体内精原干细胞转染法建立转基因小鼠

将人Bcl-2 cDNA与小鼠乳清酸蛋白(WAP)5’上游调控序列融合后,与脂质体按一定比例混合,再加入适量的台盼蓝制成转染液,注入到小鼠睾丸中的曲细精管中,转染精原干细胞以探讨建立转基因小鼠的可行性。共注射了3只公鼠,4天后将公鼠与发情母鼠合笼交配,共生仔鼠20只。检测结果表明,有3只呈PCR阳性,Southern blot检测,阳性鼠2只,1只公鼠,1只母鼠,其中,公鼠意外死亡;Western blot证实,1只母鼠的乳腺组织表达了Bcl-2蛋白,其F1代的16只小鼠中,有7只呈PCR阳性。证实了体内精原干细胞转染建立转基因动物的可行性。     

Polymer-based delivering of shRNA to rabbit aortic smooth muscle cells suppressed the expression of IGF-1R in vitro and in vivo

Restenosis is one of clinical limitations for vein graft in coron- ary bypass graft. It has been proved that signal pathway IGF-1 and its receptor （IGF-1R） activated by hemodynamic mechanical stretch are responsible for the vascular smooth muscle cells proliferation in vein graft neointima formation. Unfortunately, there is no routinely successful method to resolve this problem. Gene delivering to vein graft possesses great therapeutic potential to prevent neointima formation. Polymer is one kind of nanoparticles, which can activate the process of endocytosis of cells. In this study, we evaluated the transfeetion efficiency and therapeutic potential of polymer- based transfection of plasmids expressing GFP and shRNAs targeting IGF-1R （pGFPshlGF-1Rs） to smooth muscle cells and rabbit external jugular vein graft. Results showed that polymer-based transfection provided high efficiency of trans- gene expression in smooth muscle cells in vitro. In vitro, IGF- 1R-specific shRNA transfected by polymer inhibited IGF-1R protein expression by 52 ±3.6%, when compared with mock transfected cells. In vivo delivering efficiency of pGFPshlGF-IR plasmid into the rabbit external jugular vein graft was significantly high in the polymer-based trans- fection group, when compared with negative control group. In vivo, polymer-based transfection IGF-1R-specific shRNA efficiently inhibited the expression of IGF-1R protein by 77 ± 3.6%, 65.6 ± 4.9%, and 76.7 ± 4.3% at 24, 48, and 72 h, respectively, when compared with negative control group. Our findings indicated that polymer-based transfec- tion may be a promising technique that allows the targeting of gene therapy for vein graft restenosis.     

The key residue for SSB-RecO interaction is dispensable for Deinococcus radiodurans DNA repair in vivo

The RecFOR DNA repair pathway is one of the major RecA-dependent recombinatorial repair pathways in bacteria and plays an important role in double-strand breaks repair. RecO, one of the major recombination mediator proteins in the RecFOR pathway, has been shown to assist RecA loading onto single-stranded binding protein （SSB） coated single-stranded DNA （ssDNA）. However, it has not been characterized whether the protein-protein interaction between RecO and SSB contributes to that process in vivo. Here, we identified the residue arginine-121 of Deinococcus radiodurans RecO （drRecO-R121） as the key residue for RecO-SSB interaction. The substitution of drRecO-R121 with alanine greatly abolished the binding of RecO to SSB but not the binding to RecR. Meanwhile, SSB-coated ssDNA annealing activity was also compromised by the mutation of the residue of drRecO. However, the drRecO-R121A strain showed only modest sensitivity to DNA damaging agents. Taking these data together, arginine-121 of drRecO is the key residue for SSB-RecO interaction, which may not play a vital role in the SSB displacement and RecA loading process of RecFOR DNA repair pathway in vivo.     

AAV-ITR单链DNA微载体在小鼠骨骼肌中的表达

AAV-ITR单链DNA微载体是一种基于腺相关病毒(AAV)倒置末端重复序列(ITR)的基因表达载体(AAV-ITR ss DNA mini vector)。前期研究已证明AAV-ITR单链DNA微载体在HEK 293T细胞中具有较高的转染、表达效率。本文中将相同拷贝数的AAV-ITR单链DNA微载体、3?-ITR末端错配的AAV-ITR单链DNA微载体(AAV-ITRmm ss DNA mutant vector)、AAV-ITR双链DNA和质粒分别用Turbo Fect转入小鼠骨骼肌中,比较检测AAV-ITR单链DNA微载体与其他基因表达载体在小鼠体内1周、1个月及3个月的表达效率。组织切片经荧光显微镜观察及荧光灰度值分析表明,相同分子摩尔数的AAV-ITR单链DNA微载体比AAV-ITR双链DNA和质粒在不同时期表达效率都要高且更稳定。提取注射3个月后的肌肉组织的DNA,用荧光定量PCR分析比较各载体的存留分子数。RT-PCR的结果显示AAV-ITR单链DNA微载体在注射3个月后的存留分子数较其他载体高。综合结果显示AAV-ITR单链DNA微载体在动物体内具有表达效率高和长久稳定的优势,有可能开发为基因治疗的一种高效、稳定的新型载体。     

