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The PCR products were examined by agarose gel electrophoresis. The target gene fragments were purified by gel extraction kit and ligated to cloning vector pMD18-T. The recombinant vectors were transformed into host strain E. coli K802 by lithium chloride method, screened and identified with PCR and restrictive enzymatic digestion. Their sequences were confirmed by DNA sequencing.(2) sTWEAK1 gene was subcloned into expression vector pProEx HTb and transformed into E. coli BL21. sTWEAK2 gene was subcloned into expression vector pMAL-C2x and transformed into E. coli TB1. The recombinant vectors were screened and identified with PCR and restrictive enzymatic digestion. The recombinant fusion proteins were induced to express with IPTG, detected by coomassie brilliant blue-stained SDS-polyacrylamide gel electrophoresis , and confirmed by Western blot analysis.(3) The sTWEAK1 fusion protein was purified with Ni-NTA Spin Kit.(4) The biological activity was assayed on transformed and tumor cells by microplate photometer after crystal violet or sulfur rodamine B staining.(5) The contents of IL-8 in the supernatant of 1990 cell cultures were determined by ELISA.(6) The morphological changes of the sensitive cells were observed by light and transmission electron microscopies.(7) The cell cycle and apoptotic rate were assayed by flow cytometry in 1990 and M85 cells.(8) The effect of fusion proteins on induction of NF-κB in 1990 and LOVO cells was detected with Dual-Luciferase Reporter Assay system.(9) The TWEAK gene was subcloned into Adeno-X Viral DNA with pShuttle vector and transfected into HEK293 cells by lipofectamine method.

(1)本研究用RT-PCR方法，从人组织细胞总RNA中扩增可溶性TWEAK胞外区（sTWEAK1和sTWEAK2）的cDNA序列及全长编码序列，用琼脂糖凝胶电泳分析PCR产物，胶回收目的基因片段，连接到pMD18-T克隆载体中，转化大肠杆菌K802，PCR和酶切筛选阳性克隆，全自动DNA测序验证序列；(2)sTWEAK1和sTWEAK2分别亚克隆到pProEx HTb和pMAL-C2x表达载体中，分别转化大肠杆菌BL21和TB1，PCR筛选和酶切鉴定，阳性克隆用IPTG诱导表达，表达产物用SDS-PAGE分析和Western blot验证融合蛋白；(3)用NTA-Ni Spin试剂盒初步分离纯化sTWEAK1融合蛋白；(4)用体外培养的肿瘤细胞和正常对表达产物进行活性检测，贴壁细胞用结晶紫染色法，悬浮细胞用磺酰罗丹明B染色法，酶标仪检测OD值；(5)敏感细胞用ELISA法检测细胞培养上清中IL-8的含量；(6)用光镜和电镜观察敏感细胞死亡和细胞凋亡情况；(7)用流式细胞仪分析表达产物对敏感细胞凋亡率和细胞周期的影响；(8)用双荧光素酶报告基因检测法，测定表达产物对敏感细胞NF-κB的影响；(9)用pShuttle穿梭质粒将TWEAK重组到腺病毒载体上，用脂质体转染法转染HEK293细胞，PCR鉴定重组质粒。

BMSCs were isolated, depurated, cultivanted, and identified,then incubated with the concentration of 25μg Fe per milliliter at 37℃in 5% CO2. The labeled cells were stained by Prussian blue/trypan blue,and observed under fluorescent microscope.2. The labeled cells of different density (1×104/ml,5×104/ml,1×105/ml,5×105/ml,1×106 /ml,5×106/ml)were imaged by MRI with T1WI, T2WI and T2*WI sequences;and the same density (5×104/ml,1×105/ml)labeled cells were imaged by T2*WI sequences at different time.Then the signal intensities were measured and statistically analyzed.3. The model of rabbit renal ischemia-reperfusion injury was set up and treated. Then BMSCs(5×105)were injected into 16 recipient rabbits(1abeled cells in 10,unlabeled cells in 6)from ear vein.MR images of kidneys were obtained respectively at the time points of 0,1,3,5, 8 days after transplantation and before transplantation. MR imaging findings were analyzed,which were correlated with histological findings.

