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Establishment and Application of a TaqMan Real-Time Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Rubella Virus RNA

中国遗传学会“发育、遗传和疾病”研讨会

作者： Qiang Wang~1 Zheng Huang~2 Huiling Xue~2 Chengcheng Jin~1 Xiu-Li Ju~3 Jing-Dong J.Han~2 Ye-Guang Chen~(1#) 1 State Key Laboratory of Biomembrane and Membrane Biotechnology,department of Biological Sciences and Biotechnology,Tsinghua university,Beijing 100084,china 2 CAS Key Laboratory of Molecular Developmental Biology and Center for Molecular Systems Biology,Institute of Genetics and Developmental Biology,the Chinese Academy of Sciences,Beifing 100101, china 3 department of pediatrics,qilu hospital of shandong university,jinan 250012,china

MicroRNAs have been suggested to modulate a variety of cellular *** we report that miR-24 regulates erythroid differentiation by influencing the expression of human activin type I receptor ALK4(bALK4).Ectopic expression of miR-24 reduces the mRNA and protein levels of bALK4 by targeting its 3'-untranslated region and interferes with activin-induced Smad2 phosphorylation and reporter ***,miR-24 represses the activin-mediated accumulation of hemoglobin,an erythroid differentiation marker,in erythroleukemic K562 cells and decreases erythroid colony-forming and burst-forming units of CD34+ hematopoietic progenitor ***4 expression is reversely correlated with miR-24 expression during erythroid ***,our findings define a regulation mode of miR-24 on erythropoiesis by impeding ALK4 expression.

关键词： ALK receptor ALK4 MicroRNA miR-24 inhibits erythropoiesis by targeting activin type miR

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Acta Biochimica et Biophysica Sinica 2006年第10期38卷 731-736页

作者： Li-Hong ZHAO~1* Yu-Yan MA~2 Hong WANG~1 Shu-Ping ZHAO~3 Wei-Ming ZHAO~1 Hua LI~2 Lei-Yi WANG~2 1 department of Microbiology,School of Medicine,shandong university,jinan 250012,china 2 qilu hospital,shandong university,jinan 250012,china 3 Tai'an central hospital,Tai'an 271000,china 4 department of Virology,School of Public Health,shandong university,jinan 250012,china department of Microbiology School of Medicine Shandong UniversityJinan 250012 China qilu hospital Shandong UniversityJinan 250012 China Tai'an central hospital Tai'an 271000 China

The aim of this study was to establish and apply a real-time quantitative reverse transcription-polymerase chain reaction （RT-PCR） for rubella virus （RV） ***,the primer and TaqMan probeconcentrations,as well as reaction temperatures were optimized to establish an efficient real-time quantitativeRT-PCR assay for RV ***,an RV-specific PCR amplicon was made as an external standard toestimate the linearity,amplification efficiency,analytical sensitivity and reproducibility of the real timequantitative ***,the assay was applied to quantify RV RNA in clinical samples for rubella *** RV-specific PCR amplicon was prepared for evaluation of the assay at 503 bp,and its original concentrationwas 2.75×10～9 copies/μ*** real time quantitative assay was shown to have good linearity （R2=0.9920）,highamplification efficiency （E=1.91）,high sensitivity （275 copies/ml）,and high reproducibility （variation coefficientrange,from 1.25% to 3.58%）.Compared with the gold standard,the specificity and sensitivity of the assayin clinical samples was 96.4% and 86.4%,***,the established quantitative RT-PCRmethod is a simple,rapid,less-labored,quantitative,highly specific and sensitive assay for RV RNA.

