pcDNA3.1(-)/PSMA7真核表达质粒的构建及其在A549细胞中的表达

目的 构建pcDNA3.1(-)/PSMA7真核表达质粒并建立人肺腺癌细胞株A549的稳定表达株.方法 采用RT-PCR法从正常人支气管上皮细胞株HBE135-E6E7中克隆得到PSMA7 cDNA全长序列,将之与pMD18-T载体连接.双酶切和测序鉴定后,再将PSMA7片段亚克隆到真核表达载体pcDNA3.1(-)上.构建好的pcDNA3.1(-)/PSMA7真核表达质粒经双酶切和测序鉴定后,经脂质体转染法转入A549细胞株中,经CA18筛选,得到阳性克隆细胞株,再用RT-PCR及Westem blotting法检测转染后PS-MA7的mRNA及蛋白表达水平.结果 pcDNA3.1(-)/PSMA7质粒经双酶切鉴定和DNA测序证实.目的 基因PSMA7的序列完全正确,真核表达质粒构建成功.RT-PCR检测显示,转染pcDNA3.1(-)/PSMA7组的PSMA7 mRNA表达水平(2.698±0.661)明显高于未转染组(1.243±0.176)和转染pcDNA3.1(-)组(1.198±0.514,P<0.05),后两者间无明显差异.Western blotting检测同样显示,转染pcDNA3.1(-)/PSMA7组的蛋白表达水平(1.978±0.661)明显高于未转染组(0.891±0.234)和转染pcDNA3.1(-)组(0.782±0.375,P<0.05),后两组间亦无明显差异.结论 成功构建了PSMA7基因的真核表达质粒,并建立了稳定表达PSMA7的A549细胞株,为后续研究奠定了基础.     

凋亡相关斑点样蛋白ASC真核表达质粒的构建及表达

目的构建凋亡相关的斑点样蛋白（apoptosis-associated speck-like protein containing CARD,ASC）真核表达质粒。方法从人胚肾细胞系HEK293T细胞的总RNA中经过逆转录和PCR获得ASC的全长cDNA双链片段,经过酶切后连接到真核表达载体pcDNA3/flag中,挑选出转化生成的阳性克隆测序,将序列正确的重组质粒命名为pcDNA3/flag-ASC。脂质体转染pcDNA3/flag-ASC质粒到HEK293T细胞中,用Western印迹检测目的蛋白的表达。同时用免疫沉淀和免疫印迹方法检测ASC蛋白与前半胱天冬酶（pro-caspase）-1的相互作用,并用ELISA方法检测ASC蛋白对IL-1β分泌的影响。结果 Western印迹实验证明pcDNA3/flag-ASC可以在HEK293T细胞中表达ASC,并且可以与pro-caspase-1结合,使IL-1β分泌明显降低。结论构建了重组质粒pcDNA3/flag-ASC,在细胞中表达ASC后具有与pro-caspase-1结合的能力,并具有降低IL-1β分泌的生物活性。     

利用Mps1特异性siRNA抑制人宫颈癌HeLaS3细胞增殖的研究

目的验证所设计的Mps1小干扰RNA(siRNA)的特异性,为其应用于肿瘤治疗提供研究基础。方法针对Mps1基因的5’端设计合成一个siRNA(简称为siMps1),利用Western印迹检测siMps1的干扰效果,以及干扰后对肿瘤细胞的增殖和有丝分裂的影响。进一步构建稳定表达siMps1的细胞系SW480-YFPMps1siR/shRNA,检测过表达外源Mps1能否恢复其内源基因缺失的表型。结果转染siMps1能够降低细胞中的Mps1蛋白水平,导致有丝分裂指数降低,染色体中期排列异常;进而通过克隆形成实验发现,HeLaS3细胞转染siMps1后大量死亡,出现多核细胞;构建的稳定细胞系SW480-YFPMps1siR/shRNA能够恢复Mps1缺失后的表型。结论该设计得到的siMps1具有很高特异性,有望应用于肿瘤的治疗中。     

