Salusin-a基因真核表达载体的构建及在HEK293细胞中的表达

目的:研究增强型绿色荧光蛋白真核表达载体Salusin-a(pEG-FP-Salusin-a)的构建及在人胚胎肾细胞系293细胞(HEK293)中的表达。方法:提取人单核细胞系THP-1细胞Salusin-a的mRNA,逆转录多聚酶链反应(RT-PCR)扩增Salusin-a基因,克隆入pEGFP-N3载体,双酶切、菌落PCR及测序鉴定pEGFP-Salusin-a质粒。用脂质体转染pEGFP-Salusin-a入HEK293细胞,分为转染细胞组、质粒对照组和空白对照组,检测分析各组荧光蛋白和Salusin-a基因的表达。结果:成功构建pEGFP-Salusin-a质粒,并转染HEK293,细胞转染效率86%,转染细胞组和质粒对照组绿色荧光蛋白的表达明显高于空白对照组(P0.05);转染细胞组Salusin-amRNA的表达明显高于质粒对照组和空白对照组(P0.05)。结论:重组pEGFP-Salusin-a质粒能转染HEK293细胞并表达Salusin-a,为Salusin-a基因功能的进一步研究提供了实验基础。     

小鼠FasL基因真核表达载体的构建及在HEK293细胞中的表达

目的构建含有小鼠Fas Ligand(FasL)基因的重组真核表达载体,并检测FasL蛋白在其稳定转染的HEK293细胞中的表达情况,为构建表达小鼠Fas L的树突状细胞(DC)模型,深入研究DC联合T细胞防治移植物抗宿主病(GVHD)的新方法奠定基础。方法从小鼠脾脏中提取RNA并逆转录为cDNA,以该cDNA为模板扩增FasL基因,插入pcDNA3.1(+)载体中构建pcDNA3.1(+)-FasL重组质粒,利用脂质体将其转染HEK293细胞,用G418筛选稳定表达细胞系,Western blot确定重组蛋白的表达。结果通过酶切及测序证实FasL序列正确插入表达载体pcDNA3.1(+),Western blot检测证实转染重组质粒的细胞能正确表达FasL蛋白,G418筛选后能够得到稳定表达FasL的抗性细胞株即FasL-HEK293。结论重组质粒pcDNA3.1(+)-FasL和稳定表达FasL的HEK 293细胞成功构建,为后续FasL-DC细胞的研究打下了坚实基础。     

α-synuclein基因过度表达诱导HEK293细胞α-synuclein蛋白病理性积聚

目的观察α-synuclein过度表达诱导HEK293细胞α-synuclein蛋白病理性积聚的作用。方法将消除终止密码子α-synuclein基因扩增产物连入pGEM T-easy载体，DNA测序证实后亚克隆入增强型绿色荧光蛋白pEGFP-N1质粒，通过限制性内切酶酶切鉴定重组质粒。采用脂质2000将野生型α-synuclein-EGFP真核表达载体转染HEK293细胞，48h后采用Axiovert200型倒置荧光显微镜、荧光免疫细胞化学观察其在细胞内的表达，HE染色观察细胞胞浆内嗜酸性小体的形成。结果消除终止密码子的扩增序列经测序证实并亚克隆入pEGFP-N1质粒后，限制性内切酶酶切鉴定质粒大小、酶切位点上均符合实验设计。将野生型α-synuclein-EGFP真核表达载体转染HEK293细胞，48h后荧光显微镜下观察细胞在波长488nm蓝色激发光下发出明亮的绿色荧光，其中某些细胞出现绿色荧光物质积聚；免疫荧光细胞化学检测，EGFP阳性细胞同时也表现为α-synuclein免疫反应阳性，某些细胞胞浆内可见α-synuclein免疫阳性产物聚集；HE结果发现一些细胞胞浆内出现圆形的嗜酸性小体形成，约占细胞总数的10．8％。结论野生型α-synuclein基因过表达可诱导α-synuclein蛋白在HEK293细胞内积聚，胞浆内嗜酸性小体形成。     

人4-1BBL基因重组腺病毒载体的构建及其在HEK293细胞的表达

目的 体外构建含有人4-IBBL基因的重组腺病毒载体,并检测其在HEK293细胞中的表达.方法 设计一对含有Sfil酶切位点的4-1BBL因上下游引物,以质粒pCR4-TOP0-4-1BBL为模板,通过PCR扩增获得4-1BBL基因全序列.片段回收后经酶切处理,连接到穿梭质粒pShuttle-CMV-EGFP上,获得重...     