关键词： rubella virus PCR amplicon real-time fluorescence quantitative RT-PCR

Effects of Raloxifene on Caveolin-1 mRNA and Protein Expressions in Vascular Smooth Muscle Cells

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Acta Biochimica et Biophysica Sinica 2006年第11期38卷 747-752页

作者： Fa-Lin YANG~(1#*) Hong HE~(2#) Xian-Xi LIU~1* Bing TU~1 Xian-Wei ZENG~3 Ji-Xin SU~1 Xin WANG~2 Qin HU~2 1 Institute of Biochemistry and Molecular Biology,Medical College,shandong university,jinan 250012,china 2 department of Cardiology,qilu hospital,shandong university,jinan 250012,china 3 department of Clinical Medicine,Weifang Medical College,Weifang 260041,china Institute of Biochemistry and Molecular Biology Medical College Shandong UniversityJinan 250012 China department of Cardiology Qilu Hospital Shandong UniversityJinan 250012 China department of Clinical Medicine Weifang Medical CollegeWeifang 260041 China

Caveolin-1 is regulated by estrogen in vascular smooth muscle ***,a selectiveestrogen receptor modulator that possibly has cardioprotective properties without an increased risk of canceror other side effects of estrogen,may be used in women with risk of coronary artery ***,therelationship between raloxifene and caveolin-1 is still ***,this study was designed to seewhether raloxifene regulates caveolin-1 expression and if so,whether such regulation is mediated by *** aortic smooth muscle cells were cultured in the absence or presence of raloxifene (10-8to10-6M) for 12 or 24 *** mRNA and protein levels of caveolin-1 were increased significantly after 24 htreatment with *** increases were inhibited by estrogen receptor antagonist ICI 182780 (10-5M).Results of this study suggest that raloxifene stimulates caveolin-1 transcription and translation throughestrogen receptor mediated mechanisms.

关键词： raloxifene caveolin-1 estrogen receptor vascular smooth muscle cell

Adenovirus-mediated Expression of both Antisense Ornithine Decarboxylase and S-adenosylmethionine Decarboxylase Inhibits Lung Cancer Cell Growth

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Acta Biochimica et Biophysica Sinica 2007年第6期39卷 423-430页

作者： Hui TIAN~(1*), Xianxi LIU~2, Bing ZHANG~2, Qifeng SUN~1, and Dongfeng SUN~1 1 department of Thoracic Surgery, Qi Lu hospital, shandong university jinan 250012, china 2 department of Medicine, Medical Molecular Biology Experimental Center, shandong university jinan 250012 china department of Thoracic Surgery Qi Lu Hospital Shandong UniversityJinan 250012 China department of Medicine Medical Molecular Biology Experimental Center Shandong UniversityJinan 250012 China

Polyamine biosynthesis is controlled primarily by ornithine decarboxylase （ODC） and Sadenosylmethionine decarboxylase （AdoMetDC）. Antisense sequences of ODC and AdoMetDC genes were cloned into an adenoviral vector （named Ad-ODC-AdoMetDCas）. To evaluate the effects of recombinant adenovirus Ad-ODC-AdoMetDCas that can simultaneously express both antisense ODC and AdoMetDC, the human lung cancer cell line A-549 was infected with Ad-ODC-AdoMetDCas or the control vector. Viable cell counting, determination of polyamine concentrations, cell cycle analysis, and Matrigel invasion assays were carried out to assess the properties of tumor growth and invasiveness. Our study showed that adenovirus-mediated antisense ODC and AdoMetDC expression inhibits tumor cell growth through blocking the polyamine synthesis pathway. Tumor cells were arrested at the G1phase after gene transfer and the invasiveness was reduced. It suggested that the recombinant adenovirus Ad-ODC-AdoMetDCas might be a new anticancer reagent in the treatment of lung cancers.

关键词： ornithine decarboxylase S-adenosylmethionine decarboxylase polyamine lung cancer genetherapy

Gene Expression of Ornithine Decarboxylase in Lung Cancers and Its Clinical Significance

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Acta Biochimica et Biophysica Sinica 2006年第9期38卷 639-645页

作者： Hui TIAN~1* Qing HUANG~1 Lm LI~1 Xian-Xi LIU~2 Yan ZHANG~2 1 department of Thoracic Surgery,shandong university Qi Lu hospital,jinan 250012,china 2 department of Medicine,Medical Molecular Biology Experimental Center,shandong university,jinan 250012,china department of Thoracic Surgery Shandong University Qi Lu HospitalJinan 250012 China department of Medicine Medical Molecular Biology Experimental Center Shandong UniversityJinan 250012 China

