误差(RPE)学习算法．对一个非线性系统进行了解识，其仿真结果表明，改进的RPE算法优于BP算法．     

雷公藤红素对人肝癌细胞Hep3B中HIF-1α表达的影响

为探讨雷公藤红素对人体肝癌细胞Hep3B中缺氧诱导因子（HIF-1a）活化的影响,利用氯化钴（CoCl2）模拟肝癌细胞缺氧模式,采用双荧光素酶报告基因法检测雷公藤红素对HIF-1α的转录活性,蛋白免疫印迹实验检测雷公藤红素对HIF-1α蛋白质的表达水平,RT-PCR检测雷公藤红素对HIF-1α下游靶基因血管内皮生长因子（VEGF）mRNA的表达水平,电泳泳动度移动分析（EMSA）检测雷公藤红素对HIF-1αDNA的结合能力.结果显示：雷公藤红素呈剂量依赖性抑制了HIF-1α蛋白质的表达水平,降低了肝癌细胞中VEGFmRNA的表达水平并有效抑制了HIF-1α蛋白的DNA结合能力.这表明,雷公藤红素可以抑制肝癌细胞中HIF-1α的活性,这可能是其抗肿瘤的机理之一.     

多头绒泡菌细胞核肌动蛋白相对含量的动态变化

将自然同步化的多头绒泡菌生质团中的S期、G2早期、G2中期和G2晚期细胞核提取出来,经SDS－PAGE发现一条分子量为43KD的多肽,经Westen blot证明为肌动蛋白,凝胶染色后,发现细胞周期不同期相的肌动蛋白带着深浅不同。G2早期染色最深,G2晚期染色最浅,电泳凝胶扫描表明肌动蛋白相对含量依细胞周期相不同而具有动态变化;G2早期相对含量最高,达6．9％;G2晚期相对含量最低。为2．3％。     

基于小波分析的BP神经网络膜蛋白跨膜螺旋区段预测

基因组计划所产生的大量蛋白质序列迫切需要从理论上预测跨膜螺旋区段。提出了基于小波多分辨分析的BP神经网络膜蛋白跨膜螺旋区段的预测新方法,并把此方法称之为WnnTM。从MPtopo数据库中随机抽取80条三维结构已知的膜蛋白质序列构建数据集,把它们映射成疏水值序列,通过小波分解和重构得到小波系数,并结合BP神经网络构造小波BP神经网络预测模型,对膜蛋白跨膜螺旋区段的位置和数目进行预测。实例验证,WnnTM预测方法比单独用BP神经网络对膜蛋白跨膜螺旋区段进行预测更有效。     

从烟草悬浮细胞中分离液泡前体的方法研究

通过跟踪含有液泡前体特异标记物的组分, 利用细胞壁酶水解胞壁制造原生质体, 并用真针头挤压的方法裂解细胞, 用蔗糖密度梯度离心的方法分离液泡前体. 通过相差显微镜观察, 发现所分离的液泡前体保持完整的结构. 利用不同细胞器的标记物抗体进行免疫标记检测, 证明所纯化的液泡前体不含其他细胞器, 具有较高的纯度.     

牛γ干扰素基因的克隆及在巴斯德毕赤酵母GS115中的表达

利用RT PCR扩增奶牛γ干扰素基因bovIFN γ。测序和序列分析表明,bovIFN γcDNA由501个核苷酸组成,共编码166个氨基酸,N 端的23个氨基酸为信号肽,含有2个潜在的N 糖基化位点。理论相对分子质量为19393.48,理论等电点为9.63。构建转化载体pPIC9K/bovIFN γ,并转化至巴斯德毕赤酵母GS115中,获得重组酵母菌GS115/bovIFN γ。     

