HRB2基因对细胞增值影响的研究

目的:研究HRB2基因对细胞增值的影响.方法:设计4对HRB2 RNA寡核苷酸片段,经退火制备的双链DNA oligo,连接经双酶切的线性化PLKO.1-TRC-SP6载体上,转化后经PCR鉴定,阳性克隆测序,质粒抽提,应用Lipofectamine2000将质粒导入293T细胞,包装成慢病毒,再感染靶细胞293T,用嘌呤霉素筛选15天后,收集细胞抽提RNA,实时定量RT-PCR检测HRB2mRNA水平,评价干扰效果.Western免疫印迹进一步确认有效作用靶点.通过划痕愈合实验和绘制生长曲线研究HRB2基因对细胞增值的影响.结果:载体构建成功.包装成慢病毒感染293T细胞系,发现:2号和4号靶点敲减HRB2基因效率达84%和88%.差别有统计学意义.通过western进一步证实了2号和4号是抑制HRB2的有效作用靶点.划痕愈合实验和绘制生长曲线的结果显示2号和4号靶点细胞株细胞生长受到抑制,并且4号较2号稍明显.结论:成功构建并筛选了携带有人HRB2基因的shRNA慢病毒载体及有效靶点,研究发现HRB2基因对细胞增值产生了影响,这为进一步研究HRB2的生物学功能提供了实验平台.     

FIND:从下一代单端测序数据中快速查找Indel的方法

目的:针对下一代测序数据,尤其是单端测序数据,研究快速、准确查找Indel的方法。方法:先与全基因组参考序列进行快速比对,筛选出包含Indel的序列;再对这些序列进行双向的二次比对,确定Indel长度;最后借助长度信息在锁定范围内查找Indel的确切位置和相关信息。结果:本文成功构建FIND(Fast INDel detection system)系统,用于从单端测序数据中查找Indel信息。以模拟测序数据作为测试数据,在12X测试数据情况下,FIND的灵敏度和特异性分别为87.71%和99.66%,而且该性能还随着测序倍数的增加而提升。结论:充分利用比对过程获取的信息,在确定Indle长度的同时也确定出其大致位置,最终在局部范围内实现对单端测序数据中Indle的快速而准确的查找。     

蛋白S基因突变及多态性的研究进展

蛋白S在生理抗凝血过程中起着重要的作用.血浆蛋白S水平低下是血栓性疾病发生和发展的危险因素之一.蛋白S基因突变及多态性可增加静脉血栓形成的危险性.本文就蛋白S的理化性质、蛋白S基因突变及多态性,以及与静脉血栓的关系作一综述.     

1ω-3不饱和脂肪酸对视网膜新生血管的实验研究

目的:研究ω-3不饱和脂肪酸对鼠视网膜新生血管的作用。方法:32只7日龄Wistar白鼠分为4组。每组各8只,分别与母鼠共同饲养,制备早产儿视网膜病变(ROP)视网膜新生血管动物模型,其中A组为ROP模型对照组,B、C、D组为ω-3PUFAs喂养组,分别按母鼠公斤体重给与ω-3PUFAs2.5mg/kg/d、7.5mg/kg/d及15mg/kg/d喂养。采用免疫组化观察小鼠中突破视网膜内界膜CD31的特异性表达,Western印迹法检测视网膜标本中RGS5的表达。结果:免疫组化观察视网膜CD31的特异性表达,A、B、C、D组突破视网膜内界膜的新生血管内皮细胞核数分别为(17.25±1.04)个、(15.75±1.83)个、(9.75±1.49)个、(8.5±1.69)个;免疫印迹法检测各实验组视网膜RGS5结果:A组与C组、A组与D组比较差异均有显著意义((P<0.001),其中A、B组相比较;C、D组相比较差异无显著意义(P>0.05)。结论:一定剂量的摄入量后对于新生鼠视网膜新生血管病变将起到一定的防治作用。     

renalase在人近曲肾小管上皮细胞系的表达与分泌

目的:探讨renalase在人近曲肾小管上皮细胞系(HK-2)的表达与分泌,为进一步研究细胞水平renalase及其通路建立稳定的实验平台。方法:以HK-2细胞系作为研究材料。①应用Westernblot方法检测renalase蛋白的表达。②用real-timePCR方法检测renalasemRNA表达的变化。③用ELISA方法检测细胞上清液中renalase的浓度。结果:在mRNA水平及蛋白水平均检测到renalase表达。结论:首次在mRNA水平及蛋白水平证实了HK-2细胞能够表达renalase,为进一步研究儿茶酚胺或缺血缺氧刺激下细胞renalase的表达奠定了基础。     

硫酸长春碱聚乳酸缓释纳米粒的制剂学研究和体外释放度考察

目的:制备硫酸长春碱聚乳酸纳米粒(VLB-PLA-NPs)并考察其体外释放度.方法:采用复乳挥发法制备VLB-PLA-NPs,以包封率为主要指标评价指标,选择聚乳酸用量、超声时间、外水相浓度为考察因素,优化制备工艺,考察体外释放度.结果:优化工艺制备得VLB-PLA-NPs平均粒径为65±3.03nm,包封率为(99.68±0.30)%,载药量为3.323±0.01μg/mg,体外释放可持续20天.结论:该制备工艺操作简便,结果稳定,缓释特征明显,应用前景良好.     