Lung cancer is one of the most lethal cancers in china because of its high incidence and *** decarboxylase （ODC）,an important enzyme in polyamine biosynthesis,is increased incancer *** chemotherapeutic agents aimed at reducing ODC expression show inhibitory effects oncancer cell growth,so ODC can be useful in gene diagnosis and gene therapy of *** this study,weexamined the effect of antisense ODC on lung cancer cells.A-549 cells were infected with rAd-ODC/Ex3as,a recombinant adenovirus containing the cytomegalovirus promoter,green fluorescent protein gene and 120bp antisense *** cell cycle was evaluated by flow cytometry.A nude mouse xenograft model wasused in the tumorigenicity *** transcription-polymerase chain reaction,Western blot and immuno-histochernistry were used to study the expressions of ODC on lung *** was found that the growth ofcells infected with rAd-ODC/Ex3as was substantially inhibited and cells were arrested at *** with rAd-ODC/Ex3as can suppress tumor formation in a nude mouse xenograft *** of ODC mRNA and ODC protein levels in lung cancer tissues was significantly higher than thatin normal tissues （P3as has antitumor activity in human lung cancer *** ODC gene might playan important role in lung cancer and the overexpression of ODC might be related to the occurrence anddevelopment of lung cancer.

关键词： polyamine biosynthesis lung neoplasm ornithine decarboxylase A-549 cell line

Antitumor Effect of Antisense Ornithine Decarboxylase Adenovirus on Human Lung Cancer Cells

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Acta Biochimica et Biophysica Sinica 2006年第6期38卷 410-416页

作者： Hui TIAN~(1*),Lin LI~1,Xian-Xi LIU~2,and Yan ZHANG~2 ~1 department of Thoracic Surgery,shandong university Qi Lu hospital,jinan 250012,china ~2 Experimental Center of Medical Molecular Biology,School of Medicine,shandong university,jinan 250012,china department of Thoracic Surgery Shandong University Qi Lu HospitalJinan 250012 China Experimental Center of Medical Molecular Biology School of Medicine Shandong UniversityJinan 250012 China

Ornithine decarboxylase (ODC),the first enzyme of polyamine biosynthesis,was found toincrease in cancer cells,especially lung cancer *** chemotherapeutic agents aimed at decreasingODC gene expression showed inhibitory effects on cancer cells,ha this study,we examined the effects ofadenoviral transduced antisense ODC on lung cancer *** adenovirus carrying antisense ODC(rAd-ODC/Ex3as) was used to infect lung cancer cell line *** 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay was used to analyze the effect on cell *** of ODC and concentrationof polyamines in cells were determined by Western blot analysis and high performance liquid *** deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling was used to analyze *** expression of ODC in A-549 cells was reduced to 54%,and that of three polyamines wasalso decreased through the rAd-ODC/Ex3as ***,cell growth was substantially inhibitedand terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling showed that rAd-ODC/Ex3ascould lead to cell apoptosis,with apoptosis index of 46%.This study suggests that rAd-ODC/Ex3as has anantitumor effect on the human lung cancer cells.

关键词： polyamine putrescine biosynthesis antisense technology gene therapy A-549 lung cancer cell line

The Effect of Anti-Human CD134 Monoclonal Antibody on Phytohemagglutinin-Induced mRNA Expression of Perforin in Peripheral Blood Mononuclear Cells

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Cellular & Molecular Immunology 2005年第6期2卷 467-471页

作者： Ming Li~1 Yuanchao Zhang~(1,2) ~1department of Rheumatology and Immunology,shandong Provincial hospital,shandong university,jinan 250021,china ~2department of Rheumatology and Immunology,shandong Provincial hospital,shandong university,jinan 250021,shandong,china. department of Rheumatology and Immunology Shandong Provincial Hospital Shandong University Jinan 250021 China

CD134, a member of the tumor necrosis factor receptor superfamily, plays a crucial role in T cell survival. In this study, peripheral blood mononuclear cells （PBMCs） stimulated with phytohemagglutinin （PHA, 50 μg/ml） were treated by CD134 mAb （1μg/ml, 5 μg/ml, 10 μg/ml） for 6 h, 12 h, 24 h and 48 h. The level of perforin mRNA was measured by reverse transcription-polymerase chain reaction （RT-PCR） technique. Our data showed that the expression of perforin mRNA in PBMCs was down-regulated by CD134 mAb in a dose-dependent manner in range of 1 μg/ml to 5 μg/ml and dropped down to its minimum on 24 h （p 〈 0.05）. The level of perforin mRNA reached a plateau when the concentration of CD134 mAb exceeded 5 μg/ml. In conclusion, CD134 mAb can inhibit perforin expression, which may enhance the ability of T cells for survival. Cellular ＆ Molecular Immunology. 2005;2（6）： 467-471.