br基因的克隆及表达

细菌视紫红质(Bacteriorhodopsin,BR)是嗜盐菌细胞膜上唯一的一种光能转换色素蛋白,BR以视黄醛(retinal)作辅基,在光诱导下吸收光子后会产生一系列的光循环中间体,最后又回到原始状态．由于BR这种独特的分子结构和光循环过程,及其作为一种具有光敏特性的蛋白质生物分子,可嗵过生物学技术进行改造,正引起光子学研究及生物学研究领域的高度注意．从Genbank查询br基因的全序列,然后采用聚合酶链反应(Polymerase chain reaction,PCR)技术,采用DNA star软件辅助设计了上下游引物,以嗜盐菌(Halobactrium Halobium)的基因组DNA为模板克隆获得了完整的细菌视紫红质基因br,并且将经测序鉴定的br基因重组到原核高效表达载体pGEX-4T-2中,转化大肠杆菌E．coli．,将阳性转化子经IPTG诱导表达获得BR与GST(谷胱苷肽转移酶)的融合蛋白后,提取蛋白进行SDS-PAGE电泳、Western印迹反应,检测表达结果为阳性,且表达蛋白的大小与预想的一致．     

人APOBEC3G基因克隆、表达、纯化及多克隆抗体制备

为了获得人APOBEC3G蛋白及其多克隆抗体,从H9细胞中提取总RNA,采用反转录-聚合酶链式反应(RT-PCR)技术获得人APOBEC3G基因.将测序鉴定过的APOBEC3G基因克隆到原核表达载体pET-32a上,以包涵体的形式在E.coli BL21(DE3)中高效表达,由于APOBEC3G蛋白C端融合了6×His标签,有助于对蛋白的纯化及鉴定.应用酶切技术、SDS-PAGE及Western Blot等方法确保基因片段的正确性和蛋白的特异性.纯化后的APOBEC3G蛋白用来免疫日本大耳白兔,用间接ELISA法测定兔多克隆抗体滴度,获得了纯度超过80%的APOBEC3G融合蛋白,抗APOBEC3G多克隆抗体滴度高达1:102 400.     

Effect of heparin on high glucose induced proliferation and expression of matrix metalloproteinases in normal human mesangial cells

Background The pathogenesis of diabetic nephropathy (DN) is a complex pathophysiological process.Its precise mechanism is not fully known. In recent years it has been recognized that synthesis of various extracelluar matrix (ECM) components may increase, and that degradation of ECM may decrease in DN. It was reported heparin could inhibit mesangial cells proliferation in vitro. The main aim of this study is to explore whether heparin inhibits proliferation of mesangial cells grown in high glucose concentration and to measure the effect of heparin on matrix metalloproteinases (MMPs) expression in mesangial cells. Methods The medium contained either low glucose (5 mmol/L) or high glucose (25 mmol/L). The concentrations of heparin in the culture medium were 0, 25, 50,100, 200 or 400 μg/mL. A metabolic (WST-1) assay was used to measure mesangial cell proliferation and Western blot analysis was used to measure MMPs expression of mesangial cells. Results Normal human mesangial cell (NHMC) proliferation was higher in high glucose (HG) medium than in low glucose (LG) medium. They showed a 1.93 fold expansion after 72 h in high glucose in contrast to a 1.63 fold expansion in low glucose. In the presence of heparin, mesangial cells proliferation was inhibited, which was more obvious at high glucose concentrations than at low glucose concentrations. In high glucose, with heparin concentration of 50, 100, 200 and 400 μg/mL, the mesangial cells showed a 0. 61 fold, 0.52 fold, 0.52 fold and 0.41 fold reductions in cell number compared to cells grown without heparin. In low glucose, only concentrations of 200 μg/mL and 400 μg/mL showed reduction in cell number, namely 0.54 fold and 0.45 fold, when compared to cells grown without heparin. In Western blot analysis,MMP1, MMP2, MMP3 and MMP9 was expressed by mesangial cells expressed in both high and low glucose concentrations, which was more prominent in high glucose medium. Incubation of heparin further increased expression of MMP1, MMP2, MMP3 and MMP9. Conclusions This study suggests that glucose can accelerate mesangial cell proliferation while heparin can reduce proliferation, being more obvious at high glucose concentrations. Higher glucose concentrations led to increased MMP expression, which may take part in the regulation of mesangial matrix synthesis and degradation. Addition of heparin resulted in a corresponding increase in MMP expression, most notably at high glucose concentrations, indicating a potentially renoprotective role in DN.     