实验方法1分离、纯化、培养、鉴定兔BMSCs并以SPIO以25μg Fe/ml培养液浓度标记，对标记后不同时间的细胞行普鲁士蓝染色和台盼蓝拒染后显微镜观察。2将不同细胞浓度标记细胞管(1×104/ml、5×104/ml、1×105/ml、5×105/ml、1×106/ml、5×106/ml)，以不同扫描序列T1WI，T2WI，T2*WI(GRE进行MR成像，再选择相同细胞浓度组(5×104/ml、1×105/ml)进行不同时相MR成像，并测量信号强度，进行统计学分析。3缺血再灌注肾损伤模型建立和处理，然后将标记和未标记细胞(5×105个)经耳缘静脉移植入家兔体内(共16只：注入标记细胞者10只，注入未标记细胞者6只)，两组均于注射前、注射后第0、1、3、5、8天应用MRI对移植细胞进行活体示踪并与肾脏组织切片对照，然后对收集的信号强度进行统计学分析。

Methods:9 patients including 8 male and 1 female were included in this study.2 patients suffered from both side of ANFH,5 were righe side and 2 were left side.X-ray,CT and MRI were performed before the surgery.4 hips were classified as ARCO stage 1,7 hips were stage 2.All the patients were treated as follows:the area of NFH and cystis degeneration were detected and bouche decompression was done through X-ray with C-arm,and the necrotic bone was cleared for the pathologic diagnosis under the monitoring of arthroscopy.And then the non-cell based tissue engineering bone, which was the complex of autologous red bone marrow and osteoinductice absorbing material containing BMP,was transplanted into the body.

本组9例，男性8例，女性1例；双侧股骨头坏死2例，右单侧股骨头坏死5例，左单侧股骨头坏死2例；术前均行X线摄片、CT、MRI 检查，ARCO分期：Ⅰ期4髋，Ⅱ期7髋；C型臂X线透视下行股骨头坏死区和囊性变区定位、髓芯减压，关节镜监视下，刮除坏死骨送病理；植入由自体红骨髓与含BMP的骨诱导活性材料复合成的非细胞型组织工程骨，并用空心钛钉支撑股骨头软骨下骨质。

The following results were obtained: 1. Several multi-pore materials were compared and a nylon membrane was choosed to conduct experiments. 2. Two devices and methods for controlling negative hydraulic heads were developed. Coupled with the devices, a system for precise control of soil moisture udring pot experiment was developed. 3. Two times of pot experiment studying wheat seedling growth under precise soil moisture control were conducted. Some very important results were obtained. 4. Capillary rise, horizontal and vertical infiltration under different nagative hydraulic heads were observed. The concepts of "critical wetting distance" and "wetting coefficient" were proposed. The linear relationship between wetting front movement distance and cumulative infiltration amount was confirmed. Spatial distribution model of soil moisture and its simple derivation method were established.

通过该项目支持，获得了以下的研究成果： 1、比较和检验了不同多孔材料，初步选定了用于水分控制的多孔膜材料； 2、完成了两种负水头控制方法与装置的研究，并制作了仪器，与负水头控制装置配套，制作了盆栽实验用水分精控盆； 3、进行了两期小麦苗期水分精确控制盆栽实验，获得了一系列重要结论； 4、进行了不同负水头下土壤的毛管上升、水平入渗、垂直入渗研究，提出了临界湿润厚度、湿润系数两个概念，确定了湿润锋与入渗量的线性关系、土壤含水量的空间分布模型，土壤含水量空间分布的简单推算方法。

Objective To investigate the clinical features, causes of blindness and diagnosis of Vogt Koyanagi Harada syndrome Methods The data of 157 patients with VKH syndrome were reviewed and analyzed Patients were carefully examined with slit lamp, ophthalmoscope, three mirror lens, fundus fluorescein angiography, indocyanine green angiography and HLA typing Results Headache was noted in 73 5% of these patients Simultaneous involvement of both eyes occurred in 80 8% of these patients Chroiditis,papilledema and edema of the retina adjacent to the optic nerve were noted in 100% of these patients in the posterior uveitis stage, whereas recurrent granulomatous anterior uveitis (98 4%),"sunset glow" fundus (95 8%) and Dalen Fuchs nodules (71 2%) were the common ocular findings in the recurrent anterior uveitis stage The common causes of blindness were papillitis, exudative retinal detachment and complicated cataract in the posterior uveitis stage, anterior uveal involvement stage and its recurrent stage Poliosis (36 3%) and alopecia (35 0%) were the most common extraocular findings Early irregular patches of fluorescence, followed by localized hyperfluorescent spots were the typical findings of FFA Dilation of choroidal vessels and leakage of ICG from the choroidal vessels were the common ICGA findings The prevalence of HLA DR4 and HLA DRw53 in patients (54 9% and 71 8% respectively) was significantly higher than that in controls (14 7% and 38 2% respectively) Conclusions VKH syndrome is characterized by chroiditis, papillitis or neuroretinitis in the posterior uveitis stage, followed by a generalized uveitis with a typical recurrent granulomatous anterior uveitis Extraocular findings and relevant examinations including FFA, ICGA and HLA typing are helpful to the diagnosis of VKH syndrome

