NADPH oxidase-derived reactive oxygen species involved in angiotensin Ⅱ-induced monocyte chemoattractant protein-1 expression in mesangial cells

Objective To investigate the origin of oxidative stress induced by angiotensin H (Ang Ⅱ ) in human mesangial cells and the role of reactive oxygen species ( ROS) in Ang Ⅱ -induced monocyte chemoattractant protein-1 (MCP-1) expression.Methods MCP-1 expression was determined by real time RT-PCR.ROS production was measured by DCFDA fluorescence.Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was examined by lucigenin chemiluminescence.p47phox and p67phox translocation was assayed by Western blot.Twenty-four male mice were randomly divided into three groups; the control,the Ang Ⅱ infusion [ Ang Ⅱ 400 ng/(kg±min) ],and the apocynin treatment.Ang Ⅱ was infused by subcutaneously osmotic minipump for 14 days.Urinary albumin and 8-isoprostane excretion were measured by ELISA.Results In cultured human mesangial cells,Ang Ⅱ induced the MCP-1 expression in a dose-dependent manner with 3.56 fold increase as compared with the control.Ang Ⅱ increased intracellular ROS production as early as 3 min with the peak at 60 min and was in a time and dose-dependent.Incubation with different dosages of Ang Ⅱ ( 1μmol/L,10μmol/L,and 100μmol/L Ang Ⅱ ) for 60 min,ROS production increased at 1.82,2.92,and 4.08 folds respectively.Ang Ⅱ-induced ROS generation was sensitive to diphenyleneiodonium sulfate (DPI,10 μmol/L) and apocynin (500μmol/L) ,two structurally distinct NADPH oxidase inhibitors.In contrast,inhibitors of other oxidant- producing enzymes,including the mitochondrial complex I inhibitor rotenone,the xanthine oxidase inhibitor allopurinol,the cyclooxygenase inhibitor indomethacin,the lipoxygenase inhibitor nordihydroguiaretic acid,the cytochrome P450 oxygenase inhibitor ketoconazole and the nitric oxide synthase inhibitor G-nitro-L- arginine methyl ester were without an effect Ang Ⅱ -induced ROS generation was inhibited by the ATI antagonist losartan (10μmol/L) but not the AT2 antagonist PD123319 (10μmol/L).Ang Ⅱ treatment induced translocation of cytosolic of p47 and p67 to the membrane.The antioxidants almost abolished Ang Ⅱ -induced MCP-1 expression.Ang Ⅱ infusion increased urinary and p67 translocation by 2.69-,2.97-,and 2.67-fold,respectively.Conclusions NADPH oxidase-derived ROS is involved in Ang Ⅱ-induced MCP-1 expression.Inhibition of NADPH oxidase alleviates Ang Ⅱ -induced renal injury.     

检测FGFR3基因鉴别诊断先天性软骨发育不全

目的 建立一种基因水平上鉴别诊断先天性软骨发育不全的方法。方法 采用PCR—RFLP技术对1例临床确诊的ACH患者，1例临床怀疑为ACH患者的FGFR3基因第10外显子1138位核苷酸作突变型分析。结果 确诊的ACH患者1138核苷酸存在C→A的转换，而临床怀疑为ACH患者的1138核苷酸无任何改变。结论 检测FCFR3基因突变可从分子水平上鉴别诊断先天性软管发育不全，准确率达95％。     

代谢酶PCK2在非小细胞肺癌中的表达及功能

目的探讨PCK2在非小细胞肺癌组织中的表达及临床意义,干扰或过表达PCK2对肺癌细胞增殖和迁移能力的改变。方法构建含有75例非小细胞肺癌的组织芯片,采用免疫组化SP法检测PCK2在肺癌组织中的表达,分析PCK2过表达与临床病理特征的关系。构建PCK2-siRNA和过表达质粒,分别转染肺癌细胞,CCK8实验检测细胞增殖能力和Transwell小室实验检测细胞迁移能力。结果所有肺癌组织和癌旁非肿瘤组织中PCK2均呈不同程度表达,PCK2在肺癌组织中的过表达率为66.7%(50/75),PCK2过表达与淋巴结转移和TNM分期密切相关(P0.05)。干扰PCK2表达后肺癌细胞增殖能力改变不显著,但迁移能力明显抑制,抑制率高达70.1%(P0.000 1),过表达PCK2后肺癌细胞增殖能力无变化,但迁移能力上调2.5倍(P0.000 1)。结论 PCK2与肺癌转移和TNM分期相关,PCK2具有促进转移功能,PCK2有望成为预测肺癌转移的重要标志物,甚至可能成为肺癌治疗的潜在靶点。     

