聚合酶链反应芯片筛选5-氟尿嘧啶对乳鼠心肌细胞心脏毒性差异表达基因的作用

目的应用聚合酶链反应（polymerase chain reaction,PCR）芯片技术观察5-氟尿嘧啶（5-fluorouracil,5-FU）对乳鼠心肌细胞心脏毒性相关基因表达的调控作用。方法利用差速贴壁法分离培养乳鼠心肌细胞．分为对照组和5一FU刺激组。提取并纯化细胞RNA,紫外分光光度计检测RNA提取物浓度、变性琼脂糖凝胶电泳检测其纯度及完整性。选择包含84个已知心脏毒性相关基因的PCR芯片,采用比较阈值（△△Ct）法分析两组基因的表达差异。以表达差异（即上调或下调）大于2倍的基因为有意义的差异基因。结果共有48条基因出现差异表达,其中5条基因表达上调,43条基因表达下调。结论利用PCR芯片技术筛选相关基因,为深入阐明5-FU心脏毒性的作用机制提供了新思路。     

三七总皂苷与卡维地洛改善心肌梗死后大鼠心功能的作用

目的用不同浓度三七总皂苷（panaxnotoginsengsaponin,PNS）和卡维地洛干预急性心肌梗死（acutemyocardialinfarction,AMI）大鼠,评价其改善心功能作用的疗效。方法建立大鼠AMI模型84只,随机分为心肌梗死对照组（AMI组）,PNS治疗低（PNS．L,20mg·kg-1·d-1）、中（PNS．M,40mg·kg-1·d-1）、高（PNS—H,80mg·kg-1·d-1）3组,卡维地洛治疗低（CARV．L,2．5mg·kg-1·d-1）、中（CARV—M,5mg·kg-1·d-1）、高（CARV—H,10mg·kg-1·d-1）3组,另设假结扎组（sham组）,每组12只。灌胃给药4周后,用小动物超声心动图仪检测心功能,检测血浆心力衰竭标志物N-末端脑钠肽前体（N—terminalpro—brainnatriureticpeptide,NT—pro．BNP）浓度,并行心肌组织的苏木素-伊红染色分析。结果同sham组比较,AMI组左心室射血分数和左心室收缩百分率、左心室前壁舒张期厚度、左心室前壁收缩期厚度均明显降低,差异有统计学意义（P〈0．01）;左心室舒张末期内径、左心室收缩末期内径及血浆NT—pro—BNP浓度显著增高,差异有统计学意义（P〈0．01）。同AMI组比较,PNS和卡维地洛治疗的中、高剂量组左心室射血分数、左心室收缩百分率、左心室前壁舒张期厚度、左心室前壁收缩期厚度均明显增加,而左心室舒张末期内径和左心室收缩末期内径及血浆NT—pro．BNP浓度明显降低,差异有统计学意义（P〈0．01）。而且,各治疗组的心肌组织细胞均得到较好的保护。结论用PNS和卡维地洛治疗AMI大鼠4周均能有效保护心肌组织和改善心功能,减缓心力衰竭的发生、发展,且PNS效果略优。     

Dysregulation of microRNAs in a mouse model of diabetic myocardial fibrosis

Background Myocardial fibrosis plays a critical role in the process of diabetic cardiac remolding.MicroRNAs (miRNAs) are endogenous,small non-coding RNAs that negatively regulate gene expression in diverse biological and pathological processes.However,the roles of miRNAs in myocardial fibrosis have not been well elucidated.In the present study,miRNAs profiles in the fibrotic myocardium of db/db mice and miRNAs expression in TGF-β1-stimulated mouse cardiac myofibroblasts was examined.Methods Heart function of 18-week-old db/db mice and db/m control mice was detected by echocardiography.miRNA expression profile in diabetic myocardium was detected by miRNA microarray.Quantitative real-time PCR was used to determine the expression of fibrosis-related genes and miRNA precursors of interest.Western blot was used to detect the levels of fibrosis-related proteins,activated Smad3 and total Smad3.Results The result of echocardiography showed that left ventricular systolic and diastolic function was impaired in 18-week-old db/db mice without significant change of ejection fraction (EF) and fractional shortening (FS).Fibrosis-related genes expression was upregulated and the amount of phosphorylated Smad3 was increased significantly in the diabetic myocardium.miRNAs dysregulation was shown in diabetic myocardium,sixty-eight miRNAs,including miR-208b,miR-29b,miR-26b and miR-30e,were increased over two-fold,meanwhile,sixty-two miRNAs were decreased more than two-fold in the myocardium of db/db mice compared to db/m controls.In parallel with a significant upregulation of Col1a1,Col3a1 and CTGF miRNA expression,miR-208b,miR-29b,miR-26b and miR-30e precursors were also shown to be upregulated in TGF-β1-induced C57bl/6 mouse cardiac myofibroblasts.Conclusions microRNAs were dysregulated in diabetic myocardium,with the activation of TGF-β/smad3 pathway,contributing to diabetic myocardial fibrosis.     

