[目的]建立稳定可靠的、适合检测红豆杉(Taxus L.)TbAP2基因表达量的荧光定量PCR实验体系.对于检测该物种中基因的组织特异性表达具有重要意义.[方法]以曼地亚红豆杉细胞为试材,提取总RNA并反转录为cDNA,根据TbAP2基因序列设计多对引物,合成内参基因TBC41的引物,采用正交试验L9(34)方法分别筛选以上2个基因5μL和10μL小反应体系及20μL常用体系中的最佳组合,并通过cDNA模板用量和引物用量等方面进行优化,以确保基因扩增效率在90％105％之间.[结果]本研究建立了TBC41和TbAP2基因在5、10、20μL体系下的荧光定量最佳PCR反应体系,在优化后的5μL体系下,加入Mix(2×)Universal 2.5μL,cD-NA模板1.0μL,正反引物共1.5μL,内参基因TBC41和目的基因TbAP2的扩增效率均为94％;在优化后的10μL下,加入Mix(2×)Universal 5μL,cDNA模板1.2μL,正反引物共1.3μL,TBC41和TbAP2的扩增效率分别为95％和94％.在优化后的20μL下,加入Mix(2×)Universal 10μL,cDNA模板0.5μL,正反引物共1.5μL,TBC41和TbAP2的扩增效率分别为93％和99％,以上各扩增体系回归系数R2均大于0.980.[结论]在以上3种反应体系下,内参基因和目的基因均具有接近100％的扩增效率,表明本研究成功建立了适合检测红豆杉TbAP2基因表达量的荧光定量PCR实验体系,并为红豆杉其它基因的表达研究提供参考.

目的:检测中国人端粒酶催化亚基(hTERT)基因第2内含子VNTR多态性,探询VNTR序列是否具有潜在的蛋白结合位点,从而为进一步研究这些VNTR的功能提供线索.方法:采用PCR方法扩增健康中国人外周血淋巴细胞基因组hTERT 基因第2内含子多态性VNTR片段,克隆并鉴定,以多态性重复单元做为探针进行凝胶延迟实验,以确定该片段是否存在蛋白结合位点.结果和结论:hTERT基因第2内含子内上游的一个VNTR序列(VNTR2-1st)存在一个潜在蛋白结合位点,该蛋白能够与多态性VNTR DNA序列形成复合物,蛋白结合的关键位点处于42 bp重复单元C端的典型E-box基序(CACGTG),但是c-myc并不参与结合该VNTR2-1st重复单元.此外,利用第6内含子内上游的VNTR (VNTR6-1st)的重复单元做为探针,结果发现该38 bp VNTR6-1st重复单元不能与HeLa细胞核蛋白因子结合,从而提示了不同VNTR序列功能的多样性.

Mature miRNAs are noncoding RNAs of about 20-25 nucleotides in length.Hundreds of them have been found in the genomes of plants,invertebrates and vertebrates.The research suggested that the miRNAs play a great role in the regulation of gene expression.Bioinformatics tells us that about 1/3 of total human genes are regulated by the miRNAs,suggesting that the miRNAs are the core component of the gene regulation network.Some of them are also related to some human diseases.The research of miRNA in the recent years is reviewed.

The activity of protease from Deinococcus radiodurans R1 (DRR1) recovered from UV light and gamma-ray irradiation were characterized by SDS-PAGE zymography or FITC-labeled casein as substrate. It was demonstrated that proteases with large molecular weight were abundantly and constitutively expressed in DRR1, which consistent with the hypothesis that proteases were among the PHX (predicted highly expressed) proteins in DRR1. A broad range of gelatinolytic protease with molecular mass more than 140 kD and caseinolytic protease with molecular mass more than 120 kD were present in DRR1 cell. The prominent gelatinolytic protease of 174 kD was also active when denatured by SDS and displayed different operation dynamics during the repair process post UV-irradiation and gamma-irradiation, the protease showed its highest activity during gamma-irradiation and in the late recovery stage post-irradiation. Some proteases were demonstrated to be induced specifically by irradiation which suggests that these proteases may regulate breakdown of proteins responsible in sequential pathway, and an elaborate and fine tuning protease system may exist in D. radiodurans during the repair process post irradiation. The expression of protease was also influenced by the nutrition of the culture. A rod-shaped and radiation resistant bacterium RR533.2 isolated from soil in Beijing, China was revealed to display similar zymography pattern using gelatin or casein as substrate.

