人基因工程重组超氧化物歧化酶对骨髓造血干细胞的辐射防护作用

用照射小鼠的骨髓造血干细胞(CFU-S)外源性脾结节形成法,评价人基因工程重组SOD(rhSOD),聚乙二醇(PEG)修饰的长半衰期重组SOD(PEG-SOD)对CFU-S的辐射防护作用.结果显示:rhSOD和PEG-SOD均能明显提高CFU-S产率,其中rh SOD在照射前1h给药效果好,而PEG-SOD在照射前2～3h给药作用更为明显,化学修饰后SOD血浆半衰期延长,将使给药时机及药物作用持续时间得到改善.     

用pProEXHT载体进行人FLT3配体基因的表达与产物的亲和层析纯化

目的 :探讨寡聚组氨酸多肽与目标蛋白融合基因的表达及其产物的金属离子螯合亲和层析纯化方法。方法 :将人FLT3配体 (FL)胞外段cDNA连入 pProEXHT载体 ,转入大肠杆菌实现表达。分离包涵体并进行复性处理后 ,通过Ni2 离子螯合亲和层析纯化融合蛋白表达产物。结果和结论 :6×组氨酸 FL融合蛋白的表达量约占菌体蛋白总量的 15% ,用Ni2 离子螯合亲和层析纯化表达产物 ,一次过柱的纯度可达 90 %以上 ,操作程序简便省时 ,是日后FL基因工程产品大规模制备的可行途径     

γ线对小鼠细胞遗传学的影响及重组人超氧化物歧化酶的抗放射作用(论著)

目的：研究γ射线对小鼠遗传学的影响及重组人超氧化物歧化酶（rhSOD）的抗放作用。方法：实验用甲基纤维素缩集法计算淋巴细胞微核率和活体秋水仙素法进行骨髓细胞染色体畸变率分析。结果：（1）4．0Gyγ线能明显增加小鼠淋巴细胞微核细胞率、微核率和骨髓染色体畸变率。（2）rhSOD可减轻射线所致小鼠淋巴细胞微核细胞率、微核率及骨髓细胞染色体畸变率，尤以照射前后给药为佳。结论：rhSOD对小鼠细胞遗传学辐射损伤有明显保护作用。     

三种方法纯化生物工程抗体片段的比较

比较3种不同纯化体系纯化生物工程抗－HBsFab的效率和效果，寻求高效，经济的生物工程产品批量纯化方法，采用增菌发酵抗－HBsFab阳性克隆，超声破菌法制备Fab上清，分别缓冲平衡抗Fab -Sepharose亲合胶，链球菌蛋白G（prot.G)-Sepharose胶和Ni-NTA离子螯合胶，对Fab上清进行过柱纯化，紫外光检测目的蛋白得率，SDS－PAGE鉴定产物的纯度并用HBsAg包被ELISA检测其生物活性，结果显示，等容积不同类别的胶体对目的蛋白的纯化效率依次为：Ni-NTA离子螯合胶＞prot.G-Sepharose胶＞抗－FabSepharose亲合胶；在各胶体饱和结合能力之内，3种方法中获高纯度产品，在SDS－PAGE中呈现单一区带：HBsAg包被ELISA检测结果为3种方法纯化制备的Fab在抗原结合能力上未显示明显差别，提示，3种纯化方法各有优势和应用限制，Ni-NTA法在经济性，实用性和纯化效率上明显优于其他两种方法。     

大肠杆菌O157∶H7新蛋白Ecs4563的基因克隆、表达及纯化

目的:克隆、表达并纯化肠出血性大肠杆菌(EHEC)O157:H7新预测分子伴侣蛋白Ecs4563,以进一步开展其结构与功能的研究.方法:利用PCR方法从EHEC O157:H7基因组中扩增出分子伴侣基因ecs4563,构建重组表达质粒,在大肠杆菌BL21(DE3)中实现诱导表达,采用Western印迹法进行鉴定,通过Ni2 螯合柱纯化获得目的蛋白.结果:构建了pET-24a-ecs4563重组质粒,实现了目的蛋白的融合表达,Ni2 金属螯合法纯化了目的蛋白,通过Western印迹法验证了融合蛋白的特异性.结论:得到了新预测分子伴侣Ecs4563,为下一步研究蛋白功能,进一步研究EHEC O157:H7的致病机制奠定了基础.     

