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Results 1、The CD11b-positive microglia were dyed low brown with small bacilliform cell bodies,ramified morphology,thin and long protuberance in CON and NIC group;In LPS group,the CD11b-positive microglia were dyed chocolate brown with more big roundish cell bodies,thicker and shorter processes;Most CD11b-positive microglia were dyed brown with bacilliform cell bodies,ramified morphology and few cells were dyed deep brown with more big roundish cell bodies,thicker and shorter protuberance in NIC+LPS group.

结果 1、CON组与NIC组CD11b阳性小胶质细胞胞体小,呈杆状,突起细长,成浅棕色:LPS组小鼠脑内CD11b阳性小胶质细胞胞体增大,呈圆形,突起粗而短,成棕褐色;NIC+LPS组少量CD11b阳性小胶质细胞胞体增大,圆形,突起变粗变短,成深棕色,多数呈杆状,突起少、细长,成浅棕色。

Result In the HepG2 cells transfected by pSliencer 3.1-H1 neo-shTERT, expressions of hTERT, Bcl-2 and mitochondrial cyt C were significantly down-regulated, while Bax and cytoplastic cyt C were obviously up-regulated, and active caspase-9 was found in addition to procaspase-9 compared with that in negative control cells and untransfected cells.

结果 与未转细胞和对照细胞相比,转细胞hTERT、Bcl-2和线粒体cyt C表达明显减少,Bax和胞质cyt C表达明显增加;未转细胞及对照细胞仅见约46kD的前caspase-9条带,转细胞可见46kD前caspase-9条带和35kD caspase-9 p35亚基条带。

Suppression of metastatic hemangiosarcoma by a parvovirus MVMp vector transducing the CXCL10 chemokine into immunocompetent mice.

2.2 转细胞表达CXCL10蛋白的检测采用Western blot分别检测转CXCL10表达载体的细胞及其培养上清中CXCL10的表达,均可检测到CXCL10蛋白;而转空载体组和未转组细胞均未能检测到CXCL10存在

After the cell growth curves was recorded, RPE cells of the 3-5th passages were utilized. 2、Three different siRNA (siRNAl,siRNA2,siRNA3) targeting against human cx43 gene and one negative control siRNA were designed and transfected into cultured human RPE cells via liposome reagent. The most effective siRNA can be determined by semi-quantitative reverse transcription PCRRT-PCR. 3、To the most effective siRNA, after transfected into human RPEs with different concentration, the cellular proliferate activities were messured by MTT colorimetry ; the percentages of RPE in different cell circle phase was assayed by FCM; the changes of phenotypical properities were observed with SCM; the protein expression of cx43 was studied through immunocytochemistry stain and Weston blot; the communication intercellular was calculated with FRAP; and the ability of recovery was assessed by using an in vitro wound healing model.4、The total proteins of siRNA1 and RPE were seperated by two-dimensional gel electrophoresis and visualized by silver staining. Proteins with significant expression alterations were selected and their peptide mass fingerprints (PMFs were obtained by matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS).The PMFs were used to search NCBInr database by Auto MS-Fit software.

实验方法:1、培养原代的人RPE细胞,经过细胞角蛋白、S-100和神经胶质原纤维酸性蛋白免疫细胞化学鉴定后,通过AO/PI色技术确定培养细胞的存活率,描记其生长曲线,第3-5代用于以下细胞实验2、生物合成针对人cx43基因的三条小干扰RNA和一条阴性RNA通过脂质体转RPE细胞后,通过RT-PCR的方法确定抑制效率最高的干扰片断3、将该片段以不同浓度通过阳离子脂质体转培养的人RPE细胞后,采用MTT法观察其对细胞的增殖力的作用;通过流式细胞仪观察其对细胞周期的影响;通过扫描电镜观察其对细胞形态的影响;通过免疫细胞化学和Weston blot观察其对cx43蛋白表达的作用;采用激光共聚焦和荧光淬灭恢复技术观察荧光恢复速率平均百分率,评价其对细胞间通讯功能的影响;通过制作RPE细胞损伤模型,观察其对损伤修复能力的作用4、分离纯化转siRNA的RPE组和正常对照组RPE细胞的全部蛋白质,应用等电聚焦电泳和SDS-PAGE双向电泳技术,银显示分离出的蛋白质斑点,经凝胶图像分析软件对两个样本进行胶图分析,寻找差异蛋白点。

Methods After the wide type NCI-H446 cells and the three kinds of NCI-H446 cells transfected by three kinds of IL-15 genes respectively (Cmp transfected by the IL-15 maturation peptide gene, Cp transfected by prototypic IL-15 gene, and Csp transfected by modified IL-15 gene whose signal peptide was replaced with the IL-2 signal peptide) were treated by mitomycin, the cells (named MCw, MCmp, MCp, and MCsp, respectively) were used as stimulator cells to stimulate PBMC from healthy volunteers.