随着工农业的不断发展,重金属污染日益严重,尤其是土壤和水源中重金属可在农作物和水生动植物中产生累积效应,对人类健康构成潜在威胁.与其他污染物有所不同的是重金属不能被化学或生物降解,因此对它的治理更加困难和复杂.传统上对重金属污染的治理主要采用物理、化学或机械的方法,这些方法不仅造成人力、物力和财力上的极大消耗,而且效果也不明显.随着生物工程技术的发展,生物治理重金属污染具有极大的潜力和广阔的应用前景,越来越引起人们的广泛关注.

放射性废弃物污染治理是一项全球性课题,耐辐射奇球菌具有超强的辐射抗性,在放射性环境中,目前已经有一系列重金属抗性/还原以及有机物降解基因在耐辐射奇球菌中得到成功表达,显示出耐辐射奇球菌在放射性环境中行使生物修复功能的诱人前景,针对目前的进展及存在问题作一综述.

Objective To review the studies on biological effects of microwave irradiation on central nervous system in recent years. Literature resource and selection Research papers and written research reports in this field. Literature quotation Papers which have been published home and abroad were cited. Literature synthesis It was summarized into the biological effects of central nervous system caused by microwave irradiation,especially the nonthermal effect,and some new study points have been emphasized. Conclusions Central nervous system could be easily affected by microwave irradiation. After low-level exposure to microwave radiation repeatedly for a long time,the negative effect would appear.

为获得端粒酶阳性肿瘤细胞特异表达载体用于癌症的基因治疗,克隆并构建了人端粒酶催化亚基(hTERT)基因启动子调控的萤光素酶报告载体.用脂质体转染法将其分别转染肿瘤细胞和正常细胞,检测其在肿瘤细胞和正常细胞中的转录活性. hTERT启动子在所检测的4种端粒酶阳性的肿瘤细胞中具有明显的转录活性,平均为阳性对照的44.3%;而在端粒酶阴性的正常人胚肺成纤维细胞中则无明显的转录活性.提示hTRET启动子的转录活性在端粒酶阳性的肿瘤细胞中明显上调,由hTERT启动子构建的载体可能是一种新颖和有前景的肿瘤细胞特异性表达的基因治疗载体.

目的:探讨用光谱测定法对蛋白质的S-亚硝基化修饰进行检测.方法:以牛血清白蛋白(BSA)作目标蛋白,在酸性条件下用亚硝酸盐使BSA发生S-亚硝基化修饰,Hg2+离子解离亚硝基化蛋白的NO基团,所生成的NO与磺胺形成的重氮化物,再与乙二胺形成显色产物,其光密度值在一定的范围内与NO基团的浓度成线性关系,用亚硝酸盐所制作的标准曲线可定量检测目标蛋白的S-亚硝基化修饰.结果与结论:亚硝酸盐在4.0×10-3～6×10-2 mmol/L浓度范围与显色产物的光密度值存在良好线性关系,在总体蛋白中可检测到的S-亚硝基化蛋白的比率为1/10.光谱测定法是检测蛋白质S-亚硝化修饰的一种简便易行的方法.

ObjectiveTo observe the killing effect on hepatocellular carcinoma(HCC) cell lines by growth response-1(Egr-1) promoter activated herps simplex virus thymidine kinase (tk). MethodsPlasmid pET was constructed by fusing of Egr-1 promoter to the upstream of tk gene and transfect human hepatocellular carcinoma cell lines SMMC-7721(SMMC/ET) with lipofectamine as a delivery system. The cloned cells, after selected with G418, and exposure to γ-radiation by a 60Co source, were added with prodrug GCV. The viability of cell lines was observed. ResultsAfter irradiation, transfected cell lines (0.07±0.03) was killed by prodrug GCV at higher percentage compared with control group(0.88±0.12)(P0.001).The viability was 8% of control group.ConclusionsRadio-sensitized suicide gene activated by γ-ray significantly enhanced killing on HCC.