氯化钴对小鼠NIH3T3细胞增殖和凋亡的影响

目的:观察氯化钴(Cocl2)对体外培养的小鼠NIH3T3细胞增殖和凋亡的影响.方法:首先利用MTT法和流式细胞术检测不同浓度Cocl2对小鼠NIH3T3细胞增殖及凋亡的影响.然后利用免疫印迹检测HIF-1α和凋亡相关蛋白Bcl-2及Bax的表达.结果:(1)MTT结果显示,低于250μ mol/L的Cocl2作用NIH3T3细胞24 h对细胞的增殖活力影响不大,当浓度增加到500μ mol/L以上时会明显抑制细胞的增殖活力.(2)流式细胞仪检测结果显示低浓度Cocl2(≤ 250μ mol/L)并不会引起NIH3T3细胞明显的凋亡和坏死,增加浓度(≥500μmol/L)会导致细胞早期凋亡和坏死增加.3)不同浓度Cocl2作用NIH3T3细胞24 h均可诱导细胞中HIF-1α表达上调,同时Bax/Bcl-2的比值增高.结论:高浓度Cocl2可以抑制小鼠NIH3T3细胞增殖,促进凋亡发生.     

Genetic and comparative genomics mapping reveals that a powdery mildew resistance gene Ml3D232 originating from wild emmer co-segregates with an NBS-LRR analog in common wheat (Triticum aestivum L.)

Powdery mildew caused by Blumeria graminis f. sp. tritici is one of the most important wheat diseases worldwide and breeding for resistance using diversified disease resistance genes is the most promising approach to prevent outbreaks of powdery mildew. A powdery mildew resistance gene, originating from wild emmer wheat (Triticum turgidum var. dicoccoides) accessions collected from Israel, has been transferred into the hexaploid wheat line 3D232 through crossing and backcrossing. Inoculation results with 21 B. graminis f. sp. tritici races indicated that 3D232 is resistant to all of the powdery mildew isolates tested. Genetic analyses of 3D232 using an F₂ segregating population and F₃ families indicated that a single dominant gene, Ml3D232, confers resistance in the host seedling stage. By applying molecular markers and bulked segregant analysis (BSA), we have identified polymorphic simple sequence repeats (SSR), expressed sequence tags (EST) and derived sequence tagged site (STS) markers to determine that the Ml3D232 is located on chromosome 5BL bin 0.59–0.76. Comparative genetic analyses using mapped EST markers and genome sequences of rice and Brachypodium established co-linearity of the Ml3D232 genomic region with a 1.4 Mb genomic region on Brachypodium distachyon chromosome 4, and a 1.2 Mb contig located on the Oryza sativa chromosome 9. Our comparative approach enabled us to develop new EST–STS markers and to delimit the genomic region carrying Ml3D232 to a 0.8 cM segment that is collinear with a 558 kb region on B. distachyon. Eight EST markers, including an NBS-LRR analog, co-segregated with Ml3D232 to provide a target site for fine genetic mapping, chromosome landing and map-based cloning of the powdery mildew resistance gene. This newly developed common wheat germplasm provides broad-spectrum resistance to powdery mildew and a valuable resource for wheat breeding programs.     

四季竹立竹表型可塑性的林分密度效应

为了给优良的观赏和夏秋笋用竹种四季竹的丰产林分密度建立提供理论依据,开展了四季竹纯林4种林分密度(17500～27500株·hm-2,D1;37500～42500株·hm-2,D2;55000～62500株·hm-2,D3;72500～82500株·hm-2,D4)的分株构件因子调查,并对立竹主要形态特征因子进行了主成分分析。结果表明:随着林分密度的增大,四季竹立竹胸径和相同胸径下的节间长分别呈"∧"和"∨"形变化,极值分别出现在D3和D2密度。相同胸径下的立竹冠幅和枝盘数均呈"∨"形变化,并且最小值都出现在D2密度;立竹枝长和残枝率均呈倒"N"形变化,枝长最大值和残枝率的最小值均出现在D3密度。林分叶面积指数呈"∧"形变化,最大值出现在D3密度;立竹单叶面积与林分密度呈显著正相关。相同胸径下的立竹全高、胸高壁厚、枝下高和枝夹角、单叶质量、比叶面积在不同林分密度间无显著差异。经主成分分析,立竹表型形态与秆形构件关系最为密切,其次为枝条构件,最后是叶片构件。经通径分析,各林分密度立竹表型综合得分大小顺序为:D3D4D2D1。在试验林立竹胸径条件下,D3是四季竹无性系生理整合成本和效益的密度阀值,是分株形态良好建成的适宜林分密度。     

Molecular insights into nitrogenase FeMoco insertion--the role of His 274 and His 451 of MoFe protein alpha subunit

The final step of FeMo cofactor (FeMoco) assembly involves the insertion of FeMoco into its binding site in the molybdenum-iron (MoFe) protein of nitrogenase. Here we examine the role of His alpha274 and His alpha451 of Azotobacter vinelandii MoFe protein in this process. Our results from combined metal, activity, EPR, stability and insertion analyses show that mutations of His alpha274 and/or His alpha451, two of the histidines that belong to a so-called His triad, to small uncharged Ala specifically reduce the accumulation of FeMoco in MoFe protein. This observation indicates that the enrichment of histidines at the His triad is important for FeMoco insertion and that the His triad potentially serves as an intermediate docking point for FeMoco through transitory ligand coordination and/or electrostatic interaction.