pEGFP-hApelin-R重组真核表达载体的构建及在HEK293细胞中的表达

目的构建与增强型绿色荧光蛋白（enhanced green fluorescent protein,EGEP）融合的人apelin受体（Apelin receptor,apelin-R）真核表达载体。方法以质粒pcDNA3.1-hApelin-R为模板,PCR方法扩增人apelin受体。扩增的人apelin受体用EcoRⅠ和BamHⅠ双酶切,同时用这两种酶双酶切质粒peGFP-C1。然后将两种酶切产物按常规方法连接、转化大肠杆菌Top10。挑取菌落培养,提取质粒,然后进行酶切鉴定,最后进行测序。将测序正确的重组载体用脂质体法转染人胚胎肾（human embryonic kidney293,HEK293）细胞,共聚焦显微镜观察。提取转染细胞的总蛋白,进行Westernblot检测。结果扩增出一条约1200bp的片段,与预期的apelin受体大小相符。酶切结果显示,重组质粒pEGFP-hApelin-R被切成两条片段,其中一条为peGFP-C1载体大小,另一条为目的片段大小。经测序鉴定,序列与GenBank（NM_005161）中的序列高度同源。共聚焦显微镜观察显示,人apelin受体主要在细胞膜上表达。Westernblot结果显示在相对分子质量69000处有一蛋白条带,与预期大小相符。结论构建成功pEGFP-hApelin-R重组表达载体,此表达载体可用于检测apelin受体和κ型阿片受体（kappa opioid receptor,KOR）或与其他受体间的相互作用。     

Multivalent dendrimeric and monomeric adenosine agonists attenuate cell death in HL-1 mouse cardiomyocytes expressing the A3 receptor

Multivalent dendrimeric conjugates of GPCR ligands may have increased potency or selectivity in comparison to monomeric ligands, a phenomenon that was tested in a model of cytoprotection in mouse HL-1 cardiomyocytes. Quantitative RT-PCR indicated high expression levels of endogenous A₁ and A_2A adenosine receptors (ARs), but not of A_2B and A₃ARs. Activation of the heterologously expressed human A₃AR in HL-1 cells by AR agonists significantly attenuated cell damage following 4 h exposure to H₂O₂ (750 μM) but not in untransfected cells. The A₃ agonist IB-MECA (EC₅₀ 3.8 μM) and the non-selective agonist NECA (EC₅₀ 3.9 μM) protected A₃ AR-transfected cells against H₂O₂ in a concentration-dependent manner, as determined by lactate dehydrogenase release. A generation 5.5 PAMAM (polyamidoamine) dendrimeric conjugate of a N⁶-chain-functionalized adenosine agonist was synthesized and its mass indicated an average of 60 amide-linked nucleoside moieties out of 256 theoretical attachment sites. It non-selectively activated the A₃AR to inhibit forskolin-stimulated cAMP formation (IC₅₀ 66 nM) and, similarly, protected A₃-transfected HL-1 cells from apoptosis-inducing H₂O₂ with greater potency (IC₅₀ 35 nM) than monomeric nucleosides. Thus, a PAMAM conjugate retained AR binding affinity and displayed greatly enhanced cardioprotective potency.     

手动膜片钳检测HMS-01对HEK293细胞hERG通道电流的影响

目的 检测新化合物HMS-01有无心脏毒性,对其进行临床前安全评价研究,为进入临床试验做准备.方法 用手动膜片钳检测转染后hERG钾通道稳定表达的HEK293细胞的电流,西沙必利做阳性药,将HMS-01依次稀释成0.3、1、3、10、30μmol/L,依次作用于细胞,记录电流变化,计算抑制率.结果 HMS-01在实验设...     

肿瘤坏死因子相关凋亡诱导配体修饰的人羊水间充质干细胞

背景：以间充质干细胞为载体的基因治疗新方法具有广阔的应用价值。目的：利用慢病毒感染方法将肿瘤坏死因子相关凋亡诱导配体导入人羊水来源的间充质干细胞,以期获得稳定表达肿瘤坏死因子相关凋亡诱导配体的人羊水间充质干细胞。方法：首先利用多位点Gateway技术构建慢病毒表达载体pLVpuro／EFlet．肿瘤坏死因子相关凋亡诱导配体,将该表达载体与慢病毒包装质粒同时转染293FT细胞,从而获得携带肿瘤坏死因子相关凋亡诱导配体基因的慢病毒颗粒。利用重组慢病毒颗粒感染人羊水间充质干细胞,并通过抗生素筛选的方法获得稳定表达目的基因一肿瘤坏死因子相关凋亡诱导配体的羊水间充质干细胞,并对其稳定性进行鉴定。结果与结论：酶联免疫吸附法和Westernblot检测结果表明,肿瘤坏死因子相关凋亡诱导配体蛋白在感染的人羊水间充质干细胞内呈高表达,其表达量可达到72μg／L。说明实验成功制备了肿瘤坏死因子相关凋亡诱导配体修饰的人羊水间充质干细胞。     

ADAM9基因真核表达质粒的构建及表达

目的:构建人源性去整合素金属蛋白酶9(ADAM9)基因真核表达载体并表达其编码蛋白(转染293T细胞).方法:利用包含ADAM9全长编码核酸序列的质粒PCR4-TOPO作为模板,用PCR扩增其核心编码区,将其克隆至PMD18-T Vector中构建PMD18-T-ADAM9质粒,同时双酶切PMD18-T-ADAM9及P...     