p185HER-2蛋白胞外区的表达及其结合肽的筛选

目的：构建p185^HER-2蛋白胞外区的原核表达裁体，实现p185^HER-2蛋白胞外区的表达。以纯化p185^HER-2蛋白胞外区为靶，从噬菌体肽库中筛选其结合肽，用于肿瘤导向治疗。方法：从含有HER-2全长cDNA的质粒psv^2-cerbB-2中克隆了人p185^HER-2蛋白胞外区cDNA，将此cDNA克隆到pET-30a 表达栽体中，构建p185蛋白胞外区的表达栽体。表达的p185^HER-2蛋白胞外区通过Zn^2 螯合层析柱进行纯化。以纯化的p185^HER-2蛋白胞外区为靶，通过淘洗方法进行噬茵体肽库筛选。结果与结论：构建了p185^HER-2蛋白胞外区的原核表达栽体，实现了p185^HER-2蛋白胞外区的高表达。通过Zn^2 螯合层析柱实现一步纯化，p185^HER-2蛋白胞外区的纯度达到95％。从噬菌体随机环七肽库中找到了一组具有核心序列p185^HER-2蛋白胞外区结合肽。这些p185^HER-2蛋白胞外区结合肽有可能成为肿瘤治疗的新的候选分子。     

人组织型纤溶酶原激活剂（t—PA）突变体在CHO细胞中表达产？…

目的；为了从细胞株培养液中纯化生长半衰期的ｔ－ＰＡ组合突变体。方法：采用高表达克隆细胞株，进行大量培养，收集２４ｈ细胞培养液为初始材料，利用ｔ－ＰＡ Ｋ２区含有Ｌｙｓｉｎｅ结合位点的特性和Ｚｎ＾２＋与蛋白质中咪唑，巯基等基团螯合作用，经Ｌｙｉｎｅ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析和Ｚｎ＾２＋－Ｓｅｐｈａｒｏｓｅ４Ｂ金属螯合层析二步组合纯化。结果：获得高比活的ＦｒＧＧＩ，为研究该突变体生物学特性及推广     

咪唑啉受体抗血清选择性蛋白的表达、纯化及单克隆抗体制备

目的:表达和纯化咪唑啉受体抗血清选择性蛋白(imidazoline receptor antiserum-selected protein,IRAS protein),制备IRAS蛋白的单克隆抗体.方法:采用基因重组技术在大肠杆菌表达IRAS蛋白;金属镍螯合的Ni-NTA亲和层析柱进行蛋白纯化;杂交瘤技术建立分泌IRAS单克隆抗体的杂交瘤细胞株;以间接ELISA方法筛选分泌特异性IRAS单克隆抗体的杂交瘤细胞;采用蛋白免疫印迹、间接免疫荧光方法鉴定单克隆抗体的特异性.结果:成功表达并纯化了IRAS重组蛋白,纯度达到95%.共筛选出5株分泌IRAS单克隆抗体的杂交瘤细胞株,制备腹水并纯化了IRAS的单克隆抗体,腹水中单克隆抗体效价分别为1∶ 8×106,1∶ 2×106和1∶ 5×106,属于IgG1亚型.该抗体能与原核及真核系统表达的IRAS重组蛋白发生特异性反应,间接免疫荧光显示IRAS蛋白主要定位于细胞质中.结论:建立了稳定分泌IRAS单克隆抗体的杂交瘤细胞株,成功制备了特异性好的IRAS单克隆抗体,为研究IRAS的功能提供了有力的研究工具.     

