小鼠beta-防御素2真核表达载体的构建及表达

构建小鼠heta-防御素2(murie beta-defensin 2,mBD2)的真核表达载体pcDNA-mBD2,并观察其在真核细胞中的表达.从小鼠肝组织中提取mRNA通过逆转录聚合酶链反应扩增得到mBD2基因,定向克隆至真核表达栽体pcDNA3.1(+)获得pcDNA-mBD2;经酶切和测序鉴定构建正确,应用脂质体法转染siha细胞,并通过蛋白免疫印迹法检测细胞内mBD2的表达.结果显示,构建了小鼠mBD2的真核表达载体,转染siha细胞培养并采用G418稳定筛选后,获得稳定转染细胞,收集并裂解转染细胞,蛋白免疫印迹法检测到转染细胞内mBD2的表达.成功构建了真核表达裁体pcDNA-mBD2,为研究mBD2在宫颈癌发生发展过程中的作用及作用机制奠定基础.     

敲减人HPIP基因表达抑制细胞生长增殖

为了探讨人造血相关的PBX相互作用蛋白质基因（HPIP）在肿瘤发生发展中的生物学作用,构建了HPIP小干扰RNA(siRNA)的真核表达载体,验证其敲减效果并观察其对细胞生长增殖的影响.根据人HPIP的cDNA序列,设计了含有小发卡结构的寡核苷酸序列,将其克隆到siRNA表达载体上;将重组质粒转染人胚肾293T细胞,通过实时定量RT-PCR及Western 印迹分析检测HPIP基因的表达水平;结晶紫实验及流式细胞技术检测敲减HPIP基因表达对细胞生长和增殖的影响,软琼脂实验检测对肿瘤细胞非锚定依赖性生长的影响.结果显示,构建的siRNA能够有效抑制HPIP基因的表达;结晶紫实验与细胞周期分析实验显示,siRNA介导的HPIP表达沉默导致细胞生长增殖的显著抑制,软琼脂实验结果表明,稳定转染HPIP siRNA能够抑制肿瘤细胞的锚定非依赖性生长.上述结果初步表明,HPIP siRNA能明显抑制肿瘤细胞的生长与增殖,可能是一个潜在的肿瘤治疗新靶点.     

PES1慢病毒表达载体的构建及其对乳腺癌ZR75-30细胞生长的影响

目的:利用慢病毒载体表达PES1基因,研究其对乳腺癌ZR75-30细胞生长的影响.方法:以乳腺文库为模板,PCR扩增PES1基因,克隆到pCDH载体,构建成pCDH-PES1,将其与包装载体共转293T细胞,包装成Lenti-PES1慢病毒载体并测定病毒滴度,感染乳腺癌ZR75-30细胞,Western印迹鉴定病毒载体...     

The functional expression of extracellular calcium-sensing receptor in rat pulmonary artery smooth muscle cells

Background

The extracellular calcium-sensing receptor (CaSR) belongs to family C of the G protein coupled receptors. Whether the CaSR is expressed in the pulmonary artery (PA) is unknown.

Methods

The expression and distribution of CaSR were detected by RT-PCR, Western blotting and immunofluorescence. PA tension was detected by the pulmonary arterial ring technique, and the intracellular calcium concentration ([Ca²⁺]_i) was detected by a laser-scanning confocal microscope.

Results

The expressions of CaSR mRNA and protein were found in both rat pulmonary artery smooth muscle cells (PASMCs) and PAs. Increased levels of [Ca²⁺]_o (extracellular calcium concentration) or Gd³⁺ (an agonist of CaSR) induced an increase of [Ca²⁺]_i and PAs constriction in a concentration-dependent manner_. In addition, the above-mentioned effects of Ca²⁺ and Gd³⁺ were inhibited by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (specific inhibitor of PLC), 2-APB (specific antagonist of IP₃ receptor), and thapsigargin (blocker of sarcoplasmic reticulum calcium ATPase).

Conclusions

CaSR is expressed in rat PASMCs, and is involved in regulation of PA tension by increasing [Ca²⁺]_i through G-PLC-IP₃ pathway.     

Role of miR-1 and miR-133a in myocardial ischemic postconditioning

Background  

Ischemic postconditioning (IPost) has aroused much attention since 2003 when it was firstly reported. The role of microRNAs (miRNAs or miRs) in IPost has rarely been reported. The present study was undertaken to investigate whether miRNAs were involved in the protective effect of IPost against myocardial ischemia-reperfusion (IR) injury and the probable mechanisms involved.     