Characterization of novel Pannexin 1 isoforms from rat pituitary cells and their association with ATP-gated P2X channels

Our previous studies have showed that Pannexin 1 (Panx1), a member of a recently discovered family of gap junction proteins, is expressed in the pituitary gland. Here we investigated the presence and expression pattern of Panx1 isoforms in pituitary cells, their roles in ATP release, and their association with purinergic P2X receptor subtypes that are native to pituitary cells. In addition to the full-size Panx1, termed Panx1a, pituitary cells also express two novel shorter isoforms, termed Panx1c and Panx1d, which formation reflects the existence of alternative splicing sites in exons 2 and 4, respectively. Panx1c is lacking the Phe108-Gln180 sequence and P2X1d is missing the Val307-Cys426 C-terminal end sequence. Confocal microscopy and biotin labeling revealed that Panx1a is expressed in the plasma membrane, whereas Panx1c and Panx1d show the cytoplasmic localization when expressed as homomeric proteins. The three Panx1 isoforms and Panx2 form homomeric and heteromeric complexes in any combination. These splice forms can also physically associate with ATP-gated P2X2, P2X3, P2X4, and P2X7 receptor channels. The Panx1a-mediated ATP release in AtT-20 immortalized pituitary cells is attenuated when co-expressed with Panx1c or Panx1d. These results suggest that Panx1c and Panx1d may serve as dominant-negative effectors to modulate the functions of Panx1a through formation of heteromeric channels. The complex patterns of Panx1 expression and association could also define the P2X-dependent roles of these channels in cell types co-expressing both proteins.     

重组腺病毒Ad5F35-SD-EGFP的构建及其对K562细胞增殖的影响

目的构建表达SH2-DED融合基因的新型重组腺病毒Ad5F35-SD-EGFP,观察其对K562细胞增殖的抑制作用。方法重叠PCR扩增SH2-DED融合基因并克隆至腺病毒穿梭载体pAdTrack-CMV中,构建成穿梭质粒pAdT-SD-EGFP并进行酶切和测序鉴定。穿梭质粒经Pme I酶切后转化pAd5F35-GFP的BJ5183感受态,通过细菌内同源重组产生重组腺病毒质粒pAd5F35-SD-EGFP及其突变体,Pac I酶切后转染AD293细胞进行包装。重组腺病毒Ad5F35-SD-EGFP经PCR和western blot鉴定后进行病毒扩增和滴度测定。体外感染K562细胞,通过MTT和流式周期检测实验观察Ad5F35-SD-EGFP对K562细胞增殖的影响。结果 PCR、酶切和测序结果均表明pAdT-SD-EGFP和pAd5F35-SD-EGFP构建成功;PCR、EGFP检测结果证明重组腺病毒Ad5F35-SD-EGFP包装扩增成功,滴度可达1.4×1012 pfu/ml。转染K562细胞后,Western blot可检测到目的蛋白的表达,MTT实验和流式周期检测结果证明其对K562细胞的增殖有明显的抑制作用。结论成功构建并鉴定了携带SH2-DED融合基因的重组腺病毒Ad5F35-SD-EGFP,其对BCR-ABL阳性K562细胞的增殖有明显的抑制作用。     

人源5-羟色胺转运体稳定表达细胞系的建立及其功能探究

目的建立人源5-羟色胺转运体（human serotonin transporter,hSERT）稳定表达细胞系,并对细胞系hSERT的结合活性和重摄取功能进行初步研究。方法采用脂质体转染法将hSERT转染于母细胞,即人胚胎肾（human embryo kidney,HEK）293细胞,应用抗生素G418（800μg/ml）压力筛选,并采用RT-PCR和Western印迹法在基因和蛋白水平对HEK293-hSERT细胞系进行鉴定和稳定性验证;采用放射性配体结合实验检测其与氚标西酞普兰（[3H]-citalopram）的结合活性,并检测其对[3H]5-HT的重摄取功能。结果 RT-PCR和Western印迹结果表明,所建立的HEK293-hSERT细胞系在15代内（P1,P5,P10,P15）均稳定表达hSERT;放射配体结合实验表明,HEK293-hSERT细胞系与[3H]-西酞普兰具有明显的特异性结合;并且在100 nmol/L[3H]5-HT条件下,具有明显的5-HT重摄取功能,这一作用可被10μmol/L的氟西汀所阻断,而母细胞HEK293无此功能。结论本研究所建立的HEK293-hSERT细胞系可稳定表达SERT,并且具有内源性SERT的结合和重摄取功能,是一种稳定表达功能性hSERT的细胞系。     

人Neuritin真核表达载体的构建及其在细胞中的定位

目的获得人突触生长素Neuritin基因并构建pEGFP-C1-neuritin真核表达载体,观察Neuritin在人AD293细胞中的定位。方法利用PCR获得Neuritin基因,然后克隆到pEGFP-C1载体中,利用脂质体将构建的表达载体转染AD293细胞,荧光观察融合蛋白的表达。结果从人类cDNA文库中得到Neuritin的完整开放阅读框序列,将其重组到pEGFP-C1载体中并转染AD293细胞,荧光显示Neuritin蛋白定位在细胞浆中。结论成功构建Neuritin真核表达载体并在人AD293细胞表达,证明Neuritin蛋白定位于细胞浆,为进一步研究Neuritin与其他蛋白的相互作,探讨Neuritin功能及作用机制奠定理论基础。