东亚三角涡虫肌球蛋白轻链（MLC）融合蛋白原核表达载体的构建及其在大肠杆菌中的表达

构建了涡虫肌球蛋白轻链融合蛋白原核表达载体，并进行表达，根据涡虫基因文库中肌球蛋白轻链（Mlc）基因完整的ORF序列设计合成特异性51物，通过PCR扩增涡虫Mlc基因，并插入到融合蛋白原核表达载体PET-28a中，转化宿主菌B121（DE3）细胞．0．4mMol／L的IPTG诱导表达MLc蛋白．重组质粒测序和酶切结果显示Mlc基因已正确插入PET-28a中，重组蛋白经SDS—PAGE在18．2KD处有一条明显的蛋白表达条带。western blot检测得到同样大小的条带。结果表明，涡虫His-MLC融合蛋白已成功表达。     

si—DNMT1对肝门部胆管癌细胞抑癌基因甲基化的作用

目的研究RNA干扰对肝门部胆管癌细胞株QRC939抑癌基因甲基化的影响，初步探谤其在胆管癌治疗中的价值。方法构建靶向hDNMT1的发夹式siRNA表达载体；运用脂质体介导法将其转染人胆管癌细胞QBC939；RT-PCR法检测不同时间点hDNMT1、CDH1、p15的表达水平；MSP方法检测转染前后抑癌基因CDH1、p15的甲基纯状态；MTT检测各组细胞的增殖能力。结果1）hDNMT1的基因沉默恢复了抑癌基因CDH1、p15的表达水平；2）CDH1、p15的表达沉默是由启动子高甲基亿导致的；3）转染靶向hDNMT1的发夹式siRNA表达载体能有效地抑制QBC939的增殖能力。结论靶向hDNMT1的发夹式siRNA表达载体能有效、持续、稳定发挥对hDNMT1的基因沉默作用，恢复抑癌基因CDH1、p15的表达水平，从而抑制QBC939肿瘤细胞增殖。     

反义Ki-67和Bc1-2真核细胞双表达载体的构建及鉴定

目的 构建反义Ki-67和Bc1-2双表达栽体用于肿瘤基因治疗的研究.方法 从SGC-7901细胞总RNA中逆转录扩增Ki-67和Bc1-2 cDNA,T-A克隆到pMD18-T Simple栽体.Ki-67经BamH I和Cla I双酶切,Bc1-2经EcoR I和Xho I双酶切后,分别反向插入pVITR02的多克隆位点1(mcs1)和2(mcs2),构建单表达质粒pVITR02-AsKi-67扣pVITR02-AsBc1-2,再将Bc1-2EcoR I和Xho I双酶切,反向插入pVITR02-AsKi-67的mcs2,构建pVITR02-AsKi-67-AsBc1-2双表达质粒.结果限制性内切酶和测序表明单表达质粒pVITR02-AsKi-67和pVITR02-AsBc1-2以及双表达质粒pVITR02-AsKi-67-AsBc1-2构建成功.结论 本研究成功构建了pVITR02-AsKi-67和pVITR02-AsBc1-2单表达质粒,及pVITR02-AsKi-67-AsBc1-2双表达质粒.     

转录因子TCF7L2在HePG2细胞IDE表达调控中的作用

目的 TCF7L2是一种重要的转录因子,与2型糖尿病(T2DM)发生发展密切相关.本研究探讨慢病毒介导的RNA干扰抑制TCF7L2基因表达对人肝癌细胞株HePG2胰岛素降解酶(IDE)基因的影响.方法 以人TCF7L2 mRNA编码序列作为干扰靶点,构建TCF7L2特异性短发卡RNA慢病毒表达载体(LV-TCF7 L2-shRNA)感染HePG2细胞.应用实时定量PCR及Western blot检测转染后TCF7L2与IDE表达的变化.结果 成功构建TCF7L2 shRNA慢病毒载体LV-TCF7L2-shRNA.qPCR及Westem blot结果显示干扰组HePG2细胞TCF7L2和IDE mRNA及蛋白的表达水平较空白组及阴性对照组显著降低(P＜0.05).结论 LV-TCF7L2-shRNA载体有效地抑制了IDE的表达,结果证明TCF7L2是IDE表达调控中重要的转录因子,为探讨TCF7L2与IDE在2型糖尿病发病机制中的作用奠定了基础.     