细菌-酵母穿梭表达质粒pGBKT7-MIF的构建及转化

【目的】构建并转化能在酵母菌AH10 9中表达巨噬细胞游走抑制因子 (MIF)的穿梭表达质粒pGBKT7 MIF。【方法】用RT PCR方法从人T淋巴细胞中扩增MIF基因全外显子片段 ,并定向克隆入细菌 酵母穿梭表达质粒 pGBKT7中 ;用PEG/LiAC法将pGBKT7 MIF转化感受态酵母菌AH10 9。提取转化酵母菌的质粒DNA ,转化大肠杆菌并行质粒的酶切鉴定。【结果】扩增出人MIFcDNA片段 ,限制性内切酶酶切和DNA测序证实获得正确的重组质粒 pGBKT7 MIF ;获得可在色氨酸缺陷培养基 (SD/ trp)上生长含 pGBKT7 MIF的酵母菌克隆。酶切鉴定表明pGBKT7 MIF已转化入酵母菌AH10 9。【结论】成功构建穿梭质粒 pGBKT7 MIF ,并转化入酵母菌AH10 9。     

表达人血管内皮生长因子和血管生成素1的CHO细胞株的建立

目的：克隆人血管内皮生长因子(VEGFm)和血管生成素1(Ang1)基因，建立可分别表达VEGFm和Ang1的中国仓鼠卵巢细胞(CHO)株。方法：用PCR技术，分别从人心脏cDNA库中扩增VEGF165和Ang1 cDNA，并定向克隆人真核表达载体pSecTag2B中。用Lipofectamine2000将重组质粒转化入CHO细胞中，用Zeocin筛选并维持已转化重组质粒的CHO细胞株。在大肠杆菌DH10B中扩增转化入CHO细胞中的重组质粒，行DNA测序鉴定。用Westem-blot鉴定CHO细胞中重组VEGFm和Ang1的表达。结果：从人心脏cDNA库中扩增出VEGFⅢ和Ang1 cDNA片段，酶切和测序结果显示，已正确构建重组质粒pSecTag-VEGF165和pSecTag-Ang1。用Zeocin筛选到候选的CHO细胞株，DNA测序结果表明，pSecTag-VEGF165和pSecTag-Ang1已分别成功转化入CHO细胞中。Western-blot结果显示，在CHO细胞中表达出可被VEGF和Ang1单抗识别的特异蛋白。结论：克隆了VEGFm和Ang1基因，并建立了表达VEGF165和Ang1的CHO细胞株。     

Inhibitory Mechanism of Carvedilol on L-type Ca^2＋ Current in Rat Ventricular Myocytes

Objectives The effects of carvedilol on calcium current (ICa) were investigated in isolated adult rat ventricular myocytes. Methods ICa was recorded by using whole-cell patch-clamp recording technique. Results Carvedilol reversibly inhibited ICa in a concentration-dependent manner, carvedilol at 0.1,0.3, 1 and 10μmol/L in the extracellular solution decreased peak ICa by 1.52%, 18.04%, 37.34% and 72.18%,respectively. The steady-state inactivation curve of ICa was shifted to more negative potentials, while the activation curve was not altered. The recovery from inactivation was shifted to right direction, it could not be recovered completely. In addition, Pretreatment of ventricular myocytes with prazosin and propranolol couldn‘t block the carvedilol-induced reduction of ICa.Conclusions Carvedilol inhibits ICa in adult rat ventricular myocytes by mechanisms involving preferential interaction with the inactivated state of calcium channel.     