人LDLR基因的克隆及在Hepa1-6细胞中的表达

目的 :从能感染HCV的人肝癌细胞HepG2中克隆出人LDLR基因 ,构建其真核表达质粒 ,并在小鼠肝癌细胞Hepa1 6中进行表达。方法 :从HepG2中提取总RNA ,采取逆转录PCR(RT PCR)方法得到人LDLR的cDNA。将获得的人LDLR基因克隆到真核表达载体pcDNA3中 ,进行酶切和序列鉴定。将重组质粒pcDNA3 hLDLR和空载体转入Hepa1 6 ,G4 18筛选 ,并用RT PCR和FACS检测人LDLR基因转录和蛋白的表达。结果 :人LDLR的cDNA已插入载体pcDNA3构建成真核表达质粒pcDNA3 hLDLR ,双酶切和测序表明 ,克隆的人LDLR与已登录GenBank的序列存在核苷酸多态性 ,但编码的氨基酸序列完全一致 ,该序列的GenBank登录号为gi|2 16 2 96 4 7|gb|AY114 15 5 .1,并且真核表达质粒的构建正确。用脂质体介导的方法转入Hepa1 6后 ,用RT PCR和FACS检测表明 ,细胞可表达人LDLR。结论 :成功地构建了真核表达质粒pcDNA3 hLDLR ,为进一步研究HCV和人LDLR的相互作用 ,以及建立可能的HCV细胞感染模型奠定了基础。     

微丝相关蛋白hHBRK1突变体的构建、表达及纯化

目的：构建微丝相关蛋白hHBRK1截短体和突变体原核表达载体，并表达及纯化融合蛋白。方法：采用常规PCR并结合定点突变技术，对hHBrkl基因进行缺失和点突变；利用限制性内切酶将PCR产物克隆至原核表达载体pGEX-4T；IPTG诱导融合蛋白表达，通过谷胱甘肽一琼脂糖纯化技术分离纯化GST融合蛋白。结果与结论：构建了包括氨基端缺失截短体hHBRK1-AN、羧基端缺失截短体hHBRK1-AC和点突变蛋白hHBRKl-S^56G^57在内的原核表达质粒，分离获得较高纯度的重组hHBRKl突变体融合蛋白，Western杂交证实纯化蛋白为GST融合蛋白。本实验为进一步研究hHBRK1的相互作用蛋白及其可能的结合位点提供了基础。     

氧浓度非敏感性低氧诱导因子-1α表达载体的构建及其在PC12细胞系中的表达

目的 构建大鼠非氧浓度敏感性低氧诱导因子1α(HIF-1α)真核表达载体并观察其在PC12细胞系中的表达. 方法 应用RT-PCR从脊髓损伤区域的细胞总RNA中扩增HIF-1αcDNA,采用重叠延伸PCR的方法克隆获得缺失氧敏感性降解结构域(ODD)的HIF-1α突变体基因克隆,采用质粒_pEGFP-C1构建重组真核融合表达载体,将其转入培养的PC12神经细胞系中,应用所融合的绿色荧光蛋白的表达和Western blot鉴定其在细胞内的表达. 结果 通过重叠延伸PCR的方法成功扩增得到非氧浓度敏感性的HIF-1α突变体基因(HIF-1α△ODD)序列,并构建了过量表达缺失ODD的转录调控因子HIF-1α突变体的重组表达质粒()pEGFPC1-HIF-1α△ODD).将其转染PCI2细胞系后,Western blot和绿色荧光蛋白结果均表明HIF-1α△AODD蛋白能在PC12神经细胞系中正确表达. 结论 成功构建了_pEGFPC1-HIF-1α△ODD真核表达载体并在PC12细胞系中观察到融合蛋白表达.     

人PD-1分子基因克隆及其在COS-7细胞中表达的研究

目的：克隆人PD—1分子的编码序列cDNA，将其重组进真核表达载体中，并表达于COS—7细胞。方法：从活化的人T细胞cDNA文库中以PCR方法克隆编码人PD—l的编码cDNA序列，将其克隆进真核表达载体pcDNA3．1( )，构建成重组表达载体。并测序证实。用脂质体法转染COS—7细胞，流式细胞仪检测PD—1分子的膜表达。结果：PCR方法扩增出—880bp左右的基因片段，插入pcDNA3．1( )载体后构建成重组表达质粒，经测序证实扩增的片段为人PD—l编码序列。将重组质粒转染COS—7细胞，流式细胞仪检测显示有21．10％的细胞表达人PD—1分子。结论：本研究为进一步研究PD—1分子在免疫耐受、自身免疫性疾病中的作用奠定了基础。     

