共查询到20条相似文献，搜索用时 555 毫秒
1.
设计小鼠Txt5基因的特异性引物,将小鼠c DNA作为模板,用PCR扩增出m Txt5编码区,并加入HA标签序列.将这一片段插入p MD-18T载体,再亚克隆至p Adtrack-cmv穿梭载体上.线性化后,转化BJ5183感受态细胞,在BJ5183中发生同源重组获得p Ad-Txt5质粒.p Ad-Txt5线性化后转染293A细胞,包装得到含Txt5基因的病毒.将病毒溶液感染大鼠原代心肌细胞,一段时间后观察绿色荧光,然后通过RT-PCR和免疫印迹法检测HA标签蛋白的表达.结果表明成功构建小鼠Txt5腺病毒表达载体并实现在大鼠原代心肌细胞中的表达. 相似文献
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《实验技术与管理》2014,(1)
目的:构建microRNA的表达载体,再转染生物体细胞观察该microRNA的转录或表达情况,以获知它的作用机理及对生物体的影响。方法:以microRNA-21(简化为miR-21)为例说明构建表达载体的方法。根据microRNA的成熟序列以及附近约200多碱基共约470个碱基序列,设计PCR引物,PCR扩增,PCR产物和pcDNA3.1(+)质粒双酶切后,用T4连接酶进行连接反应,并转化入感受态细胞,筛选后对重组质粒进行双酶切、琼脂糖凝胶电泳鉴定及测序分析,并将其转染Hela细胞,经RT-PCR实验检测其表达情况。结果:pcDNA3.1(+)/miR-21过表达载体转染细胞后使得miR-21在细胞中过表达。结论:成功构建了miR-21的真核表达载体,为进一步研究miR-21功能及作用机制奠定实验基础。 相似文献
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为了检测miR-431-5p在食管鳞癌（Esophageal squamous cell carcinoma,ESCC）中的表达,探讨其功能,利用TCGA公共数据库分析miR-431-5p在食管癌中的表达,通过RT-qPCR检测miR-431-5p在组织和细胞中表达;再利用miR-431-5p模拟物和抑制剂,通过CCK-8实验、细胞划痕和Transwell实验以及蛋白免疫印迹实验,检测miR-431-5p对ESCC细胞增殖、侵袭和迁移的影响以及分子作用机制。结果显示：miR-431-5p在食管癌中高表达;转染miR-431-5p模拟物后,KYSE30细胞增殖率、迁移率和侵袭率分别增加了24.19%,30.86%和50.29%(P<0.05);而转染miR-431-5p抑制剂后,TE-11细胞数量、迁移率分别降低了21.43%和9.52%(P<0.05);miR-431-5p模拟物增加KYSE30细胞p-ERK表达量11.68%;miR-431-5p抑制剂降低TE-11细胞p-ERK表达量45.73%。研究结果表明：miR-431-5p通过MAPK/ERK通路促进ESCC细胞... 相似文献
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目的：克隆人Lumican基因及构建Lumican基因真核表达载体并研究其对人子宫内膜癌细胞HEC-1A增殖的影响。方法：取人新鲜正常子宫内膜组织,提取总RNA,采用逆转录-聚合酶链反应（RT-PCR）扩增Lumican基因,将该基因定向克隆到真核表达载体pEGFP-N1中,构建真核细胞表达载体pEGFP-N1-Lumican,用限制性内切酶酶切分析,DNA序列分析鉴定重组质粒;将测序正确的Lumican基因通过脂质体介导下转染人子宫内膜癌细胞HEC-1A;逆转录-聚合酶链反应（RT-PCR）和Western Blot 检测转染细胞HEC-1A的Lumican mRNA和蛋白表达。采用MTT法观察转染Lumican基因的人子宫内膜癌细胞HEC-1A的增殖能力。结果：重组质粒pEGFP-N1-Lumican经HindⅢ和BamHⅠ酶切,获得8311 bp片段和865 bp插入片段,序列分析表明插入的片段与Gene Bank发布的序列一致;荧光显微镜下可见转染的HEC-1A细胞有绿色荧光蛋白的表达;转染细胞HEC-1A的Lumican mRNA表达上调,Lumican蛋白相对上调率为74．62％（P＜0．05）。与对照组细胞比较,转染Lumican 基因的人子宫内膜癌细胞HEC-1A的抑制率为21．35％±2．78％。结论：成功构建了真核表达载体pEGFP-N1-Lumican,该载体在体外能有效抑制人子宫内膜癌细胞HEC-1A的增殖能力,为以Lumican基因为靶点研究其对人子宫内膜癌细胞HEC-1A的恶性生物学特性提供实验基础。 相似文献
5.