小鼠B16黑色素瘤细胞酪氨酸酶基因的克隆及在毕赤酵母中的表达

目的:克隆酪氨酸酶基因(TYR), 并在毕赤酵母中表达和纯化其融合蛋白, 体外测定纯化的TYR活性.方法:利用RT-PCR技术, 从小鼠B16黑色素瘤细胞中克隆全长TYR, 克隆至pMD18-T载体, 进行双链核苷酸序列测定.构建TYR毕赤酵母表达质粒pPICZaA- TYR并导入毕赤酵母Pichia pastoris GS115, 表达的重组蛋白用SDS-PAGE和Western blot进行分析, 采用Co2 离子亲和层析柱纯化TYR并采用TYR多巴速率氧化法体外测定纯化的TYR活性.结果:克隆了TYR基因.构建了重组表达质粒pPICZaA-TYR.SDS-PAGE和Western blot分析显示, 在相对分子质量(Mr)为约53 000处出现1条特异性蛋白带, 且能与兔抗TYR抗体发生反应.结论:克隆TYR基因片段与基因库所登录的序列相一致, 并在毕赤酵母中获得表达和纯化并在体外测定了纯化的TYR活性.     

人CD7 cDNA在HEK293细胞中的表达

目的 建立表达人白细胞分化抗原CD7蛋白的哺乳动物细胞模型,为深入研究CD7的功能奠定基础。方法 采用PCR方法扩增CD7 cDNA,将目的片段定向克隆于真核表达载体pcDNA3,限制性内切酶酶切分析重组质粒并进行序列测定;通过电穿孔法将重组真核表达载体pcDNA3/CD7转染于人胚肾293细胞（HEK293）,经G418筛选得到抗性克隆;利用FCM检测CD7分子在HEK293细胞的表达。结果 得到了含CD7全长cDNA的重组真核表达载体pcDNA3/CD7。FCM分析表明：转染了pcDNA3/CD7的HEK293细胞表达人CD7蛋白。结论 为深入研究CD7的作用机制提供了条件。     

AR-GFP融合基因真核表达载体的构建及在HEK293细胞中的表达

目的：构建由绿色荧光蛋白（GFP）标记的人醛糖还原酶（AR）融合基因真核表达载体并在HEK293细胞中表达。 方法:应用DNA重组技术构建AR与GFP的融合基因（AR-GFP），将AR-GFP插入pcDNA3.1/myc-HisA真核表达载体中，通过磷酸钙共沉淀转染HEK293细胞，倒置荧光显微镜评价转染效率，Western blotting 技术检测AR基因在转染细胞中的整合及表达，分光光度法（UV法）测定AR表达活性，用公认的AR抑制剂（ARI）反证活性AR的存在。结果:荧光显微镜对转染细胞的观察及Western blotting 结果证实AR-GFP融合蛋白在HEK293细胞中表达，UV法测活、ARI抑制试验均未证实其生物学活性。 结论:转染的HEK293细胞可成功地表达AR-GFP融合蛋白，但不能成为ARI真核细胞筛选模型。     

Sirt2基因的克隆及其在HEK293中的表达

目的:克隆大鼠Sirt2 基因, 构建其真核表达载体并在HEK293细胞中表达.方法:利用RT-PCR从大鼠脑组织总RNA中扩增出包含Sirt2编码区的cDNA片段, 产物纯化后T-A克隆连接至pMD20-T载体.以此为模板, 将该基因编码区克隆入真核表达载体pcDNA3.1myc-his(-)中, 转染HEK293细胞检测其表达.结果:测序证实所克隆的Sirt2编码区cDNA正确地插入pcDNA3.1myc-his(-)中, 经免疫荧光检测证实其在HEK293细胞中得到表达.结论:成功克隆了大鼠Sirt2 cDNA, 构建了其真核表达载体, 并在HEK293细胞中得到有效表达, 为进一步研究大鼠Sirt2的功能奠定了基础.     