A型肉毒毒素多表位串联体的设计、表达、纯化及鉴定

目的:设计、表达两个A型肉毒毒素多表位串联体.方法:从文献报道的A型肉毒毒素(botulinum neurotoxin type A,BoNT/A)的表位及生物信息学方法预测得到的B细胞表位中遴选表位,并加入适当的辅助性元件,设计多表位串联体A、B.对其基因进行优化后,经重叠PCR方法合成串联体A、B的全长基因.分别插入原核表达载体pQE-30,再转化E.coli M15[pREP4]感受态细胞中进行诱导表达,以金属螯合亲和层析法纯化重组蛋白,并进行鉴定.结果与结论:成功设计并构建了两个多表位串联体A、B,并在E.coli中以包涵体形式获得高效表达.Ni-NTA法纯化后分别获得纯度大于92.2%、99%的重组串联体A、B蛋白,并经透析复性法复性.Western印迹和间接ELISA显示纯化、复性后的重组蛋白与抗天然BoNT/A马血清有特异性结合反应.     

乙型肝炎病人血清中PreS1／2抗原检测方法的建立及初步应用

目的：建立乙型肝炎病毒（ｈｅｐａｔｉｔｉｓＢｖｉｒｕｓ，ＨＢＶ）ＰｒｅＳ抗原检测方法。方法：表达与纯化了ＨＢＶＰｒｅＳ１／２抗原，并用纯化蛋白免疫家兔，利用纯化抗血清包被ＥＬＩＳＡ板，吸附病人血清中包含ＰｒｅＳ１／２抗原的ＨＢＶ的病毒颗粒或病毒亚单位，特异吸附颗粒用抗ＨＢｓＡｇ的酶标单抗检测。结果：利用金属螯合柱层析的方法高度纯化了ＰｒｅＳ重组抗原，用复性的重组抗原免疫家兔３次，血清抗体滴度可达１     

子宫输卵管造影术中造影剂逆流分析

目的:分析子宫输卵管造影术(HSG)中的造影剂流,找出逆流的原因及防止的办法。材料和方法:总结48例造影剂逆流的资料,临床诊断均为不孕症,使用油质或水质碘造影剂。结果:48例中淋巴逆流25例、静脉逆流19例,混合逆流4例,二种造影剂的逆流表现基本相似,但“辐射征”是水质碘造影剂淋巴逆流的一种特殊表现。结论:HSG中用水质碘造影剂较油质碘造影剂所需时间短,即使逆流也不会出现严重的后果,但也应尽量减少逆流的发生。     

MSCT及DSA诊断消化道出血价值的实验研究

目的:探讨MSCT和DSA对诊断消化道出血的敏感性.方法:取一段动物小肠做成实验模型,经导管分别以0.1、0.2、0.3、0.4和0.5 ml/min的流率将浓度为3.0%的对比剂注入模型肠道内,然后对其进行MSCT扫描;再以同样的注射参数将未稀释的对比剂注入肠道模型内行DSA检查.观察和比较MSCT和DSA对溢出到肠管内对比剂的显示情况.结果:3.0%浓度的对比剂以0.1 ml/min的流率进入肠道时MSCT即可以显示,流率为0.2 ml/min时可清晰显示,高于0.2 ml/min的流率时显示更清晰;未经稀释的对比剂以0.1和0.2 ml/min的流率注入肠道,在DSA上不能显示,注射流率达到0.3 ml/min时DSA可显示浅淡对比剂影,流率达0.4 ml/min及以上时DSA可清晰显示.结论:MSCT显示消化道出血的敏感性高于DSA.     

Effect of degeneration of the intervertebral disk on the process of diffusion.

