FRIL蛋白体外维持脐血造血干/祖细胞的作用及细胞周期调控基因的表达

为探讨新的豆类凝集素(Flt3 receptor-interacting lectin,FRIL)体外维持脐血CD34^ 细胞的作用以及维持过程中细胞周期调控基因HTm4及HTm4S mRNA的表达及意义,我们利用FRIL维持培养脐血CD34^ 细胞,对其增殖曲线、细胞周期及集落形成能力进行常规分析,并用半定量RT—PCR法分别测定FRIL体外维持不同时间后脐血CD34^ 细胞中周期调控基因HTm4及HTm4S mRNA的表达变化。结果显示,FRIL培养的CD34^ 造血干／祖细胞的增殖趋势平缓,整个培养期间细胞增殖倍数不超过起始的3倍：14d之前,FRIL培养细胞的高增殖潜能集落形成细胞(HPP—CFC)形成集落数与FL组无差别,其后则维持高于FL的情况。细胞周期分析则显示,在28d的培养过程内,利用FRIL培养的细胞始终有80％以上维持在G0期;而周期调控基因HTm4及HTm4S在刚分离的脐血CD34^ 细胞中的表达水平较高;但培养1d后,几乎检测不到HTm4基因的表达;培养3～14d,该基因的表达回升并持续维持在高水平。而HTm4S基因的表达在第7d达最高水平,其余时间基本呈稳定表达。转染HTm4和HTm4S,亚细胞定位结果显示HTm4主要定位于核周围,而HTm4S则定位于整个胞浆,由此可能导致它们功能的区别。以上结果提示,长期培养体现出FRIL在维持造血干／祖细胞多能性上的优势;细胞周期调控基因HTm4及其新剪接子参与了FRIL体外长期维持脐血造血干／祖细胞处于静息状态的过程。     

桂林小花苣苔离体快速繁殖技术

对抗结核植物桂林小花苣苔(Chiritopsis repanda var. guilinensis)进行离体培养与快速繁殖技术研究。结果表明: 桂林小花苣苔叶片外植体的最适初代诱导培养基为MS+0.5 mg·L^–16-BA+0.05 mg·L^–1IBA, pH8.0; 最适继代增殖培养基为 MS+0.1 mg·L^–16-BA+0.05 mg·L^–1IBA, pH6.0, 繁殖系数7.0/35天; 最适生根培养基为1/2MS+0.2 mg·L^–1NAA, pH6.0, 生根率为93.6%。模拟桂林小花苣苔自然生境, 在春季对生根试管苗进行大棚移栽, 成活率达90%。根据上述快繁技术, 理论上每株试管苗每年可繁殖桂林小花苣苔种苗46万株。     

Meningitic Escherichia coli K1 penetration and neutrophil transmigration across the blood-brain barrier are modulated by alpha7 nicotinic receptor

Alpha7 nicotinic acetylcholine receptor (nAChR), an essential regulator of inflammation, is abundantly expressed in hippocampal neurons, which are vulnerable to bacterial meningitis. However, it is unknown whether α7 nAChR contributes to the regulation of these events. In this report, an aggravating role of α7 nAChR in host defense against meningitic E. coli infection was demonstrated by using α7-deficient (α7(-/-)) mouse brain microvascular endothelial cells (BMEC) and animal model systems. As shown in our in vitro and in vivo studies, E. coli K1 invasion and polymorphonuclear neutrophil (PMN) transmigration across the blood-brain barrier (BBB) were significantly reduced in α7(-/-) BMEC and α7(-/-) mice. Stimulation by nicotine was abolished in the α7(-/-) cells and animals. The same blocking effect was achieved by methyllycaconitine (α7 antagonist). The tight junction molecules occludin and ZO-1 were significantly reduced in the brain cortex of wildtype mice infected with E. coli and treated with nicotine, compared to α7(-/-) cells and animals. Decreased neuronal injury in the hippocampal dentate gyrus was observed in α7(-/-) mice with meningitis. Proinflammatory cytokines (IL-1β, IL-6, TNFα, MCP-1, MIP-1alpha, and RANTES) and adhesion molecules (CD44 and ICAM-1) were significantly reduced in the cerebrospinal fluids of the α7(-/-) mice with E. coli meningitis. Furthermore, α7 nAChR is the major calcium channel for nicotine- and E. coli K1-increased intracellular calcium concentrations of mouse BMEC. Taken together, our data suggest that α7 nAChR plays a detrimental role in the host defense against meningitic infection by modulation of pathogen invasion, PMN recruitment, calcium signaling and neuronal inflammation.     

从野皂荚提胶后的副产物中制备分离蛋白

采用碱提酸沉法从野皂荚提胶后的副产物中制备分离蛋白,研究了制备工艺条件对分离蛋白得率的影响。结果表明:当料液比1∶10~1∶12(m/V)、提取液pH8.0~9.0、浸提温度45~50℃、浸提两次、每次浸提45 m in时,分离蛋白得率可达78.45%。研究结果可为野皂荚提胶副产物中蛋白的综合利用奠定了基础。     