人CD23基因的扩增及其在HEK 293T细胞的高效表达

目的建立一个有效表达CD23的系统。方法采用RT-PCR方法,从外周血淋巴细胞中扩增人的CD23cDNA基因,并将其克隆到真核表达载体pcDNA3·1B上,构建成pcDNA3·1/CD23质粒表达载体;将pcDNA3·1/CD23质粒转染HEK293T细胞,通过流式细胞仪和Westernblot检测CD23在细胞中的表达情况。结果扩增的CD23cDNA序列与文献报道的一致;通过构建表达载体和转染细胞实现了CD23cDNA在293T细胞膜上的表达。转染效率达80%以上,并获得了较高水平的表达。结论扩增CD23cDNA基因,经过转染HEK293T细胞,实现了CD23在HEK293T细胞的高效表达。     

FBXO6基因真核表达载体的构建及其在HEK293T细胞中的表达

目的 构建F盒蛋白6(FBXO6)基因真核表达载体.方法 采用PCR方法合成含有EcoR Ⅰ和Bgl Ⅱ双酶切位点的FBXO6 cDNA全长.分别构建载体pEGFP-C1-FBXO6和pEGFP-C1-anti-FBXO6,采用菌落PCR、双酶切鉴定以及测序证实cDNA片段大小和序列正确.将载体pEGFP-C1-FBXO6和pEGFP-C1-anti-FBXO6分别转染HEK293T细胞,Western blot法检测FBXO6蛋白的表达.结果 pEGFP-C1-FBXO6和pEGFP-C1-anti-FBXO6包含大小、序列正确的FBXO6片段;FBXO6蛋白在转染pEGFP-C1-FBXO6的293T细胞中高表达;在转染pEGFP-C1-anti-FBXO6的HEK293T细胞中表达降低.结论 成功构建FBXO6基因正义真核表达载体pEGFP-C1-FBXO6和反义真核表达载体pEGFP-C1-anti-FBXO6.     

Muscle cross-section measurement by magnetic resonance imaging

Summary Muscle cross-section areas were measured by magnetic resonance imaging (MRI) in the thigh of a human cadaver,. the results being compared with those obtained by photography of corresponding anatomic macroslices. A close correlation was found between MRI and photographic evaluation, differences between the methods ranging from nil to 9.5%, depending on the scan position and the muscle groups. In vivo MRI measurements were performed on 12 female and 16 male students, the objectivity, the test-retest reliability and the variability of the MRI measurements being studied by fixing the scan position either manually or by coronary scan. The latter method appeared to be more objective and reliable. The coefficients of variation for muscle cross-section areas measured by MRI were in the range of those for the planimetry of given cross-section areas. Allowing for differentiation between several small muscle bundles in a given area, MRI proved to be a suitable method to quantify muscle cross-sections for intra- and interindividual analysis of muscle size.     

Anatomic correlation of cadaver cryomicrotomy with magnetic resonance imaging

Summary The performance of frozen sections of a thickness varying between 5 and 50 m in fresh undecalcified cadaveric human specimens was perfected in Sweden by one of the authors in 1983. This technique makes it possible to obtain anatomic images of high definition which were correlated with MRI sections made at intervals of 20 m.

---

Corrélations anatomiques entre la cryomicrotomie sur cadavre et l'IRM

Résumé La réalisation de coupes congelées dont l'épaisseur varie entre 5 et 50 m, sur prélèvements cadavériques humains frais et non décalcifiés a été mise au point en Suède par l'un des auteurs en 1983. Cette technique permet d'obtenir des images anatomiques de haute définition qui ont été corrélées avec des coupes en IRM réalisées à des intervalles de 20 m.

     

Self-assembled magnetic fluorescent polymeric micelles for magnetic resonance and optical imaging

Stable and cytocompatible hybrid PEGylated micelles with multimodal imaging capabilities are described. The F₃O₄-encapsulated polymeric micelles composed of cores containing magnetic nanoparticles and polyethylene glycol (PEG) shells are synthesized by self-assembly of amphiphilic poly(HFMA-co-VBK)-g-PEG copolymers and oleic acid stabilized Fe₃O₄ nanoparticles. The Fe₃O₄ magnetic nanoparticles in the core produce T₂-weighted MR imaging functionalities, whereas the small fluorescent monomer carbazole in the polymer shell introduces good fluorescent properties. The multifunctional micelles exhibit excellent paramagnetic properties with the maximum saturation magnetization of 9.61 emu/g and transverse relaxivity rate of 157.44 mm⁻¹ S⁻¹. In vivo magnetic resonance imaging (MRI) studies reveal enhanced contrast between the liver and spleen. Fluorescence spectra show characteristic emission peaks from carbazole at 350 nm and 365 nm and vivid blue fluorescence can be observed by 2-photon confocal scanning laser microscopy (CLSM). In vivo optical imaging demonstrates the unique fluorescent characteristics of the Fe₃O₄-encapsulated polymeric micelles in the liver and spleen and the excellent multifunctional properties suggest potential clinical use as nanocarriers in magnetic resonance imaging and optical imaging.     