Sepiapterin improves angiogenesis of pulmonary artery endothelial cells with in utero pulmonary hypertension by recoupling endothelial nitric oxide synthase

Persistent pulmonary hypertension of the newborn (PPHN) is associated with decreased blood vessel density that contributes to increased pulmonary vascular resistance. Previous studies showed that uncoupled endothelial nitric oxide (NO) synthase (eNOS) activity and increased NADPH oxidase activity resulted in marked decreases in NO bioavailability and impaired angiogenesis in PPHN. In the present study, we hypothesize that loss of tetrahydrobiopterin (BH4), a critical cofactor for eNOS, induces uncoupled eNOS activity and impairs angiogenesis in PPHN. Pulmonary artery endothelial cells (PAEC) isolated from fetal lambs with PPHN (HTFL-PAEC) or control lambs (NFL-PAEC) were used to investigate the cellular mechanisms impairing angiogenesis in PPHN. Cellular mechanisms were examined with respect to BH4 levels, GTP-cyclohydrolase-1 (GCH-1) expression, eNOS dimer formation, and eNOS-heat shock protein 90 (hsp90) interactions under basal conditions and after sepiapterin (Sep) supplementation. Cellular levels of BH4, GCH-1 expression, and eNOS dimer formation were decreased in HTFL-PAEC compared with NFL-PAEC. Sep supplementation decreased apoptosis and increased in vitro angiogenesis in HTFL-PAEC and ex vivo pulmonary artery sprouting angiogenesis. Sep also increased cellular BH4 content, NO production, eNOS dimer formation, and eNOS-hsp90 association and decreased the superoxide formation in HTFL-PAEC. These data demonstrate that Sep improves NO production and angiogenic potential of HTFL-PAEC by recoupling eNOS activity. Increasing BH4 levels via Sep supplementation may be an important therapy for improving eNOS function and restoring angiogenesis in PPHN.     

EB病毒抗体和DNA联合检测可提高儿童传染性单核细胞增多症的诊断灵敏度

目的评估EB病毒抗体VCA-IgM、VCA-IgG、EA-IgG、EBNA-1-IgG及EBV-DNA载量检测在儿童传染性单核细胞增多症(传单)中的诊断意义。方法用ELISA方法检测70例传单患儿和25例健康儿童血清中EBV四种抗体及PCR荧光定量法检测外周血单个核细胞EBV-DNA载量。结果传单患儿组EBV-DNA的阳性率为87.14%(61/70),对照组阳性率为8.00%(2/25),传单组与对照组EBV-DNA的阳性率比较差异有统计学意义(P<0.01)。EBV抗体检测中,传单组的VCA-IgM阳性率最高,达91.43%(64/70),对照组VCA-IgM全部阴性。传单组EB病毒VCA-IgM和EBV-DNA联合检测的阳性率97.1%。结论 EBV抗体和EBV-DNA载量检测对儿童传单的诊断有极高的价值,尤其是VCA-IgM抗体和EBV-DNA联合检测,可提高儿童传单的临床诊断的敏感性。     

PCR相关技术在植物病毒检疫检测中的应用

PCR和建立在PCR基础上的分子生物学技术以其灵敏、快速、简便等优点广泛应用于植物病毒的检测。阐述反转录聚合酶链式反应、免疫捕捉反转录PCR、PCR-单链构型多态性、实时荧光定量PCR、差异显示PCR和巢式PCR等相关技术的原理及其在植物病毒检测中的应用现状,以期为我国植物病毒的检疫检测提供有益参考。     

大肠杆菌中表达关键基因产异丁醇的研究

目的:改造大肠杆菌的代谢途径,使非生产菌株大肠杆菌具备产异丁醇的能力.方法:将乳脂乳球菌NIZO B1157的2-酮酸脱羧酶基因kdcA克隆到大肠杆菌中,使大肠杆菌产异丁醇;另外,采取两种方法过量表达alsS、ilvC、ilvD基因,增加前体物质酮酸的供应,以提高异丁醇的产量:一是与kdcA串联表达;二是在另一个相容质粒中表达.结果:大肠杆菌工程菌具备产异丁醇的能力,其中相关基因在一个质粒中串联表达的产量比其在两个相容质粒中共表达的产量高30倍,达到3g/L.结论:导入的酮酸合成途径与醇类生产途径结合,能使非生产菌株大肠杆菌生产异丁醇,并且单质粒表达代谢途径相关基因的效果优于双质粒表达.