真核表达人呼吸道合胞病毒F蛋白的纯化方法

目的 真核表达人呼吸道合胞病毒(human respiratory syncytial virus,SV)融合蛋白(fusion protein,),并完成蛋白纯化及纯度测定.方法 根据编码F蛋白的基因序列设计引物,CR方法扩增出3'端带His标签的F基因序列,克隆入pGEM-T-easy载体,经核酸序列分析后,进一步克隆到pcDNA3.1( )真核表达载体,限制性内切酶鉴定,用脂质体Lipofectamine2000转染COS-7细胞,2 h后再用Westem blot检测目的蛋白的表达.Ni柱亲和层析纯化COS-7细胞表达的F蛋白,高效毛细管电泳分析纯化后蛋白纯度.结果 核酸序列分析证实获得带His标签的RSV F基因序列,没有发生无义突变.转染COS-7细胞后,利用Western blot方法检测到F蛋白的特异性条带,纯度达99%以上.结论 初步建立了真核表达RSV F蛋白的纯化方法,为进一步优化RSV F蛋白制备条件及单克隆抗体及诊断试剂等研究奠定了基础.     

Attenuation of intracavitary applicators in 192Ir-HDR brachytherapy

Unlike the penetrating monoenergetic 662 keV gamma rays emitted by 137Cs LDR sources, the spectrum of 192Ir used in HDR brachytherapy contains low-energy components. Since these are selectively absorbed by the high-atomic number materials of which intracavitary applicators are made, the traditional neglect of applicator attenuation can lead to appreciable dose errors. We investigated the attenuation effects of a uterine applicator, and of a set of commonly used vaginal cylinders. The uterine applicator consists of a stainless steel source guide tube with a wall thickness of 0.5 mm and a density of 8.02 g/cm3, whereas the vaginal cylinders consist of the same stainless steel tube plus concentric polysulfone cylinders with a radius of 1 or 2 cm and a density of 1.40 g/cm3. Monte Carlo simulations were performed to compute dose distributions for a bare 192Ir-HDR source, and for the same source located within the applicators. Relative measurements of applicator attenuation using ion-chambers (0.125 cm3) confirmed the Monte Carlo results within 0.5%. We found that the neglect of the applicator attenuation overestimates the dose along the transverse plane by up to 3.5%. At oblique angles, the longer photon path within applicators worsens the error. We defined attenuation-corrected radial dose and anisotropy functions, and applied them to a treatment having multiple dwell positions inside a vaginal cylinder.     

重组鸡Igλ轻链真核细胞分泌表达及其单克隆抗体的制备与鉴定

目的: 构建重组载体pcDNA-λ并在COS7细胞分泌表达鸡Igλ轻链, 制备抗鸡Igλ轻链的单克隆抗体(mAb).方法: PCR扩增带有信号肽的鸡Igλ轻链基因, 限制性酶切消化后, 定向克隆于真核表达载体pcDNA3, 构建具有6×His标签、分泌表达的重组载体pcDNA-λ.重组载体转染COS7细胞后, SDS-PAGE分析真核表达载体pcDNA-λ在COS7细胞的分泌表达.用2×106个雏鸡B淋巴细胞免疫BALB/c小鼠, 取脾细胞与骨髓瘤细胞融合, 分别采用细胞免疫荧光和间接ELISA筛选阳性杂交瘤克隆.结果: 成功构建分泌表达鸡Igλ轻链的重组载体, SDS-PAGE分析表明在COS7细胞上清有目的蛋白的分泌表达.杂交瘤细胞经筛选与鉴定, 获得2株分泌抗鸡Igλ轻链mAb的杂交瘤细胞株, Western blot检测到该mAb对重组鸡Igλ轻链的反应性.结论: 构建了重组表达载体pcDNA-λ并在COS7细胞上清分泌表达了重组鸡Igλ轻链.并用淋巴细胞杂交瘤技术成功筛选出抗鸡Igλ轻链mAb.为深入研究鸡Igλ轻链的结构与功能奠定了基础.     

升压治疗急性脑缺血再灌注损伤保护作用的实验研究

目的:探讨升压治疗局灶性脑缺血的保护作用及治疗的维持时间。方法:采用大鼠局灶性脑缺血再灌注模型,40只大鼠随机分为五组:模型对照组(A)、升压维持15min组(B)、维持45min组(C)、维持90min组(D)和120min组(E),观察各组神经功能评分、脑梗死体积和病理学改变。结果:神经功能缺损评分B、C、D组明显低于A组;B、C、D、E各组脑梗死体积明显低于A组,C、D、E组明显低于B组;各治疗组组织病理学改变比对照组轻,B组最轻。结论:缺血3h再灌注时,升压维持时间在2h以内对脑损伤有保护作用,升压最佳维持时间是15min。     

An animal model of SARS produced by infection of Macaca mulatta with SARS coronavirus

