Identification of Tuberculosis Susceptibility Genes with Human Macrophage Gene Expression Profiles

Although host genetics influences susceptibility to tuberculosis (TB), few genes determining disease outcome have been identified. We hypothesized that macrophages from individuals with different clinical manifestations of Mycobacterium tuberculosis (Mtb) infection would have distinct gene expression profiles and that polymorphisms in these genes may also be associated with susceptibility to TB. We measured gene expression levels of >38,500 genes from ex vivo Mtb-stimulated macrophages in 12 subjects with 3 clinical phenotypes: latent, pulmonary, and meningeal TB (n = 4 per group). After identifying differentially expressed genes, we confirmed these results in 34 additional subjects by real-time PCR. We also used a case-control study design to examine whether polymorphisms in differentially regulated genes were associated with susceptibility to these different clinical forms of TB. We compared gene expression profiles in Mtb-stimulated and unstimulated macrophages and identified 1,608 and 199 genes that were differentially expressed by >2- and >5-fold, respectively. In an independent sample set of 34 individuals and a subset of highly regulated genes, 90% of the microarray results were confirmed by RT-PCR, including expression levels of CCL1, which distinguished the 3 clinical groups. Furthermore, 6 single nucleotide polymorphisms (SNPs) in CCL1 were found to be associated with TB in a case-control genetic association study with 273 TB cases and 188 controls. To our knowledge, this is the first identification of CCL1 as a gene involved in host susceptibility to TB and the first study to combine microarray and DNA polymorphism studies to identify genes associated with TB susceptibility. These results suggest that genome-wide studies can provide an unbiased method to identify critical macrophage response genes that are associated with different clinical outcomes and that variation in innate immune response genes regulate susceptibility to TB.     

人LRG1基因的原核表达及生物信息学分析

为了探讨LRG1基因结构与功能,对该基因进行克隆并构建到原核表达载体上,并对其进行表达及生物信息学分析。用Trizol法提取人肝癌HepG2细胞总RNA后,PCR扩增得到LRG1片段,经鉴定后将目的基因与原核表达载体pET28a连接,经诱导表达获得His-LRG1蛋白。LRG1基因cDNA片段大小为1 044 bp,编码347个氨基酸;成功构建pET28a原核表达载体,经多次不同条件诱导后,得到大小约40 kD目的蛋白;利用软件对LRG1蛋白的一级、二级结构进行了预测,分析总结得LRG1基因编码的蛋白是一个不稳定且具有亲水性的蛋白,可与多种信号开关相互作用。LRG1属于高度保守的富亮氨酸重复家族成员,其原核表达载体不易诱导产生大量目的蛋白,克隆表达该基因有利于验证其结构与功能关系。     

番茄 SlDP1 基因的克隆、生物信息学及表达特性分析简

DP基因属于DP/E2F转录因子家族,其编码的蛋白是E2F蛋白的二聚化分子伴侣,可能参与调控细胞周期、DNA复制、生长、分化、凋亡等多种细胞进程。根据SGN数据库登录的番茄序列,通过RT-PCR方法从野生型番茄中克隆了一个DP基因,命名为SlDP1。生物信息学预测,SlDP1蛋白定位于细胞核,具有保守的DNA结合域、二聚化区域和C-末端及多个磷酸化位点。蛋白质二级结构预测SlDP1蛋白含51.50%的环状结构,34.55%的α螺旋和2.66%的β折叠。荧光定量PCR分析外源激素对野生型番茄SlDP1基因表达量的影响,发现该基因受外源性乙烯前体ACC的诱导。对环境因子应答的RT-PCR结果表明,在伤害和盐处理的叶中该基因表达不受诱导,而盐处理的根中该基因表达量提高。SlDP1基因表达模式分析表明,该基因在根、花、萼片及成熟时期果实中表达量较高。这些结果为进一步研究SlDP1基因在番茄生长发育过程的功能奠定了基础。     

慢性酒精中毒大鼠在学习记忆方面差异基因的生物信息学分析

借助基因芯片获取慢性酒精中毒大鼠海马相关基因的表达数据集,通过生物信息学的分析方法对差异表达基因进行筛选与分析。从分子水平揭示慢性酒精中毒对大鼠大脑海马体的影响,为慢性酒精中毒的损伤机制以及相关疾病发病机制的基础研究与临床治疗提供新的方向。同时,还通过Y迷宫实验对实验大鼠的学习记忆功能进行了检测,借助电镜拍摄其线粒体。结果显示,我们一共筛选出208个差异表达基因,其中51个表达上调,157个表达下调。其中涉及的主要信号通路有氧化磷酸化通路、D-谷氨酰胺和谷氨酸代谢通路、阿尔茨海默病信号通路、帕金森病信号通路、膀胱癌信号通路、B细胞受体信号通路和亨廷顿病信号通路等。由此我们得出结论,慢性酒精中毒可能影响了海马多个基因的表达,其中包括Rpsa、Wdr31、Rps11、Rps9、Ndufa2、Mrto4、Rpl6、Dap3、Ndufb8、Ndufb6、Ephb2、Cox6c、Prkcd、Rela、Raf1、Ubd、Mrps28、Mrpl35等关键基因,进而损伤了电子传递链复合体Ⅰ,最终损伤线粒体,导致大鼠学习记忆能力的损伤。     