目的探讨Vogt-Koyanagi-Harada综合征患者的临床特征、盲目原因及诊断等有关问题。方法对在1996年1月至2000年12月间就诊资料完整的157例VKH综合征患者进行回顾性分析，并对裂隙灯、眼底镜、三面镜、荧光素眼底血管造影(fundus fluorescein angiography，FFA)、吲哚青绿血管造影(indocyanine green angiography，ICGA)及人类白细胞抗原分型等检查结果进行分析。结果 VKH综合征最常见的前驱症状为头痛(102例，73.5%)，双眼同时患病118例(80.8%)；后葡萄膜炎期眼部主要表现为脉络膜炎、视乳头及附近视网膜水肿(100.0%)；前葡萄膜炎反复发作期眼部表现为复发性肉芽肿性前葡萄膜炎(128例，98.4%)、晚霞状眼底改变(95.8%)及Dalen-Fuchs结节(71.2%)；后葡萄膜炎期、前葡萄膜受累期及前葡萄膜炎反复发作期导致盲目的主要原因分别为视乳头炎、视网膜脱离及并发性白内障；毛发变白(36.3%)及脱发(35.0%)是最常见的眼外表现；炎症活动期FFA典型表现为斑驳状高荧光，ICGA发现脉络膜血管扩张、通透性增高等改变；VKH综合征患者HLA-DR4及HLA-DRw53的阳性率(54.9%及71.8%)显著高于正常对照组(14.7%及38.2%)。结论 VKH综合征患者在后葡萄膜炎期眼部典型表现为双侧脉络膜炎、视乳头炎或神经视网膜炎，随后出现以反复发作的肉芽肿性前葡萄膜炎为特征的全葡萄膜炎。眼外症状及相关的辅助检查包括FFA、ICGA 及HLA分型等有助于VKH综合征的诊断。

The present studies showed that two cell populations were found in haemocytes: large cell with high granularity and small cell with low granularity by flow cytometry FCM on light scanttering pattern. Two distinct cell types were identified based on phase contrast microscope: one type of cell was dark and dioptric aberration, while the other was bright and dioptric strong. By Giemsa and H.E staining, cytoplasmic staining were heterogeneous and internal particles were obvious in one type of cell, while cytoplastic staining were homogeneous and internal particles were inexistent in the other type of cell. By transmission electron microscope, we found that the mitochondria, Golgi apparatus organelles were rich and internal particles were obvious in one type of cells, and contrary to the another cells.

流式细胞术光散射图谱显示血细胞被分两类，一类为颗粒度高的大细胞，另外一类为颗粒度低的小细胞；相差显微镜观察显示，血细胞可分为胞体暗、折光性差和胞体明亮、折光性强的两类； Giemsa和H.E染色显示细胞分为胞质染色不均一、胞内颗粒明显和胞质染色均一、胞内颗粒不明显的两类；透射电镜超薄切片观察显示，颗粒明显的细胞胞质内线粒体、高尔基体等细胞器较丰富，颗粒不明显的细胞胞质内细胞器较少；负染结果表明血细胞主要分为表面不光滑、突起明显和细胞表面光滑、突起较不明显的两类。

Material and Methods:From March,2008 to January,2009,250 patients(146 Males and 104 Females,ranged from 18 to 82 years old,mean age was 48 years old) enrolled in the First Affiliated Hospital of Kunming Medical College were performed dual energy scan(Group A,200 patients) and Neuro-DSA scan(Group B,50 patients) using DSCT.The patients were randomly divided into Group A and Group B.Regarding to the justification of scan protocol,100 patients in Group A were selected as sub-group to explore the scan protocol.The image quality Was analyzed both in four categories(4.0/60、4.5/60、4.0/70、4.5/70) of flow rate and total amount of contrast-enhanced material and in three categories(100—150HU、150—200HU、200-250HU) of CT value of left common carotid artery.The comparison of two scan approach was based on image quality,radiation does, postprocessing methods,time of scanning and subtraction and data storage amount.To validate the diagnostic capability of DE-CTA virtual unenhanced images,100 patients in Group A were undergoing head scan both in routine non-contrast program and DE-CTA virtual unenhanced program.Mean CT values,signal-to-noise ratio,image quality,sensitivity of lesion identification,and radiation does were applied to comparison.According to diagnostic results of dual energy of cerebral arterial angiography,62 patients out of 200 patients in Group A were confirmed with cerebral vessel lesions,the results were complied with DSA findings(the time between the two methods were within 1 week).