Flag-Tinagl1真核表达载体的构建及功能验证

目的 构建带Flag标签的肾小管间质性肾炎抗原样蛋白1(Tinagl1)基因真核表达载体,检测其对肝癌细胞HepG2 ERK/AKT磷酸化、生长和迁移的影响.方法 以人乳腺cDNA文库为模板,PCR扩增Tinagl1基因片段,将其插入pcDNA3.0载体,经双酶切和测序验证后,将重组质粒转染到肝癌HepG2细胞中,采用Western印迹检测重组蛋白对ERK和AKT磷酸化水平的影响,生长曲线和划痕实验检测其对肝癌细胞HepG2生长和迁移的影响.结果 双酶切和Western印迹结果表明,pcDNA3.0-Flag-Tinagl1真核表达质粒构建成功,Tinagl1可降低ERK和AKT的磷酸化水平;生长曲线和划痕实验表明Tinagl1抑制肝癌细胞HepG2生长和迁移.结论 构建了带Flag标签的Tinagl1真核表达载体,并能抑制肝癌细胞HepG2中ERK/AKT磷酸化、生长和迁移,为进一步研究Tinagl1在肝癌细胞中的功能奠定了基础.     

稳定表达人MAGE-1基因的小鼠细胞系的建立与鉴定

目的：克隆人黑素瘤抗原-1（MAGE-1）基因，构建真核表达载体，建立稳定表达人MAGE-1的小鼠细胞株。方法：提取人肝癌细胞的总RNA，RT-PCR法获得人MAGE-1cDNA，将测序正确的MAGE-1基因克隆至真核表达载体pcDNA3中，进行酶切鉴定和序列测定。将重组质粒pcDNA／MAGE和空载体pcDNA3分别转染入小鼠骨髓瘤细胞SP2／0中，经G418筛选获得稳定表达株，用RT-PCR和Western印迹检测MAGE-1基因的转录及蛋白表达。结果：从肝癌细胞中成功克隆到人MAGE-1基因，经测序证明基因正确，酶切和序列测定表明，人MAGE-1基因的真核表达质粒已成功构建。将此重组质粒转入的SP2／0细胞可稳定表达人MAGE-1基因。结论：成功构建可稳定表达人MAGE-1基因的小鼠转基因细胞，为MAGE-1的免疫治疗研究奠定了基础。     

人β-防御素3基因在COS-7细胞中的表达

目的：构建人β—防御素3(hBD3)的真核表达载体，建立稳定表达hBD3的细胞株。方法：用EcoR Ⅰ酶切合有hBD3全长基因的pGEM-hBD3重组质粒，获得其编码区全长序列，将其连接入EcoR Ⅰ预处理过的pcDNA3中。转化大肠杆菌，酶切鉴定筛选出插入方向正确的转化子。采用脂质体转染法将重组pcDNA3-hBD3真核表达载体导入COS-7细胞，用G418进行抗性筛选，用RT-PCR和Western印迹检测目的基因hBD3的mRNA和蛋白表达水平。结果与结论：构建的pcDNA3-hBD3真核表达载体转染COS-7细胞后可稳定表达hBD3的mRNA和蛋白，且蛋白主要以分泌形式存在于培养上清中。     

B7-H1在恶性肿瘤中的表达进展

B7-H1(B7 homolog 1)是免疫调节分子B7家族的一个成员,主要分布在心脏、胎盘、肺、脾、淋巴结、胸腺、肾、骨骼肌和胎肝组织中。B7-H1在多种肿瘤细胞上广泛高表达,它与免疫细胞上的程序性死亡分子 1结合后,启动caspase级联反应诱导免疫细胞的凋亡,导致肿瘤免疫逃逸的发生。近年来,有关B7-H1在某一恶性肿瘤中的表达以及它与临床病理、预后和免疫学因素的相关性报道诸多。作者通过总结分析近年来的相关文献,对B7-H1在一些常见恶性肿瘤中的表达进展作一综述。     

血清中IL-7、PGLYRP-1、SOST的表达水平对类风湿性关节炎的诊断价值

目的 探讨血清中白介素7（interleukin 7,IL-7）、肽聚糖识别蛋白1（peptidoglycan recognition protein 1,PGLYRP-1）、硬骨素（sclerostin,SOST）的表达水平对类风湿性关节炎的诊断价值。方法 纳入2019年5月至2021年5月达州市中心医院收治的类风湿性关节炎患者82例为类风湿组、非类风湿性关节炎患者82例为非类风湿组、健康体检者82例为对照组。检测3组受试人员血清中IL-7, PGLYRP-1, SOST的表达量;用受试者工作特征曲线（receiver operating characteristic curve,ROC）的曲线下面积（area under curve,AUC）评估IL-7, PGLYRP-1, SOST检测在类风湿性关节炎中的诊断意义。结果 类风湿组患者血清中的IL-7和SOST均低于非类风湿组和对照组（P<0.05）,类风湿组患者血清中的PGLYRP-1高于非类风湿组患者和对照组（P<0.05）。IL-7、PGLYRP-1、SOST单向诊断类风湿性组患者与非类风湿组患者的灵敏度分别为90%、86%、98%,特异度分别为72%、89%、61%。血清中IL-7, PGLYRP-1, SOST联合对类风湿性关节炎的诊断的的灵敏度为91.46%,特异度为75.61%,准确度为80.89%。此外,非条件logistic逐步回归分析结果显示血清中IL-7, PGLYRP-1, SOST的表达水平均是患类风湿关节炎的重要危险因素。结论 血清中IL-7、 PGLYRP-1、SOST的表达水平可作为诊断类风湿性关节炎的潜在指标。     