庆大霉素产生菌GntB基因的克隆与转化

根据小单孢菌产生庆大霉素的生物合成机理，利用基因克隆方法从棘孢小单孢菌（Micromonospora echinospora）基因组中扩增出庆大霉素生物合成的关键酶基因—2-脱氧青蟹肌糖合成酶基因（GntB），并将其通过大肠杆菌／链霉菌穿梭质粒pIJ699转化原菌株，采用硫链丝菌素抗性基因启动子带动2-脱氧青蟹肌糖合成酶基因在棘孢小单孢菌细胞中实现了转化。     

新型手性抗肿瘤药物德氮吡格（TNBG）体外抗肿瘤活性初步研究

目的观察手性旋光异构体德氮吡格（TNBG）对6株人体肿瘤细胞增殖的影响，初步探讨其抗肿瘤作用机制。方法运用MTT法检测手性TNBG对6株肿瘤细胞生长抑制情况；油红O染色法观察QGY-7701细胞中脂质聚集情况；流式细胞仪检测手性TNBG对人肝癌细胞株QGY-7701细胞周期分布和凋亡影响。结果手性TNBG能不同程度抑制肿瘤细胞增殖，且呈剂量依赖性；油红O染色提取显示QGY-7701细胞内脂质量与给药剂量呈正相关；流式细胞仪检测到有s期细胞增加，并且手性TNBG还能诱导QGY-7701细胞发生凋亡。结论手性TNBG在体外具有良好的抗肿瘤活性，其抗肿瘤作用机制可能是通过促使肿瘤细胞产生脂质聚集，抑制肿瘤细胞增殖、诱导其凋亡从而达到抗肿瘤效应。总体看（＋）TNBG体外抗肿瘤活性比（-）TNBG更强。     

全长人PPAR-γ的表达、纯化与结构确证

目的建立表达、纯化结构完整的全长人PPAR-γ的方法。方法构建pReceiver-B01-PPAR-γ质粒并转化E．coliBL21（DE3）细胞，诱导表达重组蛋白，优化细胞生长条件；利用亲和色谱和尺寸排阻色谱纯化重组蛋白；胶内酶解重组蛋白，用离子阱质谱仪分析二维AgilentHPLC-Chip纯化的酶解片段；基于MS／MS搜索IPI、Swiss．Prot、NCBInr和MSDB数据库，鉴定重组蛋白。结果在优化的细胞生长条件（TB介质、37℃、0．8mMIPTG、诱导3h），从每升TB中可获得280mg重组蛋白；两步纯化后，获得176mg、纯度为95％的均质重组PPAR-γ蛋白；经质谱分析和搜索蛋白质数据库，获得均匀地分布在整个PPAR-γ多肽链中的33个阳性肽段，覆盖率为60％，表明重组蛋白为结构完整的全长人PPAR-γ。配体结合活性位点S289、H323、H449和Y473无突变，保证全长人PPAR-γ与配体结合的生物学活性。结论本文所建立的方法能成功用于大量表达结构完整的全长人PPAR-γ，有利于进一步的结构、功能研究和活性配体的筛选。     

