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克柔念珠菌临床分离株对伊曲康唑耐药的分子表达情况

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中华传染病杂志 2015年第12期33卷 751-757页

作者：贺小圆赵明峰陈锦艳武日茂张坚磊崔蕊江嫣雨陈洁曹小立邢艺邓琦天津医科大学一中心临床学院天津市第一中心医院血液科 300192 天津市第一中心医院检验科

目的初步探讨克柔念珠菌临床分离株耐伊曲康唑的分子表达情况。方法对16株克柔念珠菌唑类靶酶基因ERG11进行PCR扩增和测序，比较伊曲康唑敏感组（3株）、剂量依赖性敏感组（8株）和耐药组（5株）的基因突变情况。同时，利用实时荧光定量... 详细信息

目的初步探讨克柔念珠菌临床分离株耐伊曲康唑的分子表达情况。方法对16株克柔念珠菌唑类靶酶基因ERG11进行PCR扩增和测序，比较伊曲康唑敏感组（3株）、剂量依赖性敏感组（8株）和耐药组（5株）的基因突变情况。同时，利用实时荧光定量PCR检测3组靶酶基因ERG11、外排泵基因ABC1和ABC2的mRNA表达差异。将数据转换后服从正态分布且方差齐，两组间比较采用双侧t检验。结果16株克柔念珠菌ERG11基因共存在7个突变位点，其中6个同义突变，1个错义突变（C44T），3组均可出现该错义突变。克柔念珠菌临床分离株伊曲康唑耐药组ERG11mRNA表达量较剂量依赖性敏感组和敏感组高（P值分别为0．015、0．002）；耐药组ABC2mRNA表达量也较剂量依赖性敏感组和敏感组高（P值分别为0．016、〈0．01），且剂量依赖性敏感组mRNA表达量高于敏感组（P=0．007）；而耐药组ABC1mRNA表达量较低。1株同时耐伊曲康唑和伏立康唑的克柔念珠菌（cK10），其职G11、ABC1和ABC2mRNA表达水平在所有耐药株中均为最高。结论ERG11基因序列存在多态性，未发现与伊曲康唑耐药有关的突变位点。ERG11和ABC2基因表达上调可能是克柔念珠菌临床分离株耐伊曲康唑的主要分子机制。

关键词：克柔念珠菌伊曲康唑抗药性多药聚合酶链反应反转录聚合酶链反应

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RT-PCR检测CPIV方法的建立与应用

新疆农业科学 2011年第10期48卷 1875-1880页

作者：简子健马素贞刘腾飞翟少华赵森新疆农业大学动物医学学院乌鲁木齐830052

【目的】建立CPIV的RT-PCR检测方法。【方法】从临床上疑似犬副流感(CPI)的犬鼻腔分泌物中提取CPIV总RNA。根据Genebank中收录的CPIV(序列号AY581307)中NP基因保守序列,设计一对特异性引物,扩增出667 bp片段,以此建立CPIV的RT-PCR检测... 详细信息

【目的】建立CPIV的RT-PCR检测方法。【方法】从临床上疑似犬副流感(CPI)的犬鼻腔分泌物中提取CPIV总RNA。根据Genebank中收录的CPIV(序列号AY581307)中NP基因保守序列,设计一对特异性引物,扩增出667 bp片段,以此建立CPIV的RT-PCR检测方法。【结果】特异性试验表明该引物不能扩增犬其它常见的传染病病毒基因。敏感性试验表明该引物能检测到10^(-6)的CPIV总RNA量。重复性试验表明该引物具有良好的稳定性和重复性。对临床采集的32份样品进行RT-PCR检测时,阳性率为53.12%。【结论】实验建立了CPI的RT-PCR检测方法,可适合于临床CPIV的早期快速诊断和小规模的分子流行病学调查。

关键词：犬副流感病毒 NP基因反转录聚合酶链反应早期快速诊断

Development of a reverse transcription quantitative polymerase chain reaction-based assay for broad coverage detection of African and Asian Zika virus lineages

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Virologica Sinica 2017年第3期32卷 199-206页

作者： Yang Yang Gary Wong Baoguo Ye Shihua Li Shanqin Li Haixia Zheng Qiang Wang Mifang Liang George F Gao Lei Liu Yingxia Liu Yuhai Bi Shenzhen Key Laboratory of Pathogen and Immunity State Key Discipline of Infectious DiseaseShenzhen Third People’s Hospital CAS Key Laboratory of Pathogenic Microbiology and Immunology Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease Institute of Microbiology Center for Influenza Research and Early-warning (CASCIRE) Chinese Academy of Sciences China-Japan Union Hospital of Jilin University Key Laboratory for Medical Virology NHFPC National Institute for Viral Disease Control and Prevention China CDC Office of Director-General Chinese Center for Disease Control and Prevention Savaid Medical School University of Chinese Academy of Sciences

