基于转录组测序分析甜菜夜蛾卵巢细胞离体培养成系进程

【目的】本研究旨在分析甜菜夜蛾Spodoptera exigua蛹卵巢细胞建立细胞系的整个过程,探究细胞由体内到体外培养过程中其基因表达在转录水平的变化,为昆虫体外培养模型的建立提供理论基础。【方法】利用Illumina Hiseq测序平台对甜菜夜蛾蛹卵巢细胞离体培养过程中各阶段的细胞分别进行转录组测序,对获得注释的差异表达基因及其相关信号通路进行分析;通过荧光定量PCR对部分细胞周期相关基因(cycd和cdk4)、调控基因(cdc20,apc1,skp2和mad1)、增殖相关分子标志物(mcm4和pcna)在甜菜夜蛾卵巢细胞离体培养过程中的转录进行验证。【结果】甜菜夜蛾蛹卵巢细胞离体培养过程包括5个阶段:解剖获得离体的卵巢组织,卵巢组织贴壁培养后游离出原代细胞,细胞转化重新具备增殖能力,成功首次传代,以及能够连续传代15代以上建立细胞系。上述5个阶段的细胞经转录组测序、数据组装后共获得46 796条unigenes序列,组装得到序列长度完整性好;转录本unigenes序列拼接长度分布合理,样本碱基Q30均在94%以上。通过KEGG数据库获得注释的unigenes有1 473条,参与细胞过程紧密相关的20条信号通路,其中有92条unigenes在细胞周期信号通路中获得注释。聚类分析表明,在体内处于快速发育状态的卵巢细胞与同样处于增殖状态的细胞系基因表达模式非常接近。原代细胞由短暂停滞生长至成簇细胞的转化关键期,筛选到差异表达基因619个,cdk4在离体培养期表达量显著降低,cycd在细胞转化关键期之后表达量显著升高,cdc20,apc1,skp2和mad1在卵巢组织和细胞系的表达量显著高于原代细胞、转化关键期细胞和首次传代细胞的。从原代细胞至传代后,cycd的表达显著升高8.7倍,显著高于mcm4和pcna的变化水平。【结论】甜菜夜蛾卵巢细胞离体培养过程中5个阶段的细胞转录组测序获得的序列质量符合数据分析的基本要求。筛选获得了甜菜夜蛾蛹卵巢细胞经离体培养过程中的差异表达基因。原代细胞逆转增殖可能与cdk4,cycd,skp2和mad1等细胞周期调控基因表达有关。另外,cycd可作为原代细胞具备传代能力的标志物。     

敲低去泛素化酶USP13抑制K562细胞的增殖*

目的:研究去泛素化酶USP13对人慢性髓系白血病细胞系K562增殖和凋亡的影响,并进行初步的机制探究。方法:构建pLKO.1-shUSP13-GFP慢病毒干涉载体,慢病毒包装后感染并建立稳定敲低USP13的K562细胞株。免疫印迹检测K562细胞中USP13蛋白的敲低效率。流式细胞术分析敲低USP13对K562细胞增殖和凋亡的影响。免疫共沉淀和蛋白质泛素化实验探究USP13调控K562细胞的分子机制。结果:成功构建pLKO.1-shUSP13-GFP慢病毒干涉载体,同时利用慢病毒体系获得稳定敲低USP13的K562细胞株。流式细胞术结果显示,敲低USP13促进K562细胞凋亡、抑制细胞增殖。分子机制研究发现,敲低USP13通过增强c-Myc泛素化进而导致其蛋白质水平降低。结论:初步揭示了USP13调控K562细胞增殖和凋亡的分子机制,为治疗慢性髓系白血病提供了潜在的靶点。     

Moderate mechanical stimulation rescues degenerative annulus fibrosus by suppressing caveolin-1 mediated pro-inflammatory signaling pathway