泛素样蛋白ISG15抗病毒机制研究进展

ISG15(Interferon stimulated gene 15,ISG15)蛋白是由干扰素诱导产生的一种泛素样蛋白分子,分子量大小约为15kD。ISG15同泛素分子相类似可以被共价结合于其他蛋白分子上,这种现象称为ISG化(ISGylation)现象。ISG化系统包括ISG15、UBE1L、UBCH8和HERC5四类蛋白分子,协同完成ISG化过程。ISG15及ISG化系统在抗病毒反应中具有重要作用。近几年对于ISG15的抗病毒作用和机制的研究已经有了很大的突破,ISG15的抗病毒作用也越来越受到人们重视,了解清楚ISG15抗病毒机制对于研制新的抗病毒药物及提出新的抗病毒策略具有重要意义。本文对ISG15在不同种病毒中的抗病毒机制研究进展进行了简要综述。     

丝状真菌瑞氏木霉外源基因表达系统的构建

采用PCR技术体外扩增获得了瑞氏木霉外切葡聚糖纤维二糖水解酶Ⅰ (CBHⅠ )启动子和终止子序列 .并以大肠杆菌质粒pUC1 9为骨架 ,在该启动子和终止子序列间加入多克隆位点 ,构建了瑞氏木霉强表达整合型载体pTRIL .以质粒pAN7 1为模板 ,体外扩增了带有潮霉素磷酸转移酶(hph)基因的DNA片段 ,将hph插入pTRIL的cbh1启动子和终止子序列之间 ,构建了Pcbh1 hph Tcbh1表达盒 .用此表达盒转化瑞氏木霉C30原生质体 ,在潮霉素平板上得到 1 5株抗性转化子 .对其中的H1转化子进行了PCR和Southern印迹分析 ,证实hph基因确实整合到转化子染色体DNA上 ,并在Pcbh1 启动子控制下进行高效表达 .转化子H1对潮霉素抗性达 1 5 0mg L ,比出发菌株提高 2倍 .瑞氏木霉强表达整合型载体pTRIL的构建成功为开展瑞氏木霉分子生物学研究以及进一步的工程菌株构建工作奠定了基础     

Red重组系统及在微生物基因敲除中的应用

在完成了对各种微生物基因组的测序以后,功能基因学的研究变得尤为重要。研究基因功能最直接的方法便是将待研究的基因失活。最初构建基因突变体是采用大肠杆菌的RecA系统,但是RecA重组系统操作复杂,重组效率低。最近建立了Red重组系统,该系统由3个蛋白组成：α蛋白(即λ核酸外切酶),β蛋白,Gam蛋白。应用Red系统进行基因敲除,可以直接利用线性打靶DNA,两侧同源臂长度在35~60 bp即可发生同源重组,且重组效率高。 Abstract:Since many DNA-sequencing projects of varied microorganisms have been completed,studies on their functional genomics become more important.Inactivation of an interesting gene is a direct method to characterize its function.Though the Esherichia coli RecA recombination system can be used to produce gene mutants,it needs a complex manipulation process.Furthermore,its efficiency is very low.Recently a Red recombination system was developed.This recombination system consists of three proteins:α protein（λ exonuclease）,β protein and Gam protein.In this system,the linear targeting DNA which contains a selectable marker flanked with a homologous region as short as only 35~60 bp can be directly targeted for gene knock-out with a higher efficiency.     

Conditioned medium from hypoxia-treated adipocytes renders muscle cells insulin resistant

Adipose tissue hypoxia is an early phenotype in obesity, associated with macrophage infiltration and local inflammation. Here we test the hypothesis that adipocytes in culture respond to a hypoxic environment with the release of pro-inflammatory factors that stimulate macrophage migration and cause muscle insulin resistance. 3T3-L1 adipocytes cultured in a 1% O2 atmosphere responded with a classic hypoxia response by elevating protein expression of HIF-1α. This was associated with elevated mRNA expression and peptide release of cytokines TNFα, IL-6 and the chemokine monocyte chemoattractant protein-1 (MCP-1). The mRNA and protein expression of the anti-inflammatory adipokine adiponectin was reduced. Conditioned medium from hypoxia-treated adipocytes (CM-H), inhibited insulin-stimulated and raised basal cell surface levels of GLUT4myc stably expressed in C2C12 myotubes. Insulin stimulation of Akt and AS160 phosphorylation, key regulators of GLUT4myc exocytosis, was markedly impaired. CM-H also caused activation of JNK and S6K, and elevated serine phosphorylation of IRS1 in the C2C12 myotubes. These effects were implicated in reducing propagation of insulin signaling to Akt and AS160. Heat inactivation of CM-H reversed its dual effects on GLUT4myc traffic in muscle cells. Interestingly, antibody-mediated neutralization of IL-6 in CM-H lowered its effect on both the basal and insulin-stimulated cell surface GLUT4myc compared to unmodified CM-H. IL-6 may have regulated GLUT4myc traffic through its action on AMPK. Additionally, antibody-mediated neutralization of MCP-1 partly reversed the inhibition of insulin-stimulated GLUT4myc exocytosis caused by unmodified CM-H. In Transwell co-culture, hypoxia-challenged adipocytes attracted RAW 264.7 macrophages, consistent with elevated release of MCP-1 from adipocytes during hypoxia. Neutralization of MCP-1 in adipocyte CM-H prevented macrophage migration towards it and partly reversed the effect of CM-H on insulin response in muscle cells. We conclude that adipose tissue hypoxia may be an important trigger of its inflammatory response observed in obesity, and the elevated chemokine MCP-1 may contribute to increased macrophage migration towards adipose tissue and subsequent decreased insulin responsiveness of glucose uptake in muscle.