绿色荧光蛋白基因在转基因金鱼中的整合与表达 总被引：2，自引：0，他引：2
通过分子克隆技术将鲤鱼β-肌动蛋白启动子(carpβ—actin promoter A)与编码绿色荧光蛋白(GFP)的cD-NA融合构建成能在真核生物体内表达的质粒载体pAGFP。质粒pAGFP经Pvu线性化后采用显微注射法导入金鱼受精卵，在胚胎发育初期，经紫外光激发能观察到少数胚胎发绿色荧光。在通过PCR实验检测出的5个阳性个体染色体DNA中，经点杂交实验证实有2个个体染色体基因组上整合了GFP基因。 相似文献
6.
为优化细胞生物学实验教学内容、让学生及时掌握先进的科研技术、实现研究型与创新型人才培养的目标,设计了一套简便的、适合教学的动物细胞培养和细胞转染实验方法. 即:无菌环境下培养CHO(Chinese Hamster Ovary)细胞,然后将绿色荧光蛋白基因作为报告基因以质粒为载体导入CHO细胞中,实现表达后利用荧光倒置显微镜观察美丽的荧光显微世界.同时,探讨了综合型和研究型两类细胞培养与转染实验课程的具体实施方案.该实验内容和教学方法使学生综合分析与研究问题的能力得到锻炼与提高,为培养适应科研快速发展、具有较强竞争力的创新人才奠定了基础. 相似文献
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《大连大学学报》2016,(6):62-65
本实验旨在构建人核糖核酸酶抑制因子稳定干涉载体pLKO.1-ds RI,为后续观测人核糖核酸酶抑制因子干涉载体的表达对肿瘤细胞的影响奠定基础。用亚克隆法,将针对人核糖核酸酶抑制因子的干涉片段从Simple T-ds RI质粒克隆到pLK-0.1-TRC质粒,用酶切法筛选得到阳性重组质粒pLKO.1-ds RI,用测序法鉴定克隆序列正确。转染时设干扰组、空载体组和空白组三组,每组三次重复。用脂质体CellfectinR将pLKO.1-ds RI(干扰组)、pLKO.1-TRC(空载体组)转染进人肝癌细胞HepG2细胞中,未处理的细胞作空白组。转染后72hr用Western blotting检测细胞中核糖核酸酶抑制因子表达的变化。双酶切鉴定及测序鉴定结果均正确;Western blotting结果表明,对比空白组(1.1578±0.015)和空载体组(1.1216±0.027),干扰组RI表达(0.6119±0.048)明显下调(P<0.05)。结果表明人核糖核酸酶抑制因子干涉载体pLKO.1-ds RNH1构建成功。 相似文献
9.
目的:研究mi R-718和早期生长反应蛋白3(EGR3)在肝癌细胞系Hep G2及SMMC-7721中对细胞恶性行为的作用。创新点:首次在肝癌细胞系Hep G2和SMMC-7721中发现miR-718作为抑癌基因及EGR3作为促癌基因起到调控肿瘤恶性行为的作用。方法:采用聚合酶链反应(PCR)方法构建mi R-718和EGR3的过表达及敲降质粒,并采用实时定量PCR(q RT-PCR)方法验证质粒有效性;采用MTT法和克隆形成实验检测肝癌细胞系HepG 2和SMMC-7721的生长增殖能力;采用Transwell迁移侵袭实验检测肝癌细胞系Hep G2和SMMC-7721的迁移和侵袭能力;采用增强型绿色荧光蛋白(EGFP)荧光报告载体实验验证miR-718的靶基因;采用qR T-PCR和蛋白质印迹(Western blot)检测相关基因表达水平的变化。结论:mi R-718在肝癌细胞系HepG 2和SMMC-7721中起到抑癌基因的作用;EGR3在肝癌细胞系Hep G2和SMMC-7721中起到促癌基因的作用;miR-718是通过靶定EGR3 m RNA 3'UTR下调EGR3的表达起到抑癌基因的作用。 相似文献
10.