牦牛MEF2C基因克隆、生物信息学及表达谱分析

本试验旨在分析牦牛(Bos grunniens)肌细胞增强因子2C(myocyte enhancer factor 2C,MEF2C)基因的分子特征和表达规律,探索其影响牦牛肌肉发育的作用机制.试验以大通牦牛肌肉组织cDNA为模板,采用PCR扩增技术扩增牦牛MEF2C基因,用DNAStar,ExPASy,ABCpred等生物信息学软件分析MEF2C基因序列和其编码的蛋白质结构,利用实时荧光定量PCR技术(RT-qPCR)检测了 MEF2C基因的表达情况.试验克隆获得的牦牛MEF2C基因编码区全长1 302 bp,编码433个氨基酸;蛋白质结构预测结果显示,MEF2C蛋白具有30h的半衰期,为亲水性碱性蛋白,没有信号肽但拥有跨膜结构.系统关系中牦牛与普通牛的亲缘关系最为接近,与小鼠亲缘关系较远.组织表达谱结果显示,MEF2C基因在牦牛7个组织中都有表达且在臀二头肌组织中表达量最高;不同时期MEF2C基因表达情况为胎牛＞成年牛＞6月龄牛.研究结果将为进一步探讨MEF2C基因在牦牛肌肉发育中的作用提供科学依据,同时也为解析牦牛肌肉发育的分子机制提供数据支撑.     

FASN基因的原核表达及生物信息学分析

脂肪酸合成酶(FASN)在生物体内起着重要的作用,主要参与恶性肿瘤的数量调控。本研究旨在构建pET28a-FASN原核表达载体,并表达重组His-FASN蛋白,对该基因进行结构与功能的生物信息学分析。设计FASN基因特异性引物,通过PCR扩增获得的目的基因与原核表达载体pET28a连接,经IPTG诱导表达His-FASN蛋白。获得基因片段大小为1 320 bp,编码440个氨基酸;成功构建至pET28a原核表达载体,通过优化表达,确定在温度为35℃、IPTG浓度0.5 mmol/L、诱导时间为6 h的条件下融合蛋白表达量较高,获得蛋白大小约为53 kD;生物信息学分析结果表明FASN基因编码的蛋白是一个不稳定且具有亲水性的蛋白,不存在信号肽及跨膜区,可成为蛋白激酶磷酸化位点有12个Ser、5个Thr、3个Tyr。此外,从蛋白相互作用网络中发现,相互作用的蛋白包括主要酰基辅酶A合成酶长链家族成员及乙酰辅酶A羧化酶家族成员,为开发抑制剂药物提供了理论依据。     

应用大规模测序技术和生物信息学研究造血干/祖细胞的基因表达及新基因的识别和克隆

从新生儿脐血和成人骨髓中分选出造血干／祖细胞（ＨＳＣ／ＨＰＣ）,构建成ｃＤＮＡ文库,对其进行大规模表达序列标签（ＥＳＴ）测序,通过生物信息学等手段分析基因表达谱,并进行新基因的全长ｃＤＮＡ克隆。在所测的１０５１２条可分析ＥＳＴ序列中,有９８６６条来自脐血ＣＤ３４＋细胞,其中４６９７条（４７６％）为已知基因,２６０３条（２６４％）为已知ＥＳＴ,１４１５条（１４３％）代表未知ＥＳＴ。在已知基因中,８２％基因与造血相关,２２７％涉及细胞代谢、结构和迁移,１３０％与细胞分裂和防御相关,２６２％与ＲＮＡ、蛋白质的合成相关,１０６％和细胞信号传递有关。对一些已知和未知的ＥＳＴ,综合测序、生物信息学等方法,进行全长克隆,已获得２３个新基因的全长ｃＤＮＡ。     