拟南芥基因组中注释为小檗碱桥环酶基因的分子克隆及异源表达

小檗碱桥环酶(BBE)催化(S)-牛心果碱((S)-reticuline)中N-CH3与分子内苄基部分中羟基的邻位芳香碳之间C-C键的形成,该酶属于双共价黄素蛋白家族,是苄基异喹啉类生物碱向小檗碱类生物碱转化的关键酶.迄今,在拟南芥(Arabidopsis thaliana)中尚未发现复杂生物碱,但其基因组测序结果表明拟南芥含有众多可能与复杂生物碱生物合成相关的基因,其中与BBE类似的基因有12个.基于与已知功能的BBE及拟南芥中BBE序列的分析,选定拟南芥中4个注释为BBE的编码基因为目的基因,设计特异引物,从拟南芥cDNA中扩增并克隆到pGM-T载体中,筛选重组子,测序并分析,获得了4个BBE目的基因,分别为AT2G34810、AT5G44400、AT5G44410和AT5G44440.将上述基因克隆至表达载体pET-28a或pET-30a中,分别转入大肠杆菌Rosetta(DE3)中,IPTG诱导实现了上述基因的异源表达.     

儿茶素对晚期糖基化终末产物诱导的内皮细胞凋亡的干预作用

目的探讨儿茶素对晚期糖基化终末产物（AGEs）诱导内皮细胞凋亡的干预作用。方法糖孵育法制备晚期糖基化终末产物，分离肾血管内皮细胞，将内皮细胞分成空白对照组、AGE组、浓度分别为10、15、20μg／ml的儿茶素组共五组。实验末，采用AlamarBlue还原法测定同内皮细胞增生活力，TUNEL法测定细胞凋亡率，生化法测定培养上清中·OH、MDA浓度，RT-PCR测定Bcl-2mRNA表达，Western-Blotting测定Bcl-2蛋白活性。结果与对照组相比，AGE组内皮细胞增生活性、Bcl-2基因与蛋白活性显著降低（P均〈0．01）；凋亡率、·OH与MDA浓度显著增加（P均〈0．01）；与AGE组相比，儿茶素各浓度组内皮细胞增生活力与Bcl-2基因与蛋白活性表达增高，凋亡率、·OH与MDA浓度降低；儿茶素各剂量组之间呈浓度梯度效应。结论儿茶素可降低AGEs引起的血管内皮细胞凋亡，其机制可能是通过有效清除活性氧自由基、上调Bcl-2mRNA与活性蛋白表达，阻断内皮细胞内过氧化物的堆积二种途径实现的。     

darbufelone对胃癌SGC-7901细胞裸鼠移植瘤血管生成的影响

目的观察环氧合酶-2／5-脂氧合酶双重抑制剂darbufelone对人胃癌皮下移植瘤血管生成的影响，并初步探讨其机制。方法建立裸鼠实体瘤模型，随机分为darbufelone组和对照组，darbufelone及生理盐水分别连续灌服4周。测量肿瘤质量、体积，计算抑瘤率；免疫组化检测CD34并计算微血管密度；RT—PCR法及Western blot法分析移植瘤组织中MMP-9、VEGF的表达。结果darbufelone可明显抑制裸鼠移植瘤的生长，质量抑瘤率为58．42％，体积抑瘤率为67．13％。darbufelone组的微血管密度（MVD）（15．36±0．30）明显低于对照组（29．47±0．63）（P〈0．05）；darbufelone组肿瘤组织中VEGF及MMP-9在基因水平及蛋白水平的表达（P〈0．05）。结论darbufelone能有效抑制裸鼠移植瘤的生长，减少移植瘤组织中VEGF及MMP-9的表达，抑制肿瘤的微血管生成，具有抗血管生成的作用。     