目的:探讨外源性EGFP基因转染修饰兔骨髓间充质干细胞(m esenchymal stem cells,MSCs)的可行性。方法:用增强型荧光绿色蛋白(enhanced green fluorescent protein,EGFP)作为报告基因,以脂质体法转染骨髓间充质干细胞,观察转染结果、表达情况及对靶细胞活力的影响。结果:经荧光显微镜下观察证实:成功地对骨髓间充质干细胞实现了基因转染。结论:兔骨髓间充质干细胞能够在体外适宜的条件下进行长期培养;骨髓间充质干细胞可直接作为基因靶细胞,建立稳定表达EGFP的骨髓间充质干细胞系具有可行性,为进一步做标记的MSCs细胞的移植研究奠定基础。 相似文献
11.
罗艳萍 《读与写:教育教学刊》2008,5(6)
The communication of people partially is the communication of cultures. Culture has a direct effect on international commercial activities in all aspects. Different conceptions about time, space, equality, law and the like, lead people to deal with things in different ways. So to know cultures of the counterpart is to facil-itate our enterprises so as to have a smooth and successful communication in commercial activity. 相似文献
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Richard Denner 《海外英语》2007,(4):14-15
Rosco and I wait for the fishermen to return.I sit at a wooden bench near the store at Mt.Baker Resort and watch the clouds change shape. Rosco has my belt around his neck and an eight foot tow chain hooked to a tree. Dogs must be on a leash. Ducks and rabbits are loose. 相似文献
13.
Given a graph G,a subgraph C is called a clique of G if C is a complete subgraph of G maximal under inclusion and |C|≥2. A clique-transversal set S of G is a set of vertices of G such that S meets all cliques of G. The clique-transversal number, denoted as TC (G), is the minimum cardinality of a clique-transversal set in G. The clique-graph of G, denoted as K (G), is the graph obtained by taking the cliques of G as vertices, and two vertices are adjacent if and only if the corresponding cliques in G have nonempty intersection. Let F be a class of graphs G such that F={G|K(G) is a tree}. In this paper the graphs in F having independent clique-transversal sets are shown and thus TC (G)/|G|≤1/2 for all G ∈ F. 相似文献
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邱崇丙 《唐山师范学院学报》2014,(1):21-23
汉字的六书,《说文》对“转注”一类,语焉不详。后世学者提及转注,也仅限于许慎所举出的例字。《汉字例说》一文,作者从转注的角度综合考虑,对部分现代常用字作了分析,跟传统的解释有所不同。希望能抛砖引玉,互相切磋,以推进学术研究。 相似文献
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陈美曦 《山东教育学院学报》2008,23(2):103-107
Different nations have different social cultures, and the social culture of every nation is developing with the development of the nation, language is greatly influenced by social culture, which enables language to possess national and historical characteristics. Word is the smallest, independent, meaningful linguistic unit of language. It can well reflect the national and historical characteristics. From synchronic aspect, the social and cultural influence on word meaning is mainly embodied in conceptual and associative meanings of word. The incorrespondence of word meaning is a phenomenon that exists in both conceptual and associative meanings of word among different languages. From diachronic aspect, the development of social culture causes some changes of word meaning to some extent. Therefore, social culture plays a key role in vocabulary learning and mastery of a language. 相似文献
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Rachel Zupek 《海外英语》2007,(11):40-41
我们绝大部分人都同意一件事情——我们喜欢金钱。但是你对金钱的喜欢之情足够到让你创立一份事业么?.财富通常被人忽视,主要在于人们不能真正了 相似文献
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《海外英语》2007,(4):36
There are numbers of crossroads on our long and unpredictable life journey where we totally have no idea about which direction to choose. No matter what our decision is, we should not turn back, but face the music and go ahead instead. I am this kind of girl who always does try without regretting, one example is how I dealt with my love. 相似文献
19.
YANGShiguo 《重庆大学学报(英文版)》2004,3(1):86-88
The problem on the geometrc inequalities involving an n-dimensional simplex and its inscribed simplex is studied. Aninequality is established, which reveals that the difference between the squared circumradius of the n-dimensional simplex andthe squared distance between its circumcenter and barycenter times the squared circumradius of its inscribed simplex is not lessthan the 2(n-1)th power of n times its squared inradius, and is equal to when the simplex is regular and its inscribed siplex is atangent point one. Deduction from this inequality reaches a generalization of n-dimensional Euler inequality indicating that thecircumradius of the simplex is not less than the n-fold inradius. Another inequality is derived to present the relationship betweenthe circumradius of the n-dimensional simplex and the circumradius and inradius of its pedal simplex. 相似文献