Slice-by-slice B(1) (+) shimming at 7 T

Parallel transmission has been used to reduce the inevitable inhomogeneous radiofrequency fields produced in human high‐field MRI greater than 3 T. Further improvements in the transmit homogeneity and efficiency are possible by leveraging the additional degree of freedom permitted by multislice acquisitions. Compared to simple scaling of the flip angle to compensate for B₁⁺ falloff along the radiofrequency coil, calculation of B₁⁺ shim solutions on a slice‐by‐slice basis can markedly improve homogeneity and/or reduce transmitted power and global SAR. Performance measures were acquired at 7 T with a 15‐channel head‐only transceive array featuring elements distributed over all three logical axes, facilitating B₁⁺ shimming over arbitrary orientations. Compared to a circularly polarized volume mode of the same coil, shimming to maximize excitation efficiency on a slice‐by‐slice basis yielded improvements in mean B₁⁺ by 12.8 ± 2.4% and a reduction in standard deviation of B₁⁺ of 16.3 ± 6.8%, while reducing relative SAR by 6.2 ± 3.1%. When shimming for greater uniformity, the mean and standard deviation of B₁⁺ were further improved by 15.9 ± 2.6% and 26.2 ± 10.4%, respectively, at the expense of a 135 ± 8% increase in global SAR. Robust multislice‐shim solutions are demonstrated that can be quickly calculated, applied in real time, and reliably improve on volume coil modes. Magn Reson Med, 2012. © 2011 Wiley Periodicals, Inc.     

Assessing a Dysplastic Cerebellar Gangliocytoma (Lhermitte-Duclos Disease) with 7T MR Imaging

Lhermitte-Duclos disease (LDD; dysplastic cerebellar gangliocytoma) is a rare hamartomatous lesion of the cerebellar cortex and this was first described in 1920. LDD is considered to be part of the autosomal-dominant phacomatosis and cancer syndrome Cowden disease (CS). We examined the brain of a 46-year-old man, who displayed the manifestations of CS, with 7 Tesla (T) and 1.5T MRI and 1.5T MR spectroscopy (¹H-MRS). We discuss the possible benefits of employing ultrahigh-field MRI for making the diagnosis of this rare lesion.     

RNA干涉抑制肺癌细胞DNA甲基转移酶1(DNMT1)的表达

目的：通过抑制肺癌细胞DNA甲基转移酶1(DNMT1)的表达，从而部分恢复抑癌基因的表达。方法：采用RNAi技术来抑制DNMT1的活性。应用体外转录的方法，共合成了5个针对DNMT1不同靶位的siRNA序列，并转染人肺癌细胞NCI-H157。采用MSP-PCR、COBRA等方法对抑癌基因RASSF1A进行甲基化分析，半定量RT-PCR检测DNMT1 mRNA的敲降与RASSF1A基因的表达。结果与结论：5个靶位的siRNA序列中有两个靶位能够有效地抑制DNMT1的表达，且呈剂量依赖效应。人肺癌细胞NCI-H157中抑癌基因RASSF1A发生了高度甲基化，在抑制DNMT1活性后，RASSF1A基因去甲基化而恢复表达。     

Influenza A (H1N1) virus-associated pneumonia: high-resolution computed tomography-pathologic correlation

Objective

The purpose of this study was to describe the high-resolution computed tomography (HRCT) features of fatal cases of Influenza A (H1N1) virus-associated pneumonia and to correlate them with pathologic findings.

Methods

The study included six adult patients who died following Influenza A (H1N1) virus-associated pneumonia. All patients had undergone HRCT, and the images were retrospectively analyzed by two chest radiologists, who reached decisions by consensus. Two experienced lung pathologists reviewed all pathological specimens. The HRCT findings were correlated with the histopathologic data.