关键词： CD134 perforin phytohemagglutinin

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Changes of brain neuropeptide Y and its receptors in rats with flurazepam tolerance and dependence

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Acta Pharmacologica Sinica 2005年第11期26卷 1290-1296页

作者： Li-ping ZHANG, Li WANG~2 department of pediatrics, Peking university First hospital, Beijing 100034, china department of pediatrics Peking University First Hospital Beijing China

Aim: Anticonvulsant tolerance and dependence are two obstacles that restrict the clinical use of benzodiazepines (BDZ). In order to explore the mechanism of these two adverse reactions, changes of neuropeptide Y (NPY) and its receptors in the hippocampus of rat models, in relation to flurazepam (FZP, a member of BDZ) tolerance and dependence, were investigated. Methods: The mRNA of preproNPY and its receptors (Y1, Y2, and Y5) in the hippocampus were determined by competitive RT-PCR, and the distribution of NPY in the hippocampus was examined by immunohistochemistry. Results: A decrease of preproNPY mRNA in the hippocampus was found in tolerant and dependent rats. The level of preproNPY mRNA in the hippocampus was reversely correlated with the degree of tolerance and dependence, measured by the threshold of pentylenetetrazol-induced seizures. Immunohistochemistry indicated a decrease of NPY-immunoreactive material in neurons of the CA1, CA3, and dentate gyrus regions of both tolerant and dependent rats. The mRNA of NPY receptors Y,and Y5decreased in tolerant rats but did not change in dependent rats. The mRNA of NPY receptor Y2increased in tolerant rats but decreased in dependent rats. Conclusion: A decrease of NPY in the hippocampus might be involved in anticonvulsant tolerance and dependence following long-term treatment with FZP. Y,, Y2, and Y5mRNA were also altered in FZP tolerance and dependence.

关键词： benzodiazepines neuropeptide Y flura zepam

Gum mastic increases maspin expression in prostate cancer cells

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Acta Pharmacologica Sinica 2007年第4期28卷 567-572页

作者： Mei-lan HE~(2,3,6),Wei-wen CHEN~(2,6),Peng-ju ZHANG~2,An-li JIANG~2,Wei FAN~4,Hui-qing YUAN~2,Wen-wen LIU~2,Jian-yeZHANG~(2,5)~2department of Biochemistry,Medical School,shandong university,Ji-nan 250012,china ~3Institute of Cancer Research,Life Science andTechnology School,Tongji university Shanghai 200092,china ~4Gastric Intestine department,Affiliated Qi-Lu hospital,shandong university,Ji-nan 250012,china department of Biochemistry Medical School Shandong University Ji-nan China Institute of Cancer Research Life Science and Technology School Tongji University Shanghai China Gastric Intestine department Affiliated Qi-Lu Hospital Shandong University Ji-nan China

Aim:To study whether gum mastic,a natural resin,can regulate maspin expres-sion in prostate cancer cells,and further investigate the mechanisms involved inthis regulatory ***:RT-PCR and Western blotting were used todetect maspin expression at the transcriptional and translational *** assay was used to investigate the effect of gum mastic on the maspin *** binding activity of negative androgen-responsive element (ARE) and posi-tive Spl element in the maspin promoter were studied by electrophoretic mobilityshift ***:Gum mastic induced maspin mRNA and protein expression,and the maspin promoter activity was enhanced with gum mastic ***,gum mastic inhibited the ARE binding activity and increased the Sp 1 bindingactivity in the maspin ***:Gum mastic enhances maspin pro-moter activity by suppressing ARE binding activity and enhancing Spl bindingactivity,and the increased activity in the maspin promoter finally leads to the up-regulation of both its mRNA and protein levels.

关键词： gum mastic maspin expression ARE binding activity Spl binding activity