自身代谢缓解甲氨蝶呤对小鼠体内成熟卵母细胞副作用的研究

甲氨蝶呤(methotrexate,MTX)作为抗叶酸代谢类药物,在临床上应用广泛。其副作用之一表现为损伤卵母细胞质量。那么,对于有妊娠需求的女性服药者,停药后多久才适合受孕呢?该文结合双光子荧光成像与三维重构技术研究此问题。通过单次腹腔注射生理盐水及5 mg/kg MTX建立了对照组和MTX组小鼠,随后建立了MTX注射后代谢5、10、15、20、25及30天组小鼠。研究发现,小鼠体内成熟卵母细胞的纺锤体正常形态比率与染色体正常空间排布比率,分别于代谢15天和25天后,显著高于MTX组,并恢复至对照组水平。表征卵母细胞功能的体外受精率、二细胞胚胎率及囊胚率,发现分别于代谢15、20、25天后,恢复至对照组水平。该文结果表明,自身代谢可恢复MTX所引起的卵母细胞质量受损及功能下降,所需时间约为小鼠的5个生理周期,即25天。     

人白细胞介素-11基因在家蚕培养细胞和虫体内的表达

将缺少编码信号肽序列的人白细胞介素－11（hIL-11)546核苷酸cDNA,重组于质粒pBacPAK8构建重组转移载体pBacIL-11,与经线性化修饰的家蚕核型多角体病毒（BmBacPAK)DNA共转染家蚕培养细胞株BmN,获得了插入hIL-11基因的重组病毒。Southern杂交表明重组病毒基因组中含有hIL-11基因片段,RNA斑点杂交表明hIL-11基因得到了转录。重组病毒感BmN细胞株、家蚕幼虫和蛹,在细胞培养上清、细胞抽提物、幼虫和蛹的体液样品中,SDS－PAGE电泳分析都能检测得到表达产物的特异性条带;采用IL－11依赖细胞株B9－11和MTT法测定表达产物的生物活性,表明rIL-11基因分别在培养细胞和蚕体内得到了高效表达。     

服用‘甘必康''能产生核酶吗?

五月某一天,李院士给我来电话说:2002年4月29日《科技日报》的医药、健康专版上,用三整版(10～12版)篇幅介绍治疗乙肝新药——甘必康,‘甘必康’的“原始组方是由当归、丹参、香橼等13种名贵中药……精制而成”的。文中提到,该药“一经被人服用,便可在人体环境作用下产生大量能够     

一种体内研究病原体致病机理的新方法——信号标签诱变技术

信号标签诱变技术是以整个基因组为基础的研究病原体致病机制,可在体内对毒力基因进行高通量筛选的一种新方法。近几年应用该技术已对十多种病原微生物进行了筛选。这些筛选中除了找到已知的毒力基因外,还都鉴定到了未知的毒力因子。本文就该技术的原理、优缺点、应用的必要条件、技术的改进及应用该技术鉴定到的毒力基因等作一综述。 A Novel Approach to Study Pathogenesis of Pathogens in vivo——Signature-tagged Mutagenesis YAO Xiao1,2,HUANG Liu-yu1,YANG Bo-lun2,SU Guo-fu1 1.Beijing Institute of Biotechnoloy,Beijing 100071,China; 2.College of Environmental and Chemical Engineering,Xi'an Jiaotong University,Xi'an 710049,China Abstract:Signature-tagged mutagenesis (STM) is a novel approach to study pathogenesis of pathogens and to screen virulence genes with high throughput in vivo,which is based on whole genome of pathogen in question.In resent years,more than ten species of microbial pathogens have been screened with this technology.There are also unknown virulence factors being identified with exception of known virulence genes identified in all these screens.This article reviews the principle,advantages and current limitations,the requirements,modifications of STM,and to date virulence genes identified by this technology. Key words：signature-tagged mutagenesis;virulence genes;pathogens;in vivo     

磁场与遗传免疫

人体是极为复杂的导电体,体内大量体液导电性能良好。又由于人体受地磁的影响产生磁场,但人体磁场的数量级在10^-10～10^-13的范围内,因而总的来说人体的磁场是极弱磁场。实验得知,外磁场和地球磁场对生命活动的影响,称为磁场的生物效应。磁场的生物效应的应用是磁疗,即磁场疗法。     

我国率先成功构建人体内软骨组织

国家“973”计划“组织工程的基本科学问题”项目首席科学家 ,上海第二医科大学曹谊林教授日前在接受记者采访时表示 ,在“973”计划、国家自然科学基金等的支持下 ,近年来 ,针对软骨组织工程研究中的国际性难题和关键性问题 ,我国科学家已经在软骨组织工程的基础研究和应用基础研究方面取得了一系列成果 ,并首次在世界上成功地在人体内构建出软骨组织 ,开创了软骨组织工程临床应用的先河。摘自《科学时报》2 0 0 3年 3月 10日我国率先成功构建人体内软骨组织