应用合成肽研究柯萨奇B病毒衣壳蛋白VP1共的抗原表位

对柯萨奇Ｂ４病毒（ＣＶＢ４）衣壳蛋白ＶＰ１保守区的亲水性和二级结构进行分析和预测，选择可能代表优势抗原表位的肽段进行合成（ＶＰ１－１肽，ＲＩＹＦ ＫＰＫＨＶＫ ＡＹＶ），并截取了该肽段的前１２个残基（ＶＰ１－２肽）用同样方法进行合成。用ＶＰ１－１肽免疫家兔制备出高效阶抗体，应用酶联免疫吸附法（ＥＬＩＳＡ）检测，这些抗体与ＣＶＢ１－６病毒均有结合反应，ＶＰ１－１肽与型特异性ＣＶＢ１－６抗体均有良好的     

NADPH oxidase-derived reactive oxygen species involved in angiotensin Ⅱ-induced monocyte chemoattractant protein-1 expression in mesangial cells

Objective To investigate the origin of oxidative stress induced by angiotensin H (Ang Ⅱ ) in human mesangial cells and the role of reactive oxygen species ( ROS) in Ang Ⅱ -induced monocyte chemoattractant protein-1 (MCP-1) expression.Methods MCP-1 expression was determined by real time RT-PCR.ROS production was measured by DCFDA fluorescence.Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was examined by lucigenin chemiluminescence.p47phox and p67phox translocation was assayed by Western blot.Twenty-four male mice were randomly divided into three groups; the control,the Ang Ⅱ infusion [ Ang Ⅱ 400 ng/(kg±min) ],and the apocynin treatment.Ang Ⅱ was infused by subcutaneously osmotic minipump for 14 days.Urinary albumin and 8-isoprostane excretion were measured by ELISA.Results In cultured human mesangial cells,Ang Ⅱ induced the MCP-1 expression in a dose-dependent manner with 3.56 fold increase as compared with the control.Ang Ⅱ increased intracellular ROS production as early as 3 min with the peak at 60 min and was in a time and dose-dependent.Incubation with different dosages of Ang Ⅱ ( 1μmol/L,10μmol/L,and 100μmol/L Ang Ⅱ ) for 60 min,ROS production increased at 1.82,2.92,and 4.08 folds respectively.Ang Ⅱ-induced ROS generation was sensitive to diphenyleneiodonium sulfate (DPI,10 μmol/L) and apocynin (500μmol/L) ,two structurally distinct NADPH oxidase inhibitors.In contrast,inhibitors of other oxidant- producing enzymes,including the mitochondrial complex I inhibitor rotenone,the xanthine oxidase inhibitor allopurinol,the cyclooxygenase inhibitor indomethacin,the lipoxygenase inhibitor nordihydroguiaretic acid,the cytochrome P450 oxygenase inhibitor ketoconazole and the nitric oxide synthase inhibitor G-nitro-L- arginine methyl ester were without an effect Ang Ⅱ -induced ROS generation was inhibited by the ATI antagonist losartan (10μmol/L) but not the AT2 antagonist PD123319 (10μmol/L).Ang Ⅱ treatment induced translocation of cytosolic of p47 and p67 to the membrane.The antioxidants almost abolished Ang Ⅱ -induced MCP-1 expression.Ang Ⅱ infusion increased urinary and p67 translocation by 2.69-,2.97-,and 2.67-fold,respectively.Conclusions NADPH oxidase-derived ROS is involved in Ang Ⅱ-induced MCP-1 expression.Inhibition of NADPH oxidase alleviates Ang Ⅱ -induced renal injury.     

食管贲门狭窄的内镜下扩张治疗

食管贲门良恶性狭窄常引起吞咽困难 ,严重影响病人的生活质量。我科自 1997年 6月 - 2 0 0 1年 5月在内镜介导下应用Ｓａｖａｒｙ Ｇｉｌｌｉａｒｄ扩张器 ,扩张治疗各种食管贲门狭窄 6 7例共 81次 ,取得较好临床疗效 ,现报告如下1　对象与方法1 1　临床资料 :本组 6 7例 ,男性 39例 ,女性 2 8例 ,年龄 15 - 80岁 ,平均 5 7岁 ,6 7例中 ,食管和贲门癌术后吻合口狭窄 5 0例 ,食管癌 6例 ,贲门癌 4例 (均为晚期癌失去手术机会者 ) ,贲门失迟缓症 5例 ,化学烧伤后狭窄 2例。病程最短 1月 ,最长 14年。根据临床表现和内镜下吻合口径将狭窄程度分…     

