HIF-1α/MMP9信号通路介导低氧诱导的人视网膜母细胞瘤HXO-RB44细胞侵袭

目的:探究低氧条件下人视网膜母细胞瘤细胞侵袭能力的变化及其调控机制。方法:将人视网膜母细胞瘤HXO-RB44细胞株于常氧(21%O_2)和低氧(1%O_2)条件下培养,采用Transwell侵袭实验检测细胞侵袭能力,real time-PCR和Western blot法检测细胞中HIF-1α及MMP9 m RNA和蛋白的表达水平,荧光素酶报告基因分别检测低氧对HIF-1α、HIF-1α对MMP9启动子区域的调控作用。并进一步使用HIF-1α及MMP9特异性si RNA阻断其表达,并检测低氧对细胞侵袭能力的影响。结果:低氧组Transwell下室HXO-RB44每高倍视野细胞数目、HXO-RB44HIF-1α和MMP9 m RNA和蛋白表达均较常氧组显著上调(P0.05)。转染HIF-1α过表达质粒组的MMP9活性较空载组相比明显增加(P0.05),低氧处理24小时的HXO-RB44细胞的HIF-1α启动子活性较常氧组明显增强(P0.05)。HIF-1αsi RNA组的HIF-1α及MMP9的m RNA和蛋白表达水平较NC组明显降低(P0.05),MMP9 si RNA组的HIF-1αm RNA和蛋白无明显改变,分别转染HIF-1αsi RNA组MMP9 si RNA组下室HXO-RB44每高倍视野细胞数目显著减少(P0.05)。结论:低氧能够促进HXO-RB44细胞株的侵袭转移,而可HIF-1α/MMP9轴在这一过程起关键作用。     

HIF-1α/VEGF在稽留流产患者绒毛组织中的表达及其与微血管密度的关系

目的:探讨缺氧诱导因子1α(HIF-1α)/血管内皮生长因子(VEGF)在稽留流产患者绒毛组织中的表达及其与微血管密度的关系。方法:采用免疫组织化学方法分别检测了30例人工流产和30例稽留流产患者绒毛组织的微血管密度(MVD)、HIF-1α和VEGF的表达。分别在缺氧(1%O_2、5%CO_2和94%N_2)和常氧(20%O_2、5%CO_2和75%N_2)条件下培养HTR8/SVneo细胞,并通过转染HIF-1αsi RNA来敲低HIF-1α。通过q RT-PCR和Western blot分析HTR8/SVneo细胞中HIF-1α和VEGF的m RNA和蛋白表达。此外,通过小管形成实验评价缺氧及转染HIF-1αsi RNA对HTR8/SVneo细胞小管形成的影响。结果:稽留流产组织样本中的MVD显著低于人工流产(7.22±0.55 vs 14.65±1.12,P0.05)。HIF-1α和VEGF在稽留流产组织中的表达显著低于人工流产组织(P0.05)。HIF-1α和VEGF的表达均与MVD显著正相关。与常氧相比,缺氧可显著上调HIF-1α和VEGF的m RNA和蛋白水平(P0.05)。转染HIF-1αsi RNA显著下调HIF-1α和VEGF的m RNA和蛋白水平(P0.05)。与常氧相比,缺氧可显著促进HTR8/SVneo细胞的小管形成(P0.05),而转染HIF-1αsi RNA则可显著抑制显HTR8/SVneo细胞的小管形成(P0.05)。结论:胎盘发育过程中的缺氧环境丢失及HIF-1α/VEGF的抑制可能是稽留流产发病的一项机制。     

白藜芦醇通过调控HIF-1α/NOX4/ROS通路抑制低氧诱导的大鼠肺动脉平滑肌细胞氧化应激与增殖

本文旨在明确白藜芦醇对低氧诱导的肺动脉平滑肌细胞(pulmonary artery smooth muscle cells, PASMCs)氧化应激与增殖的作用及分子机制。体外分离培养原代大鼠PASMCs,采用不同浓度的白藜芦醇(10、20和40μmol/L)或NADPH氧化酶(NADPH oxidases, NOXs)抑制剂VAS2870 (10μmol/L)预处理0.5 h,然后将细胞置于常氧(21%O_2, 5%CO_2)或低氧(2%O_2, 5%CO_2)中培养24h。采用CCK-8法和增殖细胞核抗原(proliferatingcellnuclearantigen,PCNA)的表达水平检测细胞增殖,用DCFH-DA测定细胞内活性氧(reactive oxygen species, ROS)的生成,用real-time RT-PCR和Western blot检测NOX1、NOX4和低氧诱导因子-1α(hypoxia inducible factor 1α, HIF-1α)的表达水平,通过小干扰RNAs (small interference RNAs, siRNAs)特异性沉默Hif-1α和Nox4后确定相关信号通路。结果显示,白藜芦醇和VAS2870均能显著抑制低氧诱导的大鼠PASMCs细胞增殖和ROS生成,同时白藜芦醇还能有效阻止低氧诱导的HIF-1α蛋白的聚集和NOX4的表达上调,而对NOX1没有明显的影响。沉默Hif-1α或Nox4后,低氧诱导的大鼠PASMCs细胞增殖和ROS累积均显著降低,且能被白藜芦醇进一步抑制。上述结果提示,白藜芦醇可能通过阻断HIF-1α/NOX4/ROS信号通路抑制低氧诱导的大鼠PASMCs氧化应激和增殖。     