恶性疟原虫海南株节结相关组氨酸富集蛋白基因的克隆及序列分析

目的 克隆恶性疟原虫海南株（FCC1／HN）节结相关组氨酸富集蛋白（KAHRP）基因，并进行序列分析。方法 采用PCR技术分两段从FCC1／HN株基因组A趸扩增出部分序列重叠的KAHRP基因片段，分别克隆入pMD-18T测序载体。用双脱氧链末端终止法测定这2个片段的序列，从而得到全长KAHRP基因序列。应用AnthProt、DNAstar软件辅助分析基因结构及进行同源性比较。结果 恶性疟原虫FC1/HN KAHRP全基因长2358个核苷酸，在112至564位核苷酸是内含子，全基因编码634个氨基酸残基，其中赖氨酸含量为14．35％，丙氨酸含量为9．15％，组氨酸含量为7．72％；分子量为69．37kDa。序列分析表明，我国恶性疟原虫FCC1／HN株与国外的3D7、FCR3、India-1、India-2、NF17、Palo-alto,PUPSP株KAHRP基因编码的氨基酸残残留基有52个氨基酸残基替代位点，并存在氨基酸残基序列的增加和缺失。经多参数综合分析，可能有5个潜在的抗原表位区。结论 克隆了恶性疟原虫FCC1／HN株KAHRP基因。序列测定及同源性分析表明，FCC1／HN株与其它分离株KAHRP基因编码的氨基酸序列存在一定差异。     

包虫病诊断抗原的纯化及诊断效价

[目的]建立一种简便快速、适合基层的包虫病诊断方法。[方法]采用简便的一步层析方法从包囊液内纯化具有诊断价值的脂蛋白抗原,应用酶联免疫吸附试验(ELISA)和诊断试纸法(Dipstick法),对血清进行检测,评价其效果。[结果]ELISA检测包虫病人血清48份,阳性45份,检出率93.7％,和10份羊多房棘球蚴血清中的1份起交叉反应,而和羊细颈囊尾蚴、弓形虫、血吸虫血清不起反应。用Dipstick方法检测24份包虫病人血清,19份阳性,检出率79.2％,和其它寄生虫血清无交叉反应。[结论]本试验为包虫病诊断试剂盒的研制奠定了基础。     

恶性疟原虫Pf332基因片段的克隆及表达

[目的]构建含恶性疟原虫Pf332抗原基因片段的重组质粒，并检测在大肠杆菌BL21/DE3中的表达情况。[方法]应用PCR技术从FCC1/HN株基因组DNAS中扩增Pf332基因（Pf332)片段，P332-R0、P332-R2，并分别插入pGEX4T-1原核表达载体，阳性克隆的重组质粒DNA经酶切及PCR扩增鉴定，用DNAstar软件对FCC1/HN株与3D7株Pf332和P332-R1、P332-R2片段进行同源性比较，由IPTG诱导在大肠杆菌BL21/DE3中表达重组蛋白。[结果]PCR扩增得到特异的FCC1/HN株Pf332片段，P332-R0、P332-R1、P332-R2，大小分别为489，429和393bp。酶切及PCR鉴定获得了正确的pGEX-P332-R0,pGEX-P332-R1,pGEX-P332-R2重组质粒，序列分析结果显示，与3D7株相比，FCC1/HN株P332-R1、P332-R2片段编码多肽分别缺少2、4个相应的重复单元，在大肠杆菌BL21/DE3中表达出3个重组蛋白，相对分子质量分别为46、44和42。[结论]扩增、克隆了恶性疟原虫Pf332片段，P332-R0、P332-R1、P332-R2，并在大肠杆菌BL21/DE3中成功表达，FCCl/HN株与3D7株的P332-R1、P332-R2片段编码多肽所包含的重复单元数目不同。