Modulation of Osteoclastogenesis with Macrophage M1- and M2-Inducing Stimuli

Macrophages are generated through the differentiation of monocytes in tissues and they have important functions in innate and adaptive immunity. In addition to their roles as phagocytes, macrophages can be further differentiated, in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), into osteoclasts (multinucleated giant cells that are responsible for bone resorption). In this work, we set out to characterize whether various inflammatory stimuli, known to induce macrophage polarization, can alter the type of multinucleated giant cell obtained from RANKL differentiation. Following a four-day differentiation protocol, along with lipopolysaccharide (LPS)/interferon gamma (IFNγ) as one stimulus, and interleukin-4 (IL-4) as the other, three types of multinucleated cells were generated. Using various microscopy techniques (bright field, epifluorescence and scanning electron), functional assays, and western blotting for osteoclast markers, we found that, as expected, RANKL treatment alone resulted in osteoclasts, whereas the addition of LPS/IFNγ to RANKL pre-treated macrophages generated Langhans-type giant cells, while IL-4 led to giant cells resembling foreign body giant cells with osteoclast-like characteristics. Finally, to gain insight into the modulation of osteoclastogenesis, we characterized the formation and morphology of RANKL and LPS/IFNγ-induced multinucleated giant cells.     

PI3K/Akt Signaling Pathway Modulates Influenza Virus Induced Mouse Alveolar Macrophage Polarization to M1/M2b

Macrophages polarized to M1 (pro-inflammation) or M2 (anti-inflammation) phenotypes in response to environmental signals. In this study, we examined the polarization of alveolar macrophage (AM), following induction by different influenza virus strains (ST169 (H1N1), ST602 (H3N2) and HKG9 (H9N2)). Macrophages from other tissues or cell line exert alternative responding pattern, and AM is necessary for investigating the respiratory system. AM polarized toward the M1 phenotype after 4 hours of infection by all three virus strains, and AM to presented M2b phenotype after 8 hours induction, and immunosuppressive phenotype after 24 hours of induction. Protein expression assay showed similar results as the gene expression analysis for phenotype verification. The ELISA assay showed that TNF-α secretion was up-regulated after 4 and 8 hours of infection by influenza viruses, and it returned to basal levels after 24 hours of infection. IL-10 expression was elevated after 8 and 24 hours of infection. Immunofluorescence showed that iNOS expression was up-regulated but not Arg1 expression. Influenza virus notably increased phospho-Akt but not phospho-Erk1/2 or phospho-p38, and the AM polarization pattern have been changed by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (PI3K inhibitor). In conclusion, our results demonstrate the dynamic polarization of AM induced by influenza viruses, and suggested that PI3K/Akt signaling pathway modulates AM polarization to M1/M2b.     

HDL Induces the Expression of the M2 Macrophage Markers Arginase 1 and Fizz-1 in a STAT6-Dependent Process

Our lab has previously shown in a mouse model that normalization of a low HDL level achieves atherosclerotic plaque regression. This included the shift from a pro (“M1”) to an anti-inflammatory (“M2”) phenotypic state of plaque macrophages. Whether HDL can directly cause this phenotypic change and, if so, what the signaling mechanism is, were explored in the present studies. Murine primary macrophages treated with HDL showed increased gene expression for the M2 markers Arginase-1 (Arg-1) and Fizz-1, which are classically induced by IL-4. HDL was able to potentiate the IL-4-induced changes in Arg-1, and tended to do the same for Fizz-1, while suppressing the expression of inflammatory genes in response to IFNγ. The effects of either IL-4 or HDL were suppressed when macrophages were from STAT6^-/- mice, but inhibitor studies suggested differential utilization of JAK isoforms by IL-4 and HDL to activate STAT6 by phosphorylation. Overall, our results describe a new function of HDL, namely its ability to directly enrich macrophages in markers of the M2, anti-inflammatory, state in a process requiring STAT6.     

Acidiphilium cryptum DX1-1 CO2固定相关基因的克隆及在不同营养方式下的差异表达研究

目的：研究Acidiphilium cryptum DX1-1 CO2固定相关基因的克隆及在不同营养方式下的差异表达。方法：以Acidiphilium cryptum DX1-1（CCTCCM208056）的DNA为模板,基于A．cryptum JF-5同源功能基因序列（JGI,http：／／genome．oml．gov／cgi-bin／JGI_microbial／keggcategories．cgi）设计引物,对菌株DX1-1中的CO2固定相关基因Acry_0824,Acry_082,Acry_1067,Acry_1272,Acry_0022和Acry_0827进行了克隆和序列比对分析;并对它们在不同营养条件下的基因差异表达进行了分析。结果：从菌株DX1—1成功克隆了所选择的CO2固定相关基因,其序列与菌株JF的同源功能基因序列一致性分别达到了99．8％,99．6％,99．6％,99．5％,99．3％和99．8％;Acry_0824,Acry_1272和Acry_0827三个基因在各种混合养条件下表达均上调,说明它们在DX1—1 CO2固定中起较关键的作用。在加入0．1％的葡萄糖混合养条件下,DX1—1细胞明显利用空气中的CO2来生长和累积PHB。结论：限制性葡萄糖可以促进细胞自养生长和累积PHB。