PURPOSETo test the hypothesis that diffusion of contrast medium into the intervertebral disk is affected by the integrity of the nucleus pulposus and annulus fibrosus.METHODSIn canine intervertebral disks, defects were made in the annulus fibrosus and nuclear material was removed from the disk with a nucleotome. MR imaging was performed with intravenous contrast medium at 15, 30, 60, and 90 days after the procedure. The diffusion of contrast medium in the intervertebral disk was studied by visual inspection and by measuring changes in signal intensity. The intervertebral disk were classified at each MR study as normal or abnormal on the basis of the signal intensity on T2-weighted images.RESULTSIn untreated disks after intravenous injection of contrast medium, a band of increased signal intensity was observed near the endplates that became wider with time and approached the center of the disk. In six of the 12 treated disks, the band of increased signal intensity was visibly diminished or less discrete compared with that in the control disks. Weeks later, these treated disks showed diminished signal intensity on T2-weighted images and bulging of the annulus fibrosus.CONCLUSIONSIntervertebral disks with defects in the annulus fibrosus and reduced cartilage content were characterized by abnormal diffusion of contrast medium into the disk, and changes characteristic of early disk degeneration were detected subsequently.     

The effect of susceptibility of gadolinium contrast media on diffusion-weighted imaging and the apparent diffusion coefficient

RATIONALE AND OBJECTIVES: The development of parallel magnetic resonance imaging has resulted in the frequent use of diffusion-weighted imaging (DWI) in clinical medicine, which usually involves the use of contrast medium. However, gadolinium (Gd) contrast medium may have some effect on DWI and the apparent diffusion coefficient (ADC). The present study was performed to determine whether the magnetic susceptibility of contrast medium alters the DWI signal and the value of ADC in some imaging techniques. MATERIALS AND METHODS: Nonfat suppression DWI, short-time inversion recovery (STIR) combination, and chemical shift selective (CHESS) combination DWI were performed to examine 10 phantoms with gadolinium-meglumine gadopentetate (Gd-DTPA) dissolved at concentrations from 0.0005 to 0.1 mmol in physiologic saline as a contrast medium. The average pixel value and ADC of each method were determined. RESULTS: ADC showed no differences between before and after treatment with contrast medium for all imaging techniques with Gd considered distributed over the whole tumor. The signal intensity did not change on nonfat suppression or CHESS combination DWI, but deteriorated on STIR. CONCLUSIONS: ADC was not influenced by the magnetic susceptibility of contrast medium. In addition, it was suggested that the ability of tumor detection may be reduced if STIR is used as fat suppression.     

Low-field interventional MRI in neurosurgery: finding the right dose of contrast medium

MRI is increasingly being used as an interventional tool in neurosurgery. The field strength of “intraoperative” MR systems is usually lower than that of imagers commonly used for diagnostic purposes. However, lesion enhancement and apparent lesion extent depend on field strength. The aim of this study was to compare the contrast between intracranial, contrast-enhancing space-occupying lesions and the surrounding white matter obtained with low-field (0.2 T) and high-field (1.5 T) MR imaging and to find the contrast medium dosage for low-field MRI that produces the same lesion-to-white-matter contrast as the one obtained with high-field MRI after the administration of a standard dose of the contrast medium. A total of 38 patients with intracranial metastases or high-grade glioma were enrolled in this study. T1-weighted spin-echo sequences were acquired. High-field (1.5 T) studies were performed after the i. v. administration of 0.1 mmol gadolinium-DTPA /kg body weight. For low-field MRI (0.2 T) a dose escalation technique was used. T1-weighted sequences were repeated after each of three i. v. injections of 0.1 mmol gadolinium-DTPA/kg body weight. Thus, at the low-field examinations three T1-weighted sequences with a contrast medium dosage of 0.1, 0.2 and 0.3 mmol gadolinium-DTPA /kg body weight were obtained. Lesion-to-white-matter contrasts were calculated and compared. The average lesion-to-white-matter contrast obtained with high-field MR examinations was 1.63 (standard deviation 0.32). In the low-field MR examinations the average lesion-to-white-matter contrast was 1.34 (0.2) after a single dose, 1.57 (0.2) after a double dose, and 1.71 (.19) after a triple dose of contrast medium. The lesion-to-white-matter contrast of the high-field MR examination after a single dose of contrast medium was significantly higher than that of the low-field study after a single dose (P < 0.0001), but did not differ significantly from the low-field studies after a double (P = 0.28) or a triple dose (P = 0.17) of contrast medium. In a series of patients with contrast-enhancing space occupying brain lesions low-field MRI (0.2 T) after a double dose of contrast medium yielded the same lesion-to-white-matter contrasts as high-field MRI (1.5 T) after a standard dose. This is an important finding to avoid errors in intraoperative MRI due to the immanently lower degree of lesion enhancement in low-field MR imaging. Received: 14 September 1999 Accepted: 15 February 2000     