Mechanical loading can induce or antagonize the extracellular matrix (ECM) synthesis, proliferation, migration, and inflammatory responses of annulus fibrosus cells (AFCs), depending on the loading mode and level. Caveolin-1 (Cav1), the core protein of caveolae, plays an important role in cellular mechanotransduction and inflammatory responses. In the present study, we presented that AFCs demonstrated different behaviors when subjected to cyclic tensile strain (CTS) for 24 h at a magnitude of 0%, 2%, 5% and 12%, respectively. It was found that 5% CTS had positive effects on cell proliferation, migration and anabolism, while 12% CTS had the opposite effects. Besides, cells exposed to interleukin-1β stimulus exhibited an increase expression in inflammatory genes, and the expression of these genes decreased after exposure to moderate mechanical loading with 5% CTS. In addition, 5% CTS decreased the level of Cav1 and integrin β1 and exhibited anti-inflammatory effects. Moreover, the expression of integrin β1 and p-p65 increased in AFCs transfected with Cav1 plasmids. In vivo results revealed that moderate mechanical stimulation could recover the water content and morphology of the discs. In conclusion, moderate mechanical stimulation restrained Cav1-mediated signaling pathway and exhibited anti-inflammatory effects on AFCs. Together with in vivo results, this study expounds the underlying molecular mechanisms on the effect of moderate mechanical stimulation on intervertebral discs (IVDs) and may provide a new therapeutic strategy for the treatment of IVD degeneration.     

锡林郭勒盟煤电基地开发生态脆弱性评价

内蒙古锡林郭勒盟国家大型煤电基地的建设,对我国华北和华东区域能源安全具有重要的作用。但随着锡盟大型煤电基地的开发,其对锡林郭勒盟这一具有重要生态屏障功能的典型草原区的生态环境造成不利影响。通过对区域生态敏感性、生态弹性及生态压力的系统研究,建立了基于生态敏感性、生态弹性及生态压力的生态脆弱性评价模型,对该区域生态脆弱性进行了评价。结果表明,锡盟地区总体生态环境质量较好,但呈现由东北向西南递减的特征。研究为锡林郭勒盟国家大型煤电基地的开发建设,区域协调发展与生态环境保护提供理论支持。     

Targeted overexpression of inducible 6-phosphofructo-2-kinase in adipose tissue increases fat deposition but protects against diet-induced insulin resistance and inflammatory responses

Increasing evidence demonstrates the dissociation of fat deposition, the inflammatory response, and insulin resistance in the development of obesity-related metabolic diseases. As a regulatory enzyme of glycolysis, inducible 6-phosphofructo-2-kinase (iPFK2, encoded by PFKFB3) protects against diet-induced adipose tissue inflammatory response and systemic insulin resistance independently of adiposity. Using aP2-PFKFB3 transgenic (Tg) mice, we explored the ability of targeted adipocyte PFKFB3/iPFK2 overexpression to modulate diet-induced inflammatory responses and insulin resistance arising from fat deposition in both adipose and liver tissues. Compared with wild-type littermates (controls) on a high fat diet (HFD), Tg mice exhibited increased adiposity, decreased adipose inflammatory response, and improved insulin sensitivity. In a parallel pattern, HFD-fed Tg mice showed increased hepatic steatosis, decreased liver inflammatory response, and improved liver insulin sensitivity compared with controls. In both adipose and liver tissues, increased fat deposition was associated with lipid profile alterations characterized by an increase in palmitoleate. Additionally, plasma lipid profiles also displayed an increase in palmitoleate in HFD-Tg mice compared with controls. In cultured 3T3-L1 adipocytes, overexpression of PFKFB3/iPFK2 recapitulated metabolic and inflammatory changes observed in adipose tissue of Tg mice. Upon treatment with conditioned medium from iPFK2-overexpressing adipocytes, mouse primary hepatocytes displayed metabolic and inflammatory responses that were similar to those observed in livers of Tg mice. Together, these data demonstrate a unique role for PFKFB3/iPFK2 in adipocytes with regard to diet-induced inflammatory responses in both adipose and liver tissues.     