Altered proteoglycan gene expression and the tumor stroma

Tumor stroma is a specialized form of tissue that is associated with epithelial neoplasms. Recent evidence indicates that significant changes in proteoglycan content occur in the tumor stroma and that these alterations could support tumor progression and invasion as well as tumor growth. Our main hypothesis is that the generation of tumor stroma is under direct control of the neoplastic cells and that, via a feedback loop, altered proteoglycan gene expression would influence the behavior of tumor cells. In this review, we will focus primarily on the work from our laboratory related to the altered expression of chondroitin sulfate proteoglycan and its role in tumor development and progression. The connective tissue stroma of human colon cancer is enriched in chondroitin sulfate and the stromal cell elements, primarily colon fibroblasts and smooth muscle cells, are responsible for this biosynthetic increase. These changes can be reproduced in vitro by using either tumor metabolites or co-cultures of human colon carcinoma cells and colon mesenchymal cells. The levels of decorin, a leucine-rich proteoglycan involved in the regulation of matrix assembly and cell proliferation, are markedly elevated in the stroma of colon carcinoma. These changes correlate with a marked increase in decorin mRNA levels and a concurrent hypomethylation of decorin gene, a DNA alteration associated with enhanced gene expression. Elucidation of decorin gene structure has revealed an unexpected degree of complexity in the 5 untranslated region of the gene with two leader exons that are alternatively spliced to the second coding exon. Furthermore, a transforming growth factor beta (TGF-)-negative element is present in the promoter region of decorin gene. This regulatory domain is likely to be implicated in the silencing of decorin gene by TGF- and may contribute to the regulation of this matrix gene in the tumor stroma.     

Human papillomavirus 16 E5 up-regulates the expression of vascular endothelial growth factor through the activation of epidermal growth factor receptor, MEK/ ERK1,2 and PI3K/Akt

The E5 oncoprotein of human papillomavirus (HPV) 16 plays an important role in early cervical carcinogenesis. Vascular endothelial growth factor (VEGF) plays a central role in switching on the angiogenic phenotype during early cervical carcinogenesis. However, the relationship between E5 and VEGF has not previously been examined. To clarify the regulatory role of E5 in VEGF expression, we transferred the E5 gene into various cell types. E5 increased VEGF expression. The addition of epidermal growth factor receptor (EGFR) inhibitor significantly suppressed VEGF expression, demonstrating that E5 stimulates VEGF expression through the activation of EGFR. E5-mediated EGFR activation was accompanied by phosphorylation of Akt and ERK1/2, which are also involved in VEGF expression. Furthermore, the mRNA stability of VEGF was not affected by E5, but VEGF promoter activity could be modulated by inhibitors of the EGFR, MEK-ERK1/2 and PI3K/Akt pathways in E5-expressing cells. Collectively, these novel results suggest that HPV 16 E5 increases VEGF expression by activating EGFR, MEK/ERK1/2 and PI3K/Akt. Received 23 November 2005; received after revision 10 January 2006; accepted 9 February 2006     

Beta-catenin/TCF4 transactivates miR-30e during intestinal cell differentiation

The Wnt/beta-catenin/TCF4 pathway plays critical roles in the maintenance of small intestinal epithelium; however, downstream targets of the beta-catenin/TCF4 complex are not extensively characterized. We identified miR-30e as an immediate target activated by the beta-catenin/TCF4 complex. miR-30e was detected in the peri-nuclear region of the intestinal crypt IEC-6 cells. Bioinformatics analysis revealed clustered beta-catenin/TCF4 binding sites within the miR-30e promoter region. This promoter region was cloned into pGL3-control luciferase reporter vector, with the enhancer region removed. Transfection of pCMV-SPORT6-beta-catenin expression vector dose-dependently increased luciferase activity, and co-transfection of pCMV-SPORT6-TCF4 expression vector further enhanced the promoter activity. Dexamethasone-induced IEC-6 cells differentiation caused a 2.5-fold increase in miR-30e expression, and upon beta-catenin siRNA transfection, miR-30e increased 1.3-fold. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed the binding between beta-catenin/TCF4 complexes from IEC-6 nuclear extracts and the putative sequences in the miR-30e promoter. These results demonstrate that beta-catenin/TCF4 transactivates miR-30e during intestinal cell differentiation.