Effect of heparin on high glucose induced proliferation and expression of matrix metalloproteinases in normal human mesangial cells

Background The pathogenesis of diabetic nephropathy (DN) is a complex pathophysiological process. Its precise mechanism is not fully known. In recent years it has been recognized that synthesis of various extracelluar matrix (ECM) components may increase, and that degradation of ECM may decrease in DN. It was reported heparin could inhibit mesangial cells proliferation in vitro. The main aim of this study is to explore whether heparin inhibits proliferation of mesangial cells grown in high glucose concentration and to measure the effect of heparin on matrix metalloproteinases (MMPs) expression in mesangial cells. Methods The medium contained either low glucose (5 mmol/L) or high glucose (25 mmol/L). The concentrations of heparin in the culture medium were 0, 25, 50, 100, 200 or 400 μg/mL. A metabolic (WST-1) assay was used to measure mesangial cell proliferation and Western blot analysis was used to measure MMPs expression of mesangial cells. Results Normal human mesangial cell (NHMC) proliferation was higher in high glucose (HG) medium than in low glucose (LG) medium. They showed a 1.93 fold expansion after 72 h in high glucose in contrast to a 1.63 fold expansion in low glucose. In the presence of heparin, mesangial cells proliferation was inhibited, which was more obvious at high glucose concentrations than at low glucose concentrations. In high glucose, with heparin concentration of 50, 100, 200 and 400 μg/mL, the mesangial cells showed a 0.61 fold, 0.52 fold, 0.52 fold and 0.41 fold reductions in cell number compared to cells grown without heparin. In low glucose, only concentrations of 200 μg/mL and 400 μg/mL showed reduction in cell number, namely 0.54 fold and 0.45 fold, when compared to cells grown without heparin. In Western blot analysis, MMP₁, MMP₂, MMP₃ and MMP₉ was expressed by mesangial cells expressed in both high and low glucose concentrations, which was more prominent in high glucose medium. Incubation of heparin further increased expression of MMP₁, MMP₂, MMP₃ and MMP₉. Conclusions This study suggests that glucose can accelerate mesangial cell proliferation while heparin can reduce proliferation, being more obvious at high glucose concentrations. Higher glucose concentrations led to increased MMP expression, which may take part in the regulation of mesangial matrix synthesis and degradation. Addition of heparin resulted in a corresponding increase in MMP expression, most notably at high glucose concentrations, indicating a potentially renoprotective role in DN. Foundation item: Project (30370663) supported by the National Natural Science Foundation of China     

胡椒碱对人胃癌SGC-7901细胞抗肿瘤活性的体外实验研究

胡椒碱（Piperine）是一种从胡椒属植物中提取的生物碱,具有镇静、抗炎、抗肿瘤等多种药理活性.探讨胡椒碱对人胃癌SGC-7901细胞的增殖抑制和诱导凋亡的作用.采用MTT比色法测定其对SGC-7901细胞增殖抑制率;通过免疫荧光法、流式细胞术、Western blot法对其进行抗癌机制研究.MTT结果显示Piperine处理细胞72h的IC50值为37.41 μmol·L-1,且呈现时间剂量依赖性; Hoechst33258染色荧光显微镜观察发现Piperine能诱导SGC-7901细胞核形态学改变,部分细胞呈现典型的凋亡形态学特征;Annexin V-FITC/PI荧光双染结果亦证实Piperine可以诱导SGC-7901细胞发生凋亡;DAPI和DCFH-DA染色流式细胞术分析显示Piperine诱导SGC-7901细胞G2/M期阻滞、细胞活性氧产生增加; Western blot分析发现Bcl-2蛋白表达减少,Bax蛋白表达逐渐增加,呈现剂量依赖性,且Bax/Bcl-2比例增加.因此,Piperine具有抑制SGC-7901细胞增殖和诱导凋亡的抗肿瘤活性.     