Results

The predominant HRCT findings included areas of airspace consolidation (n = 6) and ground-glass opacities (n = 3). The main pathological features consisted of diffuse alveolar damage with hyaline membrane formation (n = 5), associated with various degrees of pulmonary congestion, edema, hemorrhage, inflammatory infiltration and bronchiolitis. A patient who survived longer showed findings of organizing pneumonia.

Conclusion

Fatal cases of Influenza A (H1N1) virus-associated pneumonia can present as areas of consolidation on CT, with or without ground-glass opacities. These abnormalities can be pathologically correlated with diffuse alveolar damage. Patients with longer survival may present with findings of organizing pneumonia.     

X射线对人肺癌A549细胞CCR7表达影响的研究

目的 研究不同剂量X射线照射及照射后不同时间点对人肺癌A549细胞CC-趋化因子受体7(CCR7)表达的影响.方法 体外培养A549细胞,实验组采用直线加速器X射线一次性照射,细胞吸收剂量分别为2、4、6和8 Gy(源皮距100 cm;剂量率442.89 cGy/min),照射后4、12、24、48和72 h分别采用实时荧光定量PCR技术及Western blot方法分别进行CCR7 mRNA及蛋白质表达水平检测;对照组A549细胞除不接受x射线照射外,余处理同实验组.结果 A549细胞经2、4、6和8 Gy的X射线照射后,CCR7 mRNA及蛋白质在照射4 h后开始表达升高,达到高峰后相继出现下降;72 h后6和8 Gy组mRNA表达量仍高于对照组水平(t=6.75～7.26,P＜0.01),2和6 Gy组蛋白质表达量高于对照组(t=11.13～14.17,P＜0.01),而4和8 Gy组蛋白质表达量在48和72 h已降至对照组水平.结论 2、4、6和8 Gy的X射线照射A549细胞后,A549细胞CCR7mRNA及蛋白质的表达量明显增加,可能与一定剂量X射线辐射促进A549细胞增殖和转移有关.

Abstract：

Objective To study the effects of X-ray radiation on CC-chemokine receptor 7(CCR7) expression in human non-small cell lung cancer (NSCLC) cells.Methods Humanadenocarcinoma cells of the line A549 were cultured and irradiated by X-ray at the absorbed doses of 2,4,6,and 8 Gy respectively by linear accelerator (with the source skin distance of 100 cm and dose rate of 442.89 cGy/min).The relative levels of CCR7 mRNA and protein expression in the A549 cells were respectively detected by real time-PCR and Western blotting 4,12,24,48,and 72 h after radiation.Untreated A549 cells were used as control group.Results The expression levels of CCR7 mRNA and protein in the A549 cells began to increase since 4 h after radiation and then decreased gradually after they reached the peak.The CCR7 mRNA expression levels 72 h after radiation of the 6 and 8 Gy groups were still significantly higher than those of the control group (t = 6.75-7.26,both P < 0.01),and the CCR7 protein expression levels of the 2 and 6 Gy group were still significantly higher than those of the control group(t=11.13-14.17,both P <0.01).Then the CCR7 protein expression levels of the 4 and 8 Gy groups decreased to the control group level 48 and 72 h after radiation respectively.Conclusions The CCR7 mRNA and protein expression levels in the NSCLC cells increase after X-ray irradiation,which may be correlated with the promotion of proliferation and metastasis of NSCLC cells by X-ray irradiation at a certain dose.     

Prior irradiation results in elevated programmed cell death protein 1 (PD-1) in T cells

Purpose: In this study we addressed the question whether radiation-induced adverse effects on T cell activation are associated with alterations of T cell checkpoint receptors.

Materials and methods: Expression levels of checkpoint receptors on T cell subpopulations were analyzed at multiple post-radiation time points ranging from one to four weeks in mice receiving a single fraction of 1 or 4?Gy of γ-ray. T cell activation associated metabolic changes were assessed.

Results: Our results showed that prior irradiation resulted in significant elevated expression of programmed cell death protein 1 (PD-1) in both CD4+?and CD8+?populations, at all three post-radiation time points. T cells with elevated PD-1 mostly were either central memory or naïve cells. In addition, the feedback induction of PD-1 expression in activated T cells declined after radiation.

Conclusion: Taken together, the elevated PD-1 level observed at weeks after radiation exposure is connected to T cell dysfunction. Recent preclinical and clinical studies have showed that a combination of radiotherapy and T cell checkpoint blockade immunotherapy including targeting the programmed death-ligand 1 (PD-L1)/PD-1 axis may potentiate the antitumor response. Understanding the dynamic changes in PD-1 levels in T cells after radiation should help in the development of a more effective therapeutic strategy.