猪链球菌2型与宿主细胞体外相互作用研究进展

猪链球菌2型(Streptococcus suis serotype2,SS2)是一种世界范围内的引起猪链球菌病的重要的人兽共患病原菌,人感染SS2后多呈现败血症型和脑膜炎型。2005年猪链球菌在我国四川省的感染暴发和近年来不断出现的零散病例给我国的公共卫生和食品安全带来了威胁,但迄今为止,对SS2致病机制研究尚不够深入。SS2的致病过程和宿主细胞相互作用密切相关,SS2-体外宿主细胞相互作用模型的建立对于揭示SS2致病分子机理具有重要的意义。目前的研究报道主要是从猪链球菌黏附与侵袭上皮细胞或内皮细胞、猪链球菌抗吞噬细胞的吞噬和猪链球菌激活免疫细胞炎症反应方面研究猪链球菌与宿主细胞的相互作用,但与其他常致病性链球菌如化脓性链球菌和肺炎链球菌相比,有些SS2-体外宿主细胞相互作用模型还缺乏统一的标准,由于感染模型的实验参数不尽相同得到结果也有所不同。本文对近年来猪链球菌2型与宿主细胞体外相互作用及其致病机理研究进展作一综述。     

系统性疾病治疗与口腔病灶感染

口腔病灶被证实与许多系统性疾病的发生及发展有很大的相关关系,全身系统性疾病的治疗又会加重口腔病灶的感染,影响患者的生存质量,干扰系统性疾病的治疗,严重则造成全身感染。可能诱发或加重口腔病灶感染的系统性疾病治疗有:口服抗癫痫药物、免疫抑制剂等药物导致的药物性牙龈炎;恶性肿瘤放化疗并发的口腔黏膜炎;长期应用抑制肿瘤骨转移或预防骨质疏松的双磷酸盐造成的颌骨坏死;异基因造血干细胞移植后的口腔排异反应等。     

理气调补汤对运动性疲劳大鼠血清皮质醇和白细胞介素2影响的研究

目的:通过观察理气调补汤对运动性疲劳大鼠血清皮质醇(C)和白细胞介素2(IL-2)水平的影响,以期揭示中药理气调补汤促进运动性疲劳恢复的作用机理.方法:健康雄性SD大鼠30只,随机分为对照组(Ⅰ组)8只、单纯运动组(Ⅱ组)11只、中药运动组(Ⅲ组)11只.实验时Ⅲ组每天定时定量中药灌胃1ml/100g体重,Ⅰ组、Ⅱ组灌胃相同剂量的生理盐水.实验采用游泳运动训练方式.6周末力竭训练后24h分离血清测定C和IL-2含量.结果:Ⅲ组大鼠游泳至力竭的时间较Ⅱ组显著延长;Ⅱ组和Ⅲ组大鼠血清IL-2浓度均显著低于Ⅰ组,但Ⅲ组大鼠血清IL-2浓度显著高于Ⅱ组;血清C浓度Ⅱ组显著高于Ⅰ组,Ⅲ组较Ⅱ组显著降低.结论:大强度运动训练可能导致HPA轴释放过量皮质醇,从而使免疫功能受到抑制.理气调补汤能够调节疲劳大鼠的HPA轴适应性,降低C的过量释放,维持免疫功能稳定,延缓疲劳的发生.     

油菜花粉总黄酮的微波辅助提取及其抗氧化活性研究

采用微波辅助提取法提取油菜花粉总黄酮,通过单因素和正交试验,得到最佳提取条件：80％的乙醇、1：20的料液比、提取时间140s、微波功率242W、提取3次,总黄酮质量分数为（2．64±0．05）％。同时还研究提取物对羟自由基、DPPH自由基的清除作用,以及对小鼠肝组织脂质过氧化的抑制作用,自由基半清除质量浓度和脂质过氧化丰抑制盾奄浓度分剐为0．115、0．325和0．065mg／mL。     

扩张型心肌病动物模型的研究进展

扩张型心肌病严重危害人类健康,为了广泛深入地研究和治疗DCM,迫切需要该病的动物模型。本文按DCM的病因：家族性/遗传性,病毒性/免疫性,酒精性/中毒性及其他病因,对近年来应用较为成熟的DCM模型作一综述,分析各自的优缺点。