The Zika virus(ZIKV) is an arbovirus that has spread rapidly worldwide within recent times. There is accumulating evidence that associates ZIKV infections with Guillain-Barré Syndrome(GBS) and microcephaly in humans. The ZIKV is genetically diverse and can be separated into Asian and African lineages. A rapid, sensitive, and specific assay is needed for the detection of ZIKV across various pandemic regions. So far, the available primers and probes do not cover the genetic diversity and geographic distribution of all ZIKV strains. To this end, we have developed a one-step quantitative reverse transcription polymerase chain reaction(qRT-PCR) assay based on conserved sequences in the ZIKV envelope(E) gene. The detection limit of the assay was determined to be five RNA transcript copies and 2.94 × 10^(–3) 50% tissue culture infectious doses(TCID50) of live ZIKV per reaction. The assay was highly specific and able to detect five different ZIKV strains covering the Asian and African lineages without nonspecific amplification, when tested against other flaviviruses. The assay was also successful in testing for ZIKV in clinical samples. Our assay represents an improvement over the current methods available for the detection ZIKV and would be valuable as a diagnostic tool in various pandemic regions.

关键词：逆转录聚合酶链反应病毒检测覆盖血统亚洲非洲反转录聚合酶链反应特异性扩增

RRAS:A key regulator and an important prognostic biomarker in biliary atresia

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World Journal of Gastroenterology 2011年第6期17卷 796-803页

作者： Rui Zhao Hao Li Chun Shen Shan Zheng Department of Pediatric Surgery Children's Hospital of Fudan University Shanghai 201102 China

AIM:To characterize the differentially expressed gene profiles in livers from biliary atresia (BA) patients including,ascertain genes,functional categories and pathways that play a central role in the pathogenesis of BA,and identify the novel prognostic markers for BA.METHODS:Liver tissue samples from control patients,neonatal cholestasis patients,and BA patients at the age of 90 d were pooled for DNA microarray analysis.Bioinformatics analysis was performed using,series test cluster of gene ontology,and Pathway-Finder software.Reverse-transcription polymerase chain reaction was performed to confirm changes in selected genes.Relation between RRAS gene expression and prognosis of 40 BA patients was analyzed in a 2-year follow-up study.RESULTS:The 4 identified significant gene expression profiles could confidently separate BA liver tissue from normal and other diseased liver tissues.The included genes were mainly involved in inflammation response and reconstruction of cellular matrix.The significant pathways associated with BA were primarily involved in autoimmune response,activation of T lymphocytes and its related cytokines.The RRAS,POMC,SLC26A6 and STX3 genes were important regulatory modules in pathogenesis of BA.The expression of RRAS was negatively correlated with the elimination rate of jaundice and positively correlated with the occurrence rate of cholangitis.CONCLUSION:Autoimmune response mediated by T lymphocytes may play a vital role in the pathogenesis of BA.The RRAS gene is an important regulatory module in the pathogenesis of BA,which may serve as a novel prognostic marker for BA.

关键词： RRAS 预后闭锁胆道差异表达基因标志物调节器反转录聚合酶链反应

siRNA targeting of Cdx2 inhibits growth of human gastric cancer MGC-803 cells

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World Journal of Gastroenterology 2012年第16期18卷 1903-1914页

作者： Xiao-Tong Wang Yu-Bo Xie Qiang xiao Department of Surgery The First Affiliated HospitalGuangxi Medical UniversityNanning 530021Guangxi Zhuang Autonomous RegionChina Department of Anesthesiology The First Affiliated HospitalGuangxi Medical UniversityNanning 530021Guangxi Zhuang Autonomous RegionChina

AIM:To investigate the effects of small interference RNA(siRNA) targeting of Cdx2 on human gastric cancer MGC-803 cells in vitro and in vivo.METHODS:The recombinant pSilencer 4.1-Cdx2 siRNA plasmids were constructed and transfected into gastric cancer MGC-803 cells in vitro.The stable transfectants were selected.The effects of Cdx2 siRNA on growth,proliferation,cell cycle,apoptosis,migration and invasiveness of human gastric cancer MGC-803 cells were evaluated and the expression of phosphatase and tensin homolog(PTEN),caspase-9 and caspase-3 was observed in vitro by reverse transcription polymerase chain reaction(RT-PCR) and Western blotting analysis.We also investigated the effect of Cdx2 siRNA on growth of MGC-803 cells in nude mice in vivo.RESULTS:Cdx2 siRNA led to inhibition of endogenous Cdx2 mRNA and protein expression as determined by RT-PCR and Western blotting analysis.Cdx2 siRNA significantly inhibited cell growth and proliferation,blocked entry into the S-phase of the cell cycle,induced cell apoptosis,and reduced the motility and invasion of MGC-803 cells.Cdx2 siRNA also increased PTEN expression,and activated caspase-9 and caspase-3 in MGC-803 cells in vitro.In addition,siRNA targeting of Cdx2 inhibited the growth of MGC-803 cells and promoted tumor cell apoptosis in vivo in nude mice tumor models.CONCLUSION:Cdx2 was involved in regulating progression of human gastric cancer cells MGC-803.Manipulation of Cdx2 expression may be a potential therapeutic strategy for gastric cancer.

