SD大鼠皮下注射重组人白介素-1190天毒性研究

重组人白细胞介素-11 (rhIL-11)是一个多功能性的细胞因子,在骨髓造血调控中发挥重要作用.为了解连续给予rhIL-11后由于蓄积而对机体产生反应及其严重程度,确保其临床应用的安全,该试验对SD大鼠连续给药3个月进行了全面的安全性评价.     

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.     

基因重排检测技术操作体会及注意事项

临床表现、形态学、免疫表型以及分子遗传学特征对淋巴组织增生性病变的诊断均具有重要的意义，尤其是分子遗传学特征是确定淋巴组织增生性病变的克隆性及其细胞来源的可靠依据。近年来开展的PCR法基因重排检测技术，能从DNA水平更早期、敏感、精确地在亚病理与亚临床阶段检测出肿瘤性克隆，对淋巴造血组织疾病的良恶性判断具有重要意义。该技术目前已用于淋巴造血组织疾病良恶性诊断、肿瘤性病变的追踪及复发预测等方面。本文就基因重排检测技术和在日常工作中的操作体会、改进意见、注意事项以及与病理形态学诊断有关的一些问题做一简单总结。[第一段]     

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％。     

重组CFP-10/ESAT-6抗原ELISA法对活动性肺结核诊断的评估

目的 以rCFP-10/ESAT-6融合蛋白抗原、植物血凝素(PHA)、生理盐水分别处理活动性肺结核患者外周血致敏T淋巴细胞,测定同一标本IFN-γ释放水平,探讨其对结核感染与发病的诊断意义.方法 病例组111例为临床确诊肺结核患者,对照组292例为经胸部透视和结核菌素试验(FeD)筛选的大学生.入选对象取血3.0 ml,均分为3份,每管1.0 ml,分别加入rCFP-10/ESAT-6融合蛋白、PHA和生理盐水,孵育后ELISA法测定IFN-γ的吸光度(A)值及其抗体.结果 rCFP-10/ESAT-6融合蛋白处理:IFN-γ测定值病例组x=1.3885±0.6236,对照组x=0.2944±0.0917,组间t'=16.4259,P90%,且无论特异性或非特异性处理,活动性肺结核患者IFN-γ释放水平均明显升高,血清抗体阳性率也高于对照组.     

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。     

p75NGFR对胰腺癌细胞株SW1990生物学行为的影响

目的通过体内和体外实验，探讨p75NGFR在胰腺癌SW1990细胞生长、分化以及侵袭中的作用。方法采用脂质体转染法将含p75NGFR的真核表达质粒转染人胰腺癌细胞株SW1990，建立稳定表达p75NGFR的细胞模型；应用MTT法、平板克隆形成实验以及流式细胞技术分别检测细胞的生长曲线、克隆形成能力以及细胞周期变化；建立胰腺癌的裸鼠皮下移植瘤和胰腺原位移植瘤模型，观察p75NGFR对SW1990细胞裸鼠成瘤能力、肿瘤组织学分化、细胞侵袭和转移的影响。结果与对照组相比较，转染p75NGFR的SW1990细胞其生长速度缓慢（P〈0.01），克隆形成能力降低（P〈0.05）；转染细胞的细胞周期改变表现为G1期细胞明显增多和S期细胞明显减少（均为P〈0.01）。与对照组相比较，转染组裸鼠皮下以及胰腺原位的成瘤率以及肿瘤体积减小（P〈0.01），肿瘤的生长缓慢，组织学分化差，浸润性生长趋势不明显，未见肿瘤卫星现象。结论体外实验均发现p75NGFR通过阻滞细胞周期进程而显著抑制胰腺癌细胞株的生长，体内研究发现p75NGFR可抑制裸鼠皮下及胰腺原位移植瘤的形成、分化和侵袭，提示p75NGFR与胰腺癌的生物学行为密切相关。     

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.