材料与方法收集2008年3月到2009年1月间在昆明医学院第一附属医院行DSCT CTA检查者250例(男性146例，女性104例，年龄范围18-82岁，平均48岁)，随机分为A、B两组，A组200例行双能量头颅CTA扫描；B组50例行Neuro-DSA扫描。A组前100例按对比剂不同流率和总量分为4组(4.0/60、4.5/60、4.0/70、4.5/70)对图像质量比较；按触发时左颈总动脉内平均CT位分3组(100—150HU、150—200HU、200-250HU)，比较图像质量的差异；对A、B两组血管图像质量、辐射剂量、后处理的方法、扫描和后处理时间及数据的容量进行比较分析；A组中前100例行常规与DE-CTA虚拟平扫的平均CT值、信号噪声比、图像质量、病灶显示情况、辐射剂量的比较分析；A组中行DSA检查62例(两种方法间隔时间均1周内)，比较两种检查方法诊断结果是否符合，并对其中24例动脉瘤行动脉瘤部位、数目、形态、瘤颈显示程度、瘤颈大小及瘤体长径和短径的比较研究。

Gene mutations were detected in 25 patients and a 18-year-old girl among 16 families. Trinucleotide repeats of CAG were 73～79. The fragments of abnormal alleles were 380～402bp, and all patients were heterozygous. The copy numbers of normal alleles were 18～40, fragments from 202～270bp. SCV reduction was much obvious compared to MCV, MCV and SCV in lower limb ?ere much more slow than that in upper's. BAEP, VEP were also delayed in latency. The anticipation in parental sex bias were much more obvioius than that in matental's. Cerebellar ataxia was most severe, the next were dysarthria and bulging eyes. Amytrophy was seen only in bed ridden patients. Cerebellar atrophy was more severe than brain stem, cord atrophy was n't observed in all MJD.

结果检出10个家系25例病人及1例症状前18岁女孩有MJD基因突变，CAG三核苷酸重复73～79次，异常等位基因片段长380～402bp，均为杂合子；正常人CAG三核苷酸重复18～40次，等位片段长200～270bp，电生理发现MJD的SCV减慢比MCV明显，而下肢的MCV、SCV又较上肢明显，BAEP、VEP均有不同程度的潜伏期延长或波的异常；MJD的父亲遗传早于母亲，进展也较块，临床以小脑性共济失调为突出症状，其次为构音障碍、突眼等，肌肉萎缩仅见于晚期病人；MRI示小脑萎缩较明显，脑干萎缩并不严重，未见明显的颈髓萎缩。

A new technology, Supercritical Fluid CO_2 Extraction, was also supplied to study the plant. The technological parameter was optimal. Total extract of SFE-CO_2 were 4.86 times of the essential oil extracted by SDE, main active extracted constituents were 1.36% high than SDE. The chemical constituents of SFE-CO_2 were also analyzed by GC-MS. 57 constituents were identified, 29 constituents of them were first discovered. 24 constituents were discovered in two extract. Fingerprint is a good technology to elevate the quality of herb medicine. The main active fraction can be extracted by SDE and SFE, Fingerprint was studied respective, chromatographic parameter was acquired. 22 peaks were assigned common peaks in the spectrum of essential oil, 17 peaks were assigned to common peaks in the spectrum of SFE extract.

采用现代共性关键技术超临界CO_2萃取技术，通过正交试验，优选了超临界CO_2提取牛至挥发油的工艺，并与传统的水蒸气蒸馏法进行了比较研究，结果显示：前者提取有效部位的收率为后者的4.87倍，主要有效成分的转移率是后者的1.36倍；采用GC-MS分析方法和NIST质谱库检索，对SFE和SDE所提化学成分进行比较，SFE分离鉴定了57种化合物，占提取物总含量的86.96％，其中29种化合物为首次从该植物中鉴定；后者分离鉴定了69个化合物，占提取物总量的98.28％，其中27种化合物为首次从该植物中鉴定；两种方法的提取物中共有成分25种，包括主要有效成分麝香草酚、香荆芥酚、对聚伞花素等。

Results 8 cases of 32 patients were misdiagnosed as tuberculosis,7 cases were misdiagnosed as pneumonia,6 cases were misdiagnosed as tuberculosis pleurisy,3 cases were misdiagnosed as omarthritis,2 cases were misdiagnosed as arthritis,2 cases were misdiagnosed as transient ischemic attack,2 cases were misdiagnosed as hemicrania,2 cases were misdiagnosed as intervertebral disk hernia.

结果 32例患者中，误诊为肺结核8例，肺炎7例，结核性胸膜炎6例，肩周炎3例，关节炎2例，短暂性脑缺血发作2例，偏头痛2例，椎间盘突出2例。

But this is impossible, as long as it is engaging in a market economy, there are risks in any operation.

但是，这是不可能的，只要是搞市场经济，是有风险的任何行动。

We're on the same wavelength.

我们是同道中人。