野生型NCI-H446细胞和被3种IL-15基因分别转的3种NCI-H446细胞(Cmp:被IL-15成熟肽基因转;Cp:被原型IL-15基因转;Csp:被信号肽换为IL-2信号肽的改型IL-15基因转),用丝裂霉素处理后(分别名为MCw、MCmp、MCp和MCsp)作为刺激细胞刺激健康志愿者的PBMC。

The research and development of levelling agent for vat dyes and cationic dyes was reviewed,which provide reference for the application, repreparation and development of levelling agent in China.

剂具有缓性和移性,可有效改善料的匀性达到均匀色的结果,从而提高料的应用性能。

Part II The experiment study on anti-tumour effect of dendritic cells derived from peripheral blood monocyte of patients with oral cancer after pulsed by tumour antigen in different ways. Collected 50ml peripheral blood from each patients and volunteers to induced DC in vitro. We parted the test group and control group into three subgroups respectively, untreated group A, freezed and thawn group B, RNA transfected group C. To group A, after induced DCs for five days , we added 200U/mlTNF-α in each hole and cultured on. To group B, we added protein antigen (DC/ tumor cells = 1 : 1 ,each hole) for 4h at 5d, and then added 200U/mlTNF-α in each hole and cultured on. To group C, transfected immature DC with tumor cell RNA(DC:RNA=1×105/ml: 5ug)at 5d, operated completely with the specification of invitrogen DMRIE-C reagents. After transfection we replaced transfection agent with complete medium, and then added 200U/mlTNF-α in each hole and cultured on.

第二部分口腔癌患者外周血来源的树突状细胞体外负载抗原后抑瘤作用的实验研究无菌采集20例口腔癌患者和20例健康志愿者外周血50ml,分离单个核细胞体外诱导培养DC,并将实验组和对照组细胞各分为三组,A组为未处理组,B组为冻融组,C组为RNA转组。A组DC在培养5d后,每孔加入TNF-α200U/ml;B组DC培养第5d后,每孔中加入癌细胞蛋白抗原,使DC/肿瘤细胞=1:1,继续培养4h后,每孔加入TNF-α200U/ml;C组将培养至第5d的未成熟DC用肿瘤细胞RNA进行转(DC:RNA=1×105/ml: 5ug,按照invitrogen DMRIE-C试剂说明书进行操作),转结束后用完全培基取代转剂,每孔加入TNF-α200U/ml。

In dyeing process, because of some factors such as nonuniform temperature or nonuniform agitation of dye bath, the dye defects or streaks may be caused.

色过程中,由于某些因素的影响,如液搅拌不均匀、浴温度分布不均等都会使色不匀、出现花或疵等问题。

We observed that FuGene6 regent had the highest transfection efficiency and lowest toxicity to auditory neurons in the three groups. The transfection efficiency was about 3%-10%, variable from experiment to experiment. However, when we used recombinant adenovirus vector to transfer LacZ gene into auditory neurons,β-gal expression was detected in more than 70% of both neurons and gilas at 100M0I viral concentration..

通过对不同转方法的选择和转条件的优化,其中FuGene 6介导的质粒转,其转效率最高,可达3%-10%左右,但不同批次结果重复性较差,易受不同批次细胞来源动物的个体差异及培养条件等因素的影响。

The total proteins from TGIF transfectant and vector control cells were separated by 2D electrophorosis. 760±41 and 680±38 spots were detected in TGIF transfectant and vector control cells respectively. Locus repeat analysis of protein spots showed that the mean deviation in IEF direction in TGIF transfectant and vector control cells were 0.864±0.123 mm and 0.843±0.115 mm respectively whereas the mean deviation in SDS-PAGE direction in these two cells were 0.812±0.109 mm and 1. 125±0.123 mm respectively. So the well-resolved and reproducible 2DE patterns from TGIF transfectant and vector control cells were established.

通过双向凝胶电泳分别分离TGIF转细胞和PcDNA3.1空白质粒转细胞的总蛋白质,测得两组细胞的蛋白质斑点数分别为760±41和680±38个;位置重复性分析表明TGIF转细胞在等电聚焦方向上的平均偏差为0.864±0.123mm,在垂直板SDS-PAGE电泳方向上的平均位置偏差为0.812±0.109mm;PcDNA3.1空白质粒转细胞在IEF方向上的平均偏差为0.843±0.115mm,在垂直板SDS-PAGE电泳方向上的平均位置偏差为1.125±0.123mm。

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