A new SARS animal model was established by inoculating SARS coronavirus (SARS-CoV) into rhesus macaques (Macaca mulatta) through the nasal cavity. Pathological pulmonary changes were successively detected on days 5-60 after virus inoculation. All eight animals showed a transient fever 2-3 days after inoculation. Immunological, molecular biological, and pathological studies support the establishment of this SARS animal model. Firstly, SARS-CoV-specific IgGs were detected in the sera of macaques from 11 to 60 days after inoculation. Secondly, SARS-CoV RNA could be detected in pharyngeal swab samples using nested RT-PCR in all infected animals from 5 days after virus inoculation. Finally, histopathological changes of interstitial pneumonia were found in the lungs during the 60 days after viral inoculation: these changes were less marked at later time points, indicating that an active healing process together with resolution of an acute inflammatory response was taking place in these animals. This animal model should provide insight into the mechanisms of SARS-CoV-related pulmonary disease and greatly facilitate the development of vaccines and therapeutics against SARS.     

db-cAMP对转化细胞增殖抑制及其对CaM水平和PKⅡ活性的影响

目的　探讨双丁酰 环核苷酸 (db cAMP)对转化细胞增殖及细胞表型作用的机理。　方法　以流式细胞光度术 (flowcytometry ,FCM)、软琼脂集落形成、放射免疫、Northern印迹和激酶活性分析等方法观察db cAMP对转化的C3H1 0 T1 2 小鼠成纤维细胞增殖、细胞表型、钙调素 (calmodulin ,CaM)表达及蛋白激酶Ⅱ (proteinkinaseⅡ ,PKⅡ )活性的影响。　结果　db cAMP(1mmol L)使C3H1 0 T1 2 转化细胞增殖及软琼脂集落形成能力受到显著抑制 ,转化细胞中CaM的表达及PKⅡ活性明显高于正常细胞 ,经db cAMP处理后也受到明显抑制。　结论　细胞转化及cAMP的诱导分化作用与PKⅡ活性的改变有密切相关性     

mPEG表面修饰的PLGA嵌段共聚物的血液相容性评价

本实验室设计合成了三种不同LA／GA比例的mPEG修饰PLGA（PELGA，含15％mPEG），为了评价它们的血液相容性，我们以硅化玻璃试管为阴性对照，未硅化的试管为阳性对照，参照国际标准（ISO10993）和《中华人民共和国国家标准GB／T16886医疗器械生物学评价》方法进行了体外评价实验。试验包括溶血率实验，血小板黏附实验，动态凝血时间实验，凝血时间实验，血浆复钙时间实验和凝血酶原时间实验等综合评价指标。结果表明，合成材料具有优良的血液相容性，材料制成的纳米粒有望应用于静脉注射。     

Expression and changes of HSP70 in the rat forebrain subjected to gamma knife (100Gy) irradiation targeted on the caudate putamen and survived for different times

Forebrain heat shock protein 70 (HSP70) immunohistochemical reactivity was investigated in rats subjected to gamma knife irradiation focusing on the right caudate putamen nucleus. The forebrain sections of all experimental animals were processed with anti-HSP70 antiserum and then by avidin-biotin peroxidase complex immunohistochemistry after gamma ray irradiation with a dose of 100Gy and they each survived for different times (from 30 min to 30 days). Some neurons, glial cells, and endothelial cells were HSP70-like immunoreactivity (HSP70-LI) positive. HSP70-LI was mainly distributed in the target area of irradiation, as well as in non-target regions, e.g. the cortex, hippocampus, and hypothalamus, etc. The expression and change of HSP70-LI from 3 h to 30 days after irradiation followed the following rules: (1) Within 3 to 24 h, the dilated vessels with HSP70-LI endothelial cells were found at first, and a few lightly stained HSP70-LI neurons and glias were observed in the target and non-target regions; (2) In 3-7 days, darkly stained HSP70-LI neurons and glias were apparently increased and formed an expression peak. From 14 to 30 days, HSP70-LI cells were distinctly decreased and became weakly stained or negative. These results suggested that although the irradiation target of the gamma knife was localized, the response to irradiation occurred extensively.     

遗尿儿童和青少年情绪和行为问题及相关因素研究

目的 调查河南省儿童青少年遗尿症的情绪和行为问题的现况及其影响因素。 方法 从2019年10月—2020年3月,采用分层随机整群抽样方法抽取河南某县5所中小学校,采用一般资料调查表、长处和困难问卷及儿童睡眠调查表进行调查,对影响遗尿患儿情绪和行为的因素进行单因素、多因素分析。 结果 实际发放问卷4 500份,回收4 120份,有效问卷3 912份,有效回收率为94.95%。调查结果显示有7.82%儿童患有遗尿症。遗尿组的情绪症状、多动和困难总分得分明显高于正常组(P＜0.001),两组品行问题、同伴交往和社会行为得分的比较差异无统计学意义(P＞0.05)。Pearson相关分析结果显示,遗尿儿童和青少年的困难总分与打鼾、夜间呼吸问题、嗜睡、注意力缺陷、睡眠总分均存在正相关关系(P＜0.05)。多元线性回归结果显示睡眠呼吸紊乱、照顾者文化程度低、便秘、学习成绩较差是遗尿患儿情绪和行为问题的影响因素。 结论 遗尿患儿情绪和行为问题的检出率高,影响因素是睡眠呼吸紊乱、照顾者文化程度低、孩子学习成绩差、便秘,应引起关注。