Non-invasive temperature imaging of muscles with magnetic resonance imaging using spin-echo sequences

The application of spin-echo magnetic resonance imaging sequences on non-invasive temperature imaging for temperature mapping of human limbs is investigated. In an in vitro expriment performed on a meat sample, the equilibrium magnetisation P and the spin-lattice relaxation time T₁ are calculated from the values for the repetition time TR and the signal intensities obtained by a spin-echo sequence at different tissue temperatures tures as measured by a fibre-optic probe. T₁ is linearly correlated to the tissue temperature, and P is linearly correlated to the reciprocal value of the absolute temperature. Both effects, taken together, lead to a non-linear dependency of the signal intensity on temperature. Therefore a TR leading to maximum temperature dependency of the signal intensity is calculated and used in the futher experiments. In the in vivo experiments, the lower legs of two volunteers are cooled from outside. Images are acquired with a spin-echo sequence (1.5T, TR=1200 ms, TE=10 ms). A rise in signal intensity in the muscle with falling skin temperature is observed, particularly in more peripheral muscle layers. This study shows that spin-echo sequences can be used to monitor temperature changes and temperature differences in living muscle tissue.     

咀嚼时局部脑活动的功能性核磁共振成像

目的：应用功能性核磁共振成像（fMRI）探测人吸嚼时的大脑功能活动。方法：要求人在无任何其它躯体活动条件下咀嚼肌以10s运动20s休息的频率进行。选用8例成人冠状切面和横轴面的头部磁共振片,观察脑功能活动情况。结果：①在咀嚼时脑的广泛区域是激活的;②在相对应的咀嚼活动中有优势半球的激活区;③第I躯体感觉区激活的方式远较第I躯体运动区多样化;④在额叶中4例年轻观察对象出现了广泛的神经元激活区,但在老年人很少出现这样的激活区。结论：咀嚼活动除了它本身的功能运动外,在维持脑的活动方面具有重要的作用。同时也说明fMRI在研究活体人脑功能活动方面是一个相当有效的方法。     

应用MRI分析腰椎多裂肌-最长肌间隙入口位置

目的 应用MRI测量腰椎各椎间盘平面多裂肌-最长肌间隙入口与中线的距离,并分析其相关因素。方法 前瞻性选取2012年4月—2013年1月行腰椎MRI检查的200例患者,测量腰椎MRI各椎间盘平面双侧肌间隙入口与中线的距离（D）,并采用单因素方差分析和q检验比较节段间差异,t检验比较左右两侧及不同性别的差异,Pearson相关性检验分析各节段D间的相关性,多元线性回归分析各节段D与患者年龄、身高、BMI的相关性。结果 腰椎各椎间盘平面肌间D从L1/2水平至L5/S1水平逐渐增大,不同节段间D差异有统计学意义（左侧：F=3 614.84,P=0.00;右侧：F=3 411.34,P=0.00）,但左右侧间差异无统计学意义（P＞0.05）,D1仅与D2间存在相关性,而D2~D5间则两两正相关。男性与女性患者仅L4/5节段D差异有统计学意义（t=4.44,P＜0.01）,其他节段D男女间差异均无统计学意义（P值均＞0.05）。各节段D与患者的年龄均无相关性。男性患者D1双侧均与身高呈正相关,女性患者D1L、D1R及D2L与BMI正相关,而D4双侧与身高呈正相关。结论 腰椎多裂肌-最长肌间隙入口位置与患者年龄、性别、身高、BMI等关系不大。在L1-3水平,肌间隙距离正中线较近,适合作单个正中切口;在L4～S1水平则应根据术前测量结果应用双侧切口。     

Factors affecting perceived tumor volumes in magnetic resonance imaging

Irregularly structured brain tumors, such as glioblastomas, challenge attempts to visualize and quantify their three-dimensional structure. Magnetic resonance imaging (MRI) represents one tool for attempting to noninvasively track tumor size. MR images demonstrate widely varying perceived tumor margins. In addition, adjunct therapies, such as the administration of steroids, greatly affect the volumes perceived in images formed by certain pulse sequences. In this study tumors were grown in 15 dogs and the tumor size tracked for a period of time. The dogs were placed on dexamethasone for a week and another series of scans was obtained. No other therapies were provided. The data for visualized tumor size are provided for T₁, T₂, and proton density. Weighted images are provided and the relationships between the scans are discussed.     