关键词： siRNA 细胞周期胃癌细胞细胞生长 caspase-3 RT-PCR法反转录聚合酶链反应 PTEN基因

Effect of allogeneic umbilical cord mesenchymal stem cell transplantation in a rat model of hepatic cirrhosis

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Journal of Traditional Chinese Medicine 2015年第1期35卷 63-68页

作者： Yang Lihua Wang Yun Wang Xiaohua Liu Yanqing Institution of Traditional Chinese Medicine and Western Medicine Nanjing University of Chinese Medicine Institute of Digestive Endoscopy & Medical Center for Digestive Diseases the Second Affiliated Hospital of Nanjing Medical University

OBJECTIVE: To determine the effects of human umbilical cord mesenchymal stem cell(UCMSC) transplantation, alone or in combination with tanshinone IIA(Tan IIA) on hepatic cirrhosis in rats.METHODS: A rat model of cirrhosis was established. Rats were divided into control, UCMSC, and UCSMC plus Tan IIA groups. Rats in the UCMSC group were injected via the tail vein with 0.2 m L Di I-labeled UCMSC suspension. Intraperitoneal Tan IIA injections(20 mg/kg) were started on the day ofUCMSC transplantation in the UCMSC plus Tan IIA group, and continued for 7 consecutive days thereafter. Rats were sacrificed 1 day, 3 days, 1 month,and 3 months after transplantation and the numbers of Di I-labeled UCMSCs colonizing the liver were determined. Albumin(ALB) and alanine aminotransferase(ALT) levels were measured in venous blood, and m RNA and protein expression levels of human ALB and cytokeratin(CK)-18 in liver tissues were determined by reverse transcription-polymerase chain reaction and western blotting, respectively.RESULTS: Serum ALT levels were significantly lower and serum ALB levels significantly higher in rats in the UCMSC group compared with the control group(P < 0.05). Hepatic CK-18 and ALB m RNA and protein expression levels increased after transplantation, and were significantly higher in the UCMSC plus Tan IIA group compared with the UCMSC group(P < 0.05).CONCLUSION: Human UCMSCs transplanted into rats with liver cirrhosis can grow and differentiate into hepatocyte-like cells resulting in improved liver function in vivo. Tan IIA further influenced transplantation outcomes.

关键词：干细胞移植大鼠模型肝硬化脐带 Western印迹反转录聚合酶链反应间充质干细胞丙氨酸转氨酶

染料法实时荧光聚合酶链反应检测人外周血αβT淋巴细胞克隆的条件优化及初步应用

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中华传染病杂志 2010年第11期28卷 645-650页

作者：高海女俞海英杨介钻李敏伟何剑琴浙江大学医学院附属第一医院传染科传染病诊治国家重点实验室杭州310003

目的探讨实时荧光PCR检测人外周血αβT淋巴细胞克隆性扩增的反应条件及在监测慢性乙型肝炎（慢乙肝）患者外周血克隆特异性T淋巴细胞上的初步应用.方法染料法（SYBR Green Ⅰ）实时荧光PCR对6例健康献血者外周血单个核细胞（PBMC）... 详细信息

目的探讨实时荧光PCR检测人外周血αβT淋巴细胞克隆性扩增的反应条件及在监测慢性乙型肝炎（慢乙肝）患者外周血克隆特异性T淋巴细胞上的初步应用.方法染料法（SYBR Green Ⅰ）实时荧光PCR对6例健康献血者外周血单个核细胞（PBMC）来源的T淋巴细胞受体β链可变区（TCRBV）基因进行扩增,分别就退火温度、引物浓度、循环数等进行比较研究.优化后PCR检测12例慢乙肝患者PBMC来源的TCRBV的24个基因家族,作PCR产物的熔解曲线峰形图,并分析T淋巴细胞克隆性扩增情况.结果染料法实时荧光PCR检测 TCRBV基因家族的最佳退火温度为60.6℃,引物终浓度为0.5 μmol/L,循环数为40个,且熔解曲线分析起始温度80℃优于75℃.PCR产物熔解曲线峰形图上发现,慢乙肝患者某些TCRBV基因家族表现为单峰,测序结果表明其为单克隆PCR产物.结论本研究优化了染料法实时荧光PCR检测TCRBV基因家族方法,熔解曲线峰形图可用于检测人外周血T淋巴细胞的克隆性扩增,并可能用于检测慢乙肝患者外周血克隆特异性T淋巴细胞.