Computed tomography of the post-operative lumbar spine: the need for, and optimal dose of, intravenous contrast medium

Forty-five patients who had undergone previous lumbar spine surgery with symptoms and signs warranting further investigation were examined by computed tomography (CT). The findings in three separate subgroups of 15 patients were compared. The first randomly selected group underwent CT without the use of intravenous contrast medium; the second and third groups were examined both before and after the administration of contrast medium, using 16.25 g and 32.5 g iodine, respectively. In the latter two groups the CT studies were reported both before and after enhancement. In the 15 patients in the first group (no contrast medium), uncertainty as to whether disc material or fibrosis was responsible for the radiological abnormalities was expressed in the final report at six of the 20 post-surgical sites. Among the 30 patients who received contrast medium, 40 post-surgical sites were examined; at six of these sites there was no radiological cause for concern; at 16 sites there were unequivocal radiological appearances of either disc material or fibrosis. Enhancement resolved the radiological uncertainty at 13 of the remaining 18 sites, almost always by reinforcing the initial radiological opinion; this left five sites where uncertainty persisted in the final report. The larger dose of contrast medium caused the more marked enhancement of those abnormalities considered to be due to fibrosis, although enhancement was readily recognised using the lower dose. While intravenous enhancement assists radiological interpretation at those sites where diagnostic uncertainty persists after routine CT, it would seem that it is not essential in patients in whom routine CT studies show no serious abnormality or the characteristic appearances of disc material or fibrosis.     

超微超顺磁性氧化铁和钆对比剂增强兔动脉硬化斑块之比较

目的：比较超微超顺磁性氧化铁（USPIO）和钆对比剂在磁共振图像上增强动脉粥样硬化斑块的异同。方法：20只新西兰大白兔高脂喂养8周,存活18只,分为对照组和实验组,每组各9只。使用磁共振在心电门控下对两组兔主动脉斑块进行检测,再给对照组注射钆对比剂,实验组注射USPIO对比剂,并分别于注射后即时、24、36和48 h进行扫描。扫描序列为二维时间飞跃法、快速自旋回波T1WI、脂肪抑制的快速自旋回波序列T1WI和快速自旋回波序列T2WI。比较各组兔主动脉硬化斑块内增强前后以及增强后不同时期的脂肪抑制T1WI和T2WI,对比噪声比（CNR）值变化差异。将两组动物处死后取出胸主动脉进行大体普鲁士蓝染色和切片显微镜观察。结果：对照组主动脉硬化斑块注射钆对比剂后T1WI显著强化,CNR值明显上升,24 h后回归注射前状态;T2WICNR值轻度下降,24 h后回归正常。实验组硬化斑块注射USPIO后T1WI显著强化,CNR值明显上升,24 h后有所下降,但明显高于注射前,48 h和72 h后CNR值持续上升;T2WI注射后信号显著降低,24 h后CNR值有所上升,但仍然低于注射前,48 h和72 h CNR值持续下降。实验组兔胸主动脉和切片普鲁士蓝染色阳性,对照组显示阴性。结论：相对于钆对比剂,超顺磁性、超长时间的血浆半衰期以及能够被巨噬细胞特异性吞噬的特性,使得常规剂量的USPIO可以同时引起强化组织信号在T1WI及T2WI上显而易见的变化,并且能够保持长时间的持续强化。     

