基因治疗及其研究进展

基因治疗作为一种新型的疾病治疗手段 ,正在广泛用于血友病、糖尿病、癌症、心血管病、爱滋病等多种疾病的治疗领域 .基因治疗的一般步骤包括 :目的基因的转移和目的基因的表达两个方面 .其中目的基因的导入和目的基因表达的精确调控是两个关键步骤 .新发展的还有反义疗法以及应用核酶进行基因治疗等 .本文就基因治疗的基本步骤、基因治疗的对象、方式及基因治疗的现状和前景作一个简单介绍     

Sialyl Lewisx-liposomes as vehicles for site-directed, E-selectin-mediated drug transfer into activated endothelial cells

E-selectin, exclusively expressed on activated endothelial cells, is a potential target for site-directed delivery of agents. We and others have shown that sialyl Lewis^x-liposomes (sLe^x-liposomes) are recognized by E-selectin. We now report an approach employing sLe^x-liposomes to deliver antisense oligonucleotides (AS-ODNs) directed against the adhesion molecule ICAM-1 to activated vascular endothelial cells. ICAM-1 expression was analyzed at the protein level by immunofluorescence and a cell surface ELISA, and at the RNA level by RT-PCR. We have investigated two different AS-ODNs complementary to the 3′ untranslated region and the AUG translation initiation codon of ICAM-1 mRNA. Both inhibited protein expression, but did not influence the mRNA level, pointing to a hybridization of AS-ODNs with the mRNA in the cytoplasm. Our results demonstrate the feasibility of a novel approach for the delivery of agents to activated endothelial cells by glycoliposomes targeted to E-selectin. Received 16 October 2000; revised 29 November 2000; accepted 29 November 2000     

Z25, a novel intronic snoRNA encoded in mammalian nucleolin gene

A novel intronic small nucleolar RNA ( snoRNA) , termed Z25, was identified from mammals by-computer analysis and experimental sequence methods. Z25 is a 69 nucleotides long RNA containing typical boxC/D motifs, terminal stem and an 11-nucleotide sequence complementary to 18S rRNA. In theory, Z25 functions as an RNA guide for the 2'-0-ribose methylation of adenine at position 1678 (human 18S rRNA coordinate) in 18S rRNA. Z25 snoRNA gene was found to be located in the fifth intron of nucleolin gene of human, mouse and rat, demonstrating that the mammalian nucleoline gene is a host gene encoding multiple snoRNAs.     

OsICK1基因植物反义表达载体的构建及遗传转化

OsICK1是水稻细胞周期蛋白依赖性激酶抑制剂基因．我们构建了水稻细胞周期蛋白依赖性激酶抑制剂基因（OsICK1）的植物反义表达载体，在农杆菌的介导下尝试使antisense OsICK1整合到水稻基因组中并初步得到验证．为进一步研究OsICK1对水稻细胞周期调控的作用奠定了基础．     

AS-mCLB1重组质粒联合多西紫杉醇抗Lewis肺癌细胞增殖活性的研究

pAS-mCLB1转染Lewis肺癌(LL/2)细胞72 h后,再加用多西紫杉醇(40 nmol/L)处理细胞,MTT法测定药物对LL/2细胞的体外杀伤作用.另将LL/2细胞接种C57BL/6小鼠,成瘤后分别用pAS-mCLB1、多西紫杉醇和pAS-mCLB1+多西紫杉醇处理动物,3天1次,共4次,观察肿瘤体内增殖情况,流式细胞仪检测动物肿瘤组织的细胞凋亡.结果显示：pAS-mCLB1联合多西紫杉醇可明显降低LL/2细胞体内、外增殖活性,同时促使肿瘤组织中细胞凋亡增加,两种方法具有协同作用.pAS-mCLB1显著增强多西紫杉醇抗肿瘤效应,此增强作用可能与pAS-mCLB1诱导肿瘤细胞凋亡,提高了多西紫杉醇的细胞毒作用有关.     

肝癌相关基因MEF2D反义RNA的鉴定及分析

鉴定和分析肝癌细胞中新的天然反义RNA MEF2D-AS根据已建立的双链RNA文库筛选与肝癌相关的反义RNA.通过RT-qPCR验证其反义RNA的存在,采用cDNA末端快速扩增技术获得其全长序列,并检测HepG2细胞经5-Aza-dC处理前后MEF2D基因正反义链表达量的变化.结果显示,成功鉴定出1条全长为1 265bp的新的天然反义RNA,并预测其属于长链非编码RNA(long non-coding RNA,简称为lncRNA),命名为MEF2D-AS.经5-Aza-dC处理后,MEF2D-AS的表达量升高,而MEF2DmRNA的表达量降低.证实了MEF2D-AS的存在并预测其属于长链非编码RNA,且与MEF2D正反义RNA是反相关关系.此研究结果可为进一步探讨肝癌发生机制提供参考.     

基于表达序列标签的萝卜ACA36基因簇鉴定

利用国际公用的表达序列标签（EST）数据库资源,运用生物信息学分析方法,在萝卜（Raphanus sativus）一条表达序列标签中发现一个新的snoRNA基因簇。此基因簇含有一个box H/ACA snoRNA基因和两个box C/D snoRNA基因,分别命名为RsACA36、RssnoR66和RsSNORD88。ACA36可形成典型的box H/ACA snoRNA双茎环二级结构;snoR66和SNORD88都具有典型的C盒与D盒保守元件,末端存在反向互补序列;三个snoRNA都含有能和核糖体RNA互补配对的反向互补序列。RsACA36和RsSNORD88均为在植物中首次发现。基于EST数据库、运用生物信息学方法鉴定snoRNA基因既保留了计算机RNA组学快捷、高效的优点,又使结果因为有了实验支撑而更直接、可信。     

Identification and functional analysis of 23 novel box C／D snoRNAs from Oryza sativa

In a cDNA library generated from rice small nuclear RNAs,30box C/D small nucleolar RNAs (snoRNAs) were identiffied through preliminary screen.Except 7 known snoRNAs such as U14,all snoRNAs were identified in rice for the first time experimentally.Among the 23 novel snoRNAs,11 snoRNAs appear rice-specific,6 snoRNAs are unique to plants,the remaining 6 snoRNAs have their counterparts in both Arabidopsis and yeast or mammals according to the conserved antisense sequencs that guide 2‘-O-ribose methylation of rRNA,17 of the 23 novel snoRNAs were predicted to guide 24 2‘‘-O-ribose methylations at the specificsites of rice 5.8S,18S,25S rRNAs,among which 19 methylated sites were determined by primer extension at low dNTP concentrations.The remaining 6 snoRNAs devoid of rRNA antisense elements may represent novel snoRNA species in rice.The results show that constructing a cDNA library from small nuclear RNAs is an effective experimental approach for novel snoRNA is identification.The novel snoRNAs are important in elucidating the genomic organization and expression of plant snoRNA genes and the mechanism through which 2‘‘-O-ribose methylations took place in rRNAs.     

Introducing antisense <Emphasis Type="Italic">waxy</Emphasis> gene into rice seeds reduces grain amylose contents using a safe transgenic technique

Rice (Oryza sativa L.) eating quality is one of themost important traits. Amylose content (AC) in rice en-dosperm is a major index affecting rice eating quality[1,2].It has a negative correlation with gel consistency of rice[3].Based on amylose content, r…