一氧化氮(nitric oxide,NO)为内皮细胞舒血管因子(endo-thelium-derived relaxing factor,EDRF),它作为重要的信使分子,对平滑肌细胞的收缩与舒张,神经元兴奋与抑制,分化与增殖,凋亡与衰老等生命活动具有广泛的功能调节和信号转导作用,参与心血管、神经、呼吸、免疫、内分泌、消化、生殖等多系统的生理与病理过程.

Telomerase is a ribonucleoprotein that catalyzes telomere elongation through the addition of TTAGGG repeats in humans. Activation of telomerase is often associated with immortalization of human cells and cancer. Partially purification of telomerase was obtained by chromatography of HeLa cell extract on DEAE-Sepharose FF,Spermine-Sepharose CL-6B and Sephacryl S-300. To dissect the human telomerase enzyme mechanism, the optimal catalytic conditions of telomerase were measured and the human telomerase activity was reconstituted using a purified hTR RNA. The optimum pH is from 6^5 to 8^5, the optimum temperature is from 35℃ to 40℃, the optimum Mg 2+ concentration is from 1 mmol/L to 2^5 mmol/L,the apparent binding constant (K m app) of telomerase for the primer is about 8^6 nmol/L. Partially purified human telomerase extract was treated with MNase to remove endogenous telomerase RNA(hTR),the telomerase activity was inactivation. Telomerase activity was restored by incubating MNase-treated extract with purified hTR. These results might facilitate the development of the telomerase enzyme mechanism.

AIMTo investigate the mechanism of protective effect of SOD (superoxide dismutase) on damage of RBCs stored at 4 degrees C, the studies of erythrocyte glucose and energy metabolism were performed.METHODSwhole blood collected from healthy donors and stored at 4 degrees C in ACD, GMA and SOD solutions. Before and post storage, some parameters were assayed. Standard methods were used for the in vitro tests. The 24-hour in vivo recoveries were measured by FTTC (Fluorescein 5-isothiocyanate) from SIGMA Company.RESULTSAll parameters of red blood cell glucolysis rate without oxygen condition, ATP, PK (pyruvic kinase) and 24 h recoveries level were 86.2%, 56.4%, 64.3% and 86.2% of normal respectively stored in SOD solution at 4 degrees C for 75 days, distinctly more than in ACD and GMA groups at 75 days stored. The 24 h recovery at 75d in group SOD was near the recovery at 42d in group GMA.CONCLUSIONWhole blood in SOD solution can be stored satisfactorily for 75 days at 4 degrees C, and furnished theoretical evidence for RBCs survival.

Objective To improve the efficacy of herpes simplex virus thymidine kinase (tk) expression by the way of radiation inducible early growth response 1 (Egr 1) promoter.Methods Plasmid pET was constructed by fusing of Egr 1 promoter to the upstream of tk gene and transfect human hepatocellular carcinoma (HCC) cell line (SMMC 7721) with lipofectamine as a delivery system. The cloned cells had been selected with G418. After exposure to γ radiation by a 60 Co source, cellular tk mRNA expression in hepatoma cells was compared by RT PCR analysis.Results After irradiation, the tk mRNA expression of transfected cell lines was increased markedly compare with non irradiation cell lines (55.2±7.2), especially in 15?Gy dosage (117.2±11.1, P0.001).Conclusions As a radio inducible promoter, Egr 1 can activates tk expression significantly in transduced hepatoma cell line after irradiation. These in vitro data will provide an experimental basis for gene therapy and for further research of gene radiotherapy in HCC.

Objective: To investigate the role of telomerase activation in aging by maintaining cultures of hTRT-transduced human fetal lung fibroblasts. Methods: Following delivery of the hTRT gene to human fetal lung fibroblasts using a PCI-hTRT vector, cell clones that expressed functional telomerase activity were screened by G418. Telomerase activity, hTR RNA and hTRT mRNA in normal fibroblasts and hTRT-transduced human fetal lung fibroblasts were investigated. Results: Human fetal lung fibroblasts clones with positive telomerase were obtained, and the cellular life span of hTRT-transduced human fetal lung fibroblasts was extended. Conclusions: Ectopic expression of telomerase (hTRT) in human fetal lung fibroblasts has been shown to restore telomerase activity and to extend cellular life span.