关键词：反转录聚合酶链反应受体抗原 T细胞基因扩增肝炎乙型慢性

儿童急性淋巴细胞白血病169例遗传学异常分析及其与早期治疗反应的关系

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中华实用儿科临床杂志 2015年第15期30卷 1143-1146页

作者：岳志霞李彬赵晓曦李伟京郑胡镛首都医科大学附属北京儿童医院血液肿瘤中心、儿童血液病与肿瘤分子分型北京市重点实验室、儿科学国家重点学科、教育部儿科重大疾病研究重点实验室 100045

目的探讨儿童急性淋巴细胞白血病（ALL）常见遗传学异常及其与早期治疗反应的关系。方法应用染色体核型分析、多重巢式反转录聚合酶链反应（RT-PCR）和荧光原位杂交（FISH）技术对2010年12月至2011年12月首都医科大学附属北京儿童医院... 详细信息

目的探讨儿童急性淋巴细胞白血病（ALL）常见遗传学异常及其与早期治疗反应的关系。方法应用染色体核型分析、多重巢式反转录聚合酶链反应（RT-PCR）和荧光原位杂交（FISH）技术对2010年12月至2011年12月首都医科大学附属北京儿童医院血液肿瘤中心收治的169例初诊ALL患儿进行遗传学检查，并观察遗传学异常与早期治疗反应的关系。结果169例ALL患儿骨髓细胞标本培养成功162例，培养成功率95．9％，其中88例具有克隆性染色体异常，异常核型检出率为52．1％。接受RT-PCR检查的153例患儿中，55例检测出携带8种融合基因，异常检出率为35．9％。应用FISH技术对40例ALL患儿进行混合谱系白血病（MLL）基因重排检测，6例阳性。将常规细胞遗传学与Rt-PCR和FISH结果联合分析，105例具有遗传学异常，使异常检出率提高到62．1％。将ALL患儿遗传学异常分为6个组：t（12；21）、t（1；19）、t（9；22）、MLL基因重排、超二倍体和-6／6q-及-7／7q-，6组遗传学异常在3种不同早期治疗反应中，即第8天泼尼松诱导试验、第33天骨髓和微小残留病的结果中，差异均具有明显的统计学意义（X2=22．954、19．432、14．045，P=0．001、0．001、0．016）。结论常规细胞遗传学联合RT-PCR、FISH技术，可以提高儿童ALL遗传学异常的检出率，将遗传学异常与患儿早期治疗反应联合分析，可以更好地指导临床治疗，并进行预后评估。

关键词：急性淋巴细胞白血病细胞遗传学荧光原位杂交反转录聚合酶链反应微小残留病

机械力作用下人牙周膜细胞ODF及OCIF的表达及意义

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实用口腔医学杂志 2005年第1期21卷 85-87页

作者：王峰林珠李永明杨美祥袁璐北京海军总医院口腔科 100037 第四军医大学口腔医学院

目的 :观察周期性机械牵张力对人牙周膜细胞破骨细胞分化因子 (ODF)及破骨细胞生成抑制因子(OCIF)mRNA表达的影响 ,探讨正畸牙周组织改建的分子机制。方法 :通过体外细胞培养加载系统施于人牙周膜细胞周期性牵张力 ,利用RT PCR检测技术 ... 详细信息

目的 :观察周期性机械牵张力对人牙周膜细胞破骨细胞分化因子 (ODF)及破骨细胞生成抑制因子(OCIF)mRNA表达的影响 ,探讨正畸牙周组织改建的分子机制。方法 :通过体外细胞培养加载系统施于人牙周膜细胞周期性牵张力 ,利用RT PCR检测技术 ,观察人牙周膜细胞ODF及OCIFmRNA表达的相对强度。结果 :体外培养的人牙周膜细胞在正常情况下表达ODF及OCIF ,当间歇性牵张力作用 6、12、2 4h后 ,随着作用时间的延长 ,人牙周膜成纤维细胞ODFmRNA的表达有减弱趋势 ;而OCIFmRNA的表达有增强趋势。结论 :机械牵张力可以调节人牙周膜细胞ODF、OCIF的表达 ,从而可以调节骨吸收作用。

关键词：牙周膜细胞机械张力反转录聚合酶链反应破骨细胞分化因子破骨细胞生成抑制因子