MRI Schizas形态学分型对腰椎管狭窄症疗效的评价

目的 探讨MRI Schizas形态学B、C型退行性腰椎管狭窄症患者保守与手术治疗的临床疗效,以及Schizas形态学分型的临床应用价值。方法 回顾性分析2014年1月—2015年12月新疆医科大学第一附属医院骨科收治的62例Schizas形态学分型为B、C型腰椎管狭窄症患者的临床资料,其中男33例,女29例;年龄43～74岁。Schizas形态学B型39例,其中保守治疗20例(保守组)、手术治疗19例(手术组);C型23例,其中保守治疗13例(保守组)、手术治疗10例(手术组)。B、C型保守组和手术组患者一般资料比较差异均无统计学意义(P值均>0.05)。分别比较B、C型患者保守组和手术组治疗前及末次随访ODI、JOA评分、VAS及治疗期间住院时间、住院费用的差异。结果 所有患者治疗后随访9～16个月,平均随访12个月。Schizas B型腰椎管狭窄症的患者在保守组及手术组治疗后末次随访ODI分别为10.88%±2.84%和11.44%±2.80%,JOA评分分别为(22.41±2.26)分和(22.36±2.25)分,VAS分别为(0.84±0.52)分和(0.93±0.41)分,两组间比较差异均无统计学意义(t=-0.622、0.065、-0.646,P值均>0.05),与各组治疗前比较差异均有统计学意义(P值均<0.05);两组住院时间及住院费用比较差异均有统计学意义(Z=-3.530、-5.339,P值均<0.01)。Schizas C型腰椎管狭窄症患者在保守组和手术组治疗后末次随访腰椎ODI分别为16.72%±4.04%和10.10%±1.63%,JOA评分分别为(17.92±2.43)分和(22.75±2.99)分,VAS分别为(1.60±0.82)分和(0.70±0.25)分,两组间比较差异均有统计学意义(t=4.856、-4.271、3.713,P值均<0.01),与各组治疗前比较差异均有统计学意义(P值均<0.05);两组平均住院时间比较差异无统计学意义(Z=-1.853, P>0.05),住院费用比较差异有统计学意义(Z=-4.032, P<0.01)。结论 保守治疗及手术治疗均能不同程度地改善腰椎管狭窄症患者的症状,MRI Schizas形态学分型在治疗腰椎管狭窄症的方法选择上有参考价值。     

In utero eyeball development study by magnetic resonance imaging

The aim of this study was to measure fetal ocular development and to determine a growth curve by means of measurements in utero. Fetal ocular development was recorded by analysis of the results of magnetic resonance imaging (MRI). An anatomic study allowed definition of the best contrasted MRI sequences for calculation of the ocular surface. Biometric analysis of the values of the ocular surface in the neuro-ocular plane in 35 fetuses allowed establishment of a linear model of ocular growth curve in utero. Evaluation of ocular development may allow the detection and confirmation of malformational ocular anomalies such as microphthalmia.     

Course of cerebral lesions in a patient with periarteritis nodosa studied by magnetic resonance imaging

Summary The course is reported of a patient with periarteritis nodosa who initially presented with neurological symptoms. Multiple cerebral lesions were documented by the first magnetic resonance imaging (MRI) investigation. The majority of these had disappeared completely in the follow-up MRI studies. In contrast to neurological improvement the patient eventually died due to multiorgan failure. Postmortem histological examination revealed no pathological findings in the brain except one single necrotic area already known from MRI. Remissions of histological and angiographic alterations in periarteritis nodosa have been described as local healing leading to fibrosis and scarring. Our findings suggest that restitutio ad integrum may occur, at least in cerebral lesions.Abbreviations PAN panarteritis nodosa - MRI magnetic resonance imaging - CT computerized tomography - CNS central nervous system