北柴胡特异DNA条形码筛选及种质资源鉴定

目的 基于北柴胡Bupleurum chinense叶绿体基因组筛选特异性DNA条形码,并利用特异DNA条形码鉴定不同产地北柴胡种质资源。方法 利用Illumina HiSeq X Ten平台对北柴胡进行叶绿体基因组测序,利用mVISTA软件和核酸多样性分析筛选特异性片段,基于特异性片段进一步分析不同产区北柴胡样品单倍型。结果 不同产地3份北柴胡叶绿体基因组全长为155 557～155 959 bp,均呈现典型的环状四分体结构,均编码131个基因。trnV-UAC、trnC-GCA_petN、petN_psbM、ndhF_rpl32可以作为潜在的北柴胡种质资源鉴定的特异性DNA条形码。基于扩增效率,选择petN_psbM和ndhF_rpl32对来自4省7个产地177份北柴胡样品进行序列分析,结果表明petN_psbM和ndhF_rpl32分别有91和78个变异位点,分别鉴定到28、29个单倍型;两段序列联合分析形成40个单倍型（Hap1～Hap40）,各单倍型的遗传距离为0～0.022。6个产地拥有特有单倍型,可以将不同产地的北柴胡种质资源进行区分。结论 利用比较叶绿体基因组学筛选的特异DNA条形码petN_psbM和ndhF_rpl32可以用于北柴胡种内种质资源鉴定,为后续鉴定北柴胡的产地来源、种质资源保护利用和育种等工作奠定基础。     

妊娠HELLP综合征合并肝脏胚胎性肉瘤1例报道

1例30岁妊娠妇女,妊娠33周时因先兆子痫、HELLP综合征(溶血、肝酶升高、血小板计数下降)和呼吸困难在荷兰鹿特丹入院。妊娠31周时曾患感冒,其后一直乏力。血压为140/100mmHg(1mmHg=0.133kPa);24h尿蛋白定量13.1g;血红蛋白9.9mmol/L;尿酸0.64mmol/L;胆红素46μmol/L;LDH2036U/L;ALAT1116U/L;ASAT1509U/L;血小板34×109/L。胎儿大小与妊娠周相符,胎心监护显示变异减少,无加速及减速。因怀疑胎儿宫内窘迫,在采用双肼酞嗪(dihydralazine)控制血压稳定在130/80mmHg(1mmHg=0.133kPa)及扩充血容量后,行剖宫产术。分娩一健康男婴,体质…     

HBx蛋白羧基端30个氨基酸缺失对肝癌细胞生物学行为的影响

目的:探讨羧基端缺失了30个氨基酸的乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)突变体ΔHBx和野生型HBx对肝癌细胞Huh7生物学行为的影响。方法:脂质体介导pcDNA3ΔHBx和pcDNA3HBx重组体转染人HBV(-)的肝癌细胞Huh7。PCR扩增Neo基因及Western blot检测转染重组体。运用细胞生长曲线绘制、平板克隆形成、流式细胞仪和裸鼠成瘤实验对转染pcDNA3ΔHBx、pcDNA3HBx和pcDNA3的细胞生物学活性进行检测。结果:pcDNA3ΔHBx组细胞生长速度较pcDNA3HBx组和pcDNA3组减慢,其倍增时间延长;pcDNA3ΔHBx组克隆形成率较pcDNA3HBx组和pcDNA3组下降;细胞周期检测显示pcDNA3ΔHBx表达使Huh7细胞G0/G1期→S期的进程明显减慢。结论:HBx羧基端缺失了30个氨基酸的突变体比野生型HBx具有明显抑制细胞增殖的能力,提示该区域对于HBx功能发挥起着至关重要的作用。     

软肝煎药物血清对肝星状细胞免疫保护作用的研究

目的:探讨软肝煎药物血清对肝星状细胞(HSC)的免疫保护作用.方法:先采用不同两种种族的动物血清培养人肝星状细胞株(Lx-1),研究不同种族的血清对同一细胞的免疫损伤作用,然后再采用对Lx-1细胞株损伤小的血清组与软肝煎药物血清组进行比较来研究对肝星状细胞的免疫保护作用.结果:在测定LDH活性中,小牛血清组与大鼠血清组比较,各组浓度均有非常显著性差异(P＜0.01).药物血清组与大鼠血清组比较,各组浓度均有非常显著性差异(P＜0.01).药物血清组5%与10%比较无显著性差异(P＞0.05),浓度5%、10%两组与浓度20%比较有非常显著性差异(P＜0.01).结论:软肝煎药物血清对肝星状细胞有很好的免疫保护作用;培养细胞最好使用与培养细胞同一种族的血清.