卵巢癌病人腹水细胞的培养及其分化研究

肿瘤干细胞是指能分化出不同表型、自我更新和不断分化的肿瘤细胞群,具有与干细胞相似的特征。文章通过流式细胞术分析从人卵巢癌腹水分离所得的细胞表面蛋白的分化特征,发现这些腹水细胞具有部分干细胞表面标志(CD44+,CD90+,β2M+)蛋白,但却不表达CD133和CD34,且体外培养2周后,腹水细胞表面标志发生了变化,这些结果为卵巢癌干细胞研究提出了新的启示。     

Neural network modeling and control of proton exchange membrane fuel cell

A neural network model and fuzzy neural network controller was designed to control the inner impedance of a proton exchange membrane fuel cell (PEMFC) stack. A radial basis function (RBF) neural network model was trained by the input-output data of impedance. A fuzzy neural network controller was designed to control the impedance response. The RBF neural network model was used to test the fuzzy neural network controller. The results show that the RBF model output can imitate actual output well, the maximal error is not beyond 20 m-, the training time is about 1 s by using 20 neurons, and the mean squared errors is 141.9 m-2. The impedance of the PEMFC stack is controlled within the optimum range when the load changes, and the adjustive time is about 3 min.     

The Difference of Sensitivity between BXPC-3 and K562 Cells by Treatments with Combination of Indole-3-acetic Acid and Horseradish Peroxidase

The difference of sensitivity to indole- 3-acetic acid （ IAA ） combined with horseradish peroxidase （HRP） in K562 and BXPC- 3 cells was investigated. The cell proliferation was determined by MTF assay. The cell cycle and apoptosis of K562 and BXPC-3 cells were examined by a fluorescence flow cytometer （FCM） and terminal deoxynacleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） respectively. The experimental results show that IAA and HRP could inhibit BXPC- 3 cell proliferation greatly compared with K562 cell during the first 48 h . The cell cycle was arrested predominantly at G2/ M phase in K562 and BXPC- 3 cells. The cell apoptosis of K562 and BXPC- 3 was induced by IAA/ HRP. There was a significant difference between the two cell lines since BXPC-3 cells were more sensitive than K562 cells by treatments with combination of IAA and HRP.     

苯代谢物氢醌对人体肝细胞内DNA甲基转移酶基因和蛋白表达的影响

苯是一种具有致癌效应的环境污染物,氢醌（Hydroquinone,HQ）作为重要的苯代谢物,其介导的毒性可能是苯致癌的原因之一.越来越多的证据表明,DNA甲基化异常是致癌物质发挥毒作用的一种主要途径,HQ可以诱导人体肝细胞DNA甲基化水平改变,但其作用机制尚不清楚.为探索HQ诱导DNA甲基化改变的主要因素,本文应用RT-PCR和Western blotting技术,研究不同浓度HQ（0、5、10、25和50 μM）染毒暴露对L02肝细胞内DNA甲基化转移酶（DNMT1,DNMT3a和DNMT 3b）基因和蛋白表达的影响.实验结果表明,HQ可诱导三种酶的基因表达不同程度升高,高剂量组（≥10 μM）与对照组相比差异具有统计学意义（P ＜0.05）;0～50 μM范围内HQ可诱导DNMT1和DNMT 3b的蛋白表达呈浓度依赖性增高,25和50 μM实验组与对照组相比差异显著（P＜0.05）.这些结果表明HQ的暴露可引起L02细胞内DNA甲基化转移酶的基因和蛋白表达升高,这可能导致一些目的基因DNA甲基化发生异常,本实验从表观遗传学角度暗示了HQ影响人体早期健康的机制.