Objective To investigate the association of a variable number of tandem repeats (VNTR) polymorphism in intron 4 of the endothelial nitric oxide synthase (eNOS) gene with essential hypertension (EH) in Chinese population Methods By using the designed primers based on flanking sequences of VNTR locus, VNTR fragments were amplified by PCR from genomic DNA of normotension (NT) and EH subjects, respectively PCR products were separated through agarose gel electrophoresis for VNTR genotyping The significance of difference in the genotype and allele frequencies between NT and EH groups were assessed by statistical analysis Results Three alleles, containing 4,5 and 6 repeats, and four genotypes, namely 4/4 homozygous, 4/5 heterozygous, 5/5 homozygous and 5/6 heterozygous, were identified in both NT and EH groups The overall distributions of either allele or genotype frequencies differed significantly between the two groups, the genotypes of the 4 repeats allele(χ 2=30 80, P 0 000 1), 4/5 heterozygous (χ 2=21 45, P 0 000 1) and 4/4 homozygous (χ 2=21 45, P 0 000 1) were more frequent in the EH group than in NT group Conclusion The 4 repeats allele in eNOS gene is associated with EH and might be a risk factor of EH for the persons carrying it (χ 2=33 96, P 0 001, OR =3 2, 95%CI 2 097 4 495)

目的:利用计算机软件对端粒酶催化亚基抗原决定簇进行模拟预测分析,制备端粒酶催化亚基组合抗原决定簇重组蛋白,为制备端粒酶催化亚基单克隆抗体提供条件.方法:以Goldkey软件模拟分析端粒酶催化亚基抗原决定簇,设计抗原决定簇的PCR扩增系列引物,组合PCR扩增获得组合抗原决定簇序列并克隆到pGEM-T Easy Vector载体中,组合抗原决定簇基因序列插入pGEX-6P-1中,诱导表达融合蛋白,亲和层析获得组合融合蛋白重组抗原.结果:发现了4个可能性较大、序列较长的抗原决定簇氨基酸序列,获得了重组人端粒酶催化亚基组合融合蛋白.结论:实验结果表明,计算机辅助设计和组合扩增技术是获得含多位点抗原决定簇的端粒酶催化亚基组合抗原融合蛋白的有效方法.

Objective： To investigate the in vivo inhibitory role ofoncostatin M on the melanoma growth as well as the potential application of OSM in the gene-radiotherapy for melanoma. Methods： The OSM expression vector (pO) and the OSM radiation-inducible expression vector (pEO), in which the regulatory sequence of Egr-1 gene (Egr-1R) was linked upstream to OSM cDNA, were used to transfect mouse B16 melanoma cells, respectively. OSM secreting cells (pO-17 and pEO-1) selected and then inoculated substaneously into C57BL/6 mice. A comparison of tumor weight after cells inoculation and an investigation on inhibition rate of tumors after treatment with localized 60Co γ-ray irradiation were taken. Results： The in vivo tumor weight of pO-17 and pEO-1 cells was drastically reduced compared to either B16 or pCI-neo-transfected cells in the same period of twenty days after cells inoculation. It was further observed that pEO-1 cells, but not pO-17, demonstrated an increase of 42.4% in the inhibition rate of tumor when tumors were treated with 20Gy of 60Co γ-ray irradiation twice. Conclusion： The in vivo growth of melanoma could be controlled by OSM secreted from OSM gene-modified cells. The activated Egr-1R by radiation could enhance the expression of OSM and thereby impose an aggravated damage on tumors in vivo, namely brining a dual inhibitory effect of gene and radiation on tumors.

To establish a method for quantitative assessment of mixed hematopoietic chimerism by competitive polymerase chain reaction after nonmyeloablative allogeneic stem cell transplantation　(NAST) using highly polymorphic short tandem repeats (STRs). The feasibility of this assay and the accuracy of quantitative results were tested using serial DNA mixtures of unrelated individuals. 2　patients receiving NAST were analyzed by this method. Chromosome　was analyzed simultaneously. The results showed a linear correlation between the DNA added and the proportion found. Comparison of values obtained with chromosome　analysis showed an excellent correlation with the STR-PCR results. Taken together, this novel approach allows a rapid, sensitive, versatile, less blood , even with minuscule numbers of cells,and also sex unlimited.