The importance of serum serotonin levels in the measurement of radiation-induced bystander cell death in HaCaT cells

Abstract Purpose: The aim of this study was to investigate the importance of serum serotonin levels in the measurement of bystander cell death. The study was undertaken as part of an intercomparison exercise involving seven European laboratories funded under the European Union Sixth Framework Programme (FP6) Non-Targeted Effects (NOTE) integrated project. Materials and methods: Three batches of foetal bovine serum were tested; serum with high and low serotonin content from the intercomparison exercise as well as serum from the home laboratory. Three sets of human keratinocytes (HaCaT cell line) were cultured in DMEM:F12 medium supplemented with serum with high or low serotonin content or serum from the home laboratory and both donor and recipient HaCaT cells were plated. The donor HaCaT cells were irradiated (0.5 Gy) using a cobalt 60 teletherapy unit, the medium was harvested 1 hour post irradiation and transferred to the recipient HaCaT cells. Bystander induced cell death was measured by the clonogenic survival assay and the Alamar blue viability assay. Results: A significant reduction in cell survival, as measured by the clonogenic assay, and in cell viability, as measured by the Alamar blue assay, was observed in the recipient HaCaT cells treated with medium from irradiated cells compared to the cells treated with medium from unirradiated cells. No significant difference was found between the three batches of serum. Conclusions: The data suggest that in our cell system and with our endpoints (clonogenic assay and Alamar blue assay), serum serotonin levels do not play a role in bystander-induced cell death.     

Comparison of the safety of the standard dose and a higher dose of gadodiamide injection for MR imaging of the central nervous system

This study compared the safety and tolerability of gadodiamide injection at the standard dose (0.1 mmol/kg) and at a higher dose (0.3 mmol/kg) in 289 patients participating in a parallel group multiple independent trial program. All patients had a known or suspected central nervous system lesion necessitating investigation with contrast mediumenhanced magnetic resonance imaging. Safety assessments were made before and after injection of the contrast medium, and 1 day later. No adverse events were judged to be related to gadodiamide injection, and only 3.5% of the patients in each dose group reported adverse events that had an uncertain relationship to the contrast medium; both doses were therefore well tolerated. Headache was the most frequently reported event (2%). There were no significant injection-related changes in neurologic status, laboratory test results, or vital signs. The data obtained indicate that the higher dose of gadodiamide injection is as safe and well tolerated as the standard dose.     

MR mammography: influence of menstrual cycle on the dynamic contrast enhancement of fibrocystic disease

Magnetic resonance mammography (MRM) provides data regarding the nature of tumours based on contrast medium dynamics; fibrocystic changes in the breast, however, may lead to false-positive results. This study investigated whether the contrast medium dynamics of fibrocystic changes are dependent on the menstrual cycle. Twenty-four patients with palpable lumps but normal mammographies and ultrasound studies were examined. The MRM technique was performed during the first and second part of the menstrual cycle using a FLASH 3D sequence, both native and at 1, 2, 3 and 8 min after intravenous application of 0.15 mmol/kg body weight of gadodiamide. The calculated time–intensity curves were evaluated based on the following criteria: early percentage of contrast medium uptake in relation to the native value; formation of a plateau phenomenon after the second minute; the point of maximal contrast medium uptake; and calculation of the contrast enhancing index. During the second half of the menstrual cycle, a generally greater contrast medium uptake was observed. Nevertheless, when further diagnostic criteria, such as continuous contrast medium increase as a function of time, were considered, there was no increased rate of false-positive findings. The phase of the menstrual cycle may affect the specificity of the examination, if only the quantitative contrast medium uptake and the percentage of contrast medium uptake in the first 2 min are considered. A control MRM during the other half of the cycle may then be indicated and additional diagnostic criteria may improve specificity. Received: 14 August 1997; Revision received: 5 December 1997; Accepted: 5 October 1998