低氧条件下沉默HIF-1α与TNF-α、IL-6的相关性研究及人真皮微血管内皮细胞生物学行为的变化

该文探讨了低氧条件下沉默缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)与肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白介素-6(interleukin-6,IL-6)的相关性及人真皮微血管内皮细胞生物学行为的变化。采用三气培养箱培养人真皮微血管内皮细胞,根据时间和氧浓度分组,确定最适培养时间及缺氧浓度。同时,构建靶向HIF-1α的腺病毒并感染人真皮微血管内皮细胞,分为对照组、空载组和沉默组。应用RT-PCR和Western blot检测HIF-1α、TNF-α和IL-6的mRNA和蛋白质水平。Transwell检测细胞迁移能力,流式细胞技术观察细胞凋亡及细胞周期变化。结果显示,缺氧条件下,HIF-1α、TNF-α和IL-6的表达随缺氧时间延长而增加,随氧浓度降低而增加。与对照组相比,沉默组的细胞迁移能力(P0.01)及周期(P0.01)均明显抑制,细胞凋亡明显增加(P0.01)。与对照组相比,沉默组TNF-α和IL-6的mRNA和蛋白质水平均减少(P0.01)。由此可见,缺氧条件下,HIF-1α、TNF-α和IL-6的表达呈时间依赖性和氧浓度递减依赖性。人真皮微血管内皮细胞HIF-1α的表达与炎性因子TNF-α、IL-6具有明显的正相关。沉默HIF-1α后,能明显抑制细胞增殖,抑制细胞迁移,促进细胞凋亡。     

MicroRNA-18a通过HIF-1α调控缺氧引起的人肺动脉平滑肌细胞增殖

目的:研究microRNA-18a(miR-18a)对缺氧引起的人肺动脉平滑肌细胞(human pulmonary artery smooth muscle cells,hPASMCs)增殖的调控作用及其可能机制。方法:体外培养hPASMCs,分为未转染组、miR-18a模拟物对照组、miR-18a模拟物组、miR-18a抑制剂对照组、miR-18a抑制剂组、si RNA control组、si HIF-1α组、miR-18a抑制剂和si HIF-1α共转染组。分别于常氧(21%O2)和低氧(3%O2)作用24小时。采用CCK-8法检测细胞的增殖情况,萤光素酶报告基因系统验证缺氧诱导因子-1α(HIF-1α)是否为miR-18a的靶基因,并通过western-blot以及实时荧光定量PCR技术检测相关蛋白和基因的表达。结果:缺氧可促进hPASMCs增殖,使miR-18a表达减少;miR-18a模拟物可抑制hPASMCs增殖,而miR-18a抑制剂可促进hPASMCs增殖;抑制miR-18a可使HIF-1α的表达上调。同时抑制miR-18a和HIF-1α,可使miR-18a对hPASMCs增殖调控的能力消失。结论:缺氧通过抑制miR-18a,上调HIF-1α的表达,促进h PASMCs增殖。     

RNA干扰沉默低氧诱导因子-1α对低氧下大鼠肺动脉平滑肌细胞增殖的影响

本研究应用基因克隆技术,将合成的发卡样特异性低氧诱导因子-1α（hypoxia inducible factor-1alpha,HIF-1α）干扰寡核苷酸（siRNA）序列插入真核表达载体中,构建出特异性HIF-1α基因RNA干扰（RNAi）真核表达载体。采用组织块种植法,原代培养大鼠肺动脉平滑肌细胞（pulmonary artery smooth muscle cells,PASMCs）,将构建出的特异性HIF-1αRNAi真核表达载体转染到PASMCs;分别在常氧和低氧下进行细胞培养,采用RT-PCR检测PASMCsHIF-1αmRNA表达水平,用MTT和流式细胞仪检测细胞增殖水平,探讨低氧条件下HIF-1αRNAi真核表达载体对PASMCs增殖的影响。结果表明,低氧培养48h后,正常PASMCs和转染了HIF-1αsiRNA阴性表达载体的细胞增殖显著,HIF-1αmRNA表达水平也显著升高;而转染了HIF-1αsiRNA阳性表达质粒的细胞增殖不显著,HIF-1αmRNA表达水平较低。结果提示：HIF-1αRNAi真核表达载体能显著干扰培养的PASMCsHIF-1αmRNA表达,同时抑制低氧环境下PASMCs的增殖。     

HIF-1α修饰的骨髓间充质干细胞增强其抗PC12细胞缺氧损伤作用

该文探讨了HIF-1α修饰的大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)对缺氧损伤所致神经元样PC12细胞凋亡的作用及其可能机制。PC12细胞分别与MSCs、HIF-1α-MSCs细胞在缺氧环境下体外共培养,分为正常组、缺氧组、MSCs组及HIF-1α-MSCs组。MTT检测不同缺氧时间PC12细胞活性。HE染色观察PC12细胞形态。Hoechst 33258染色与Annexin V-FITC双染色法检测PC12细胞凋亡。RT-PCR及细胞免疫荧光检测caspase-3的m RNA及蛋白质水平。MTT结果显示,PC12细胞活性随着缺氧时间而下降,缺氧12 h细胞活性下降至45.1%。与正常组相比,缺氧组PC12细胞凋亡率明显增高,caspase-3的m RNA和蛋白质水平明显上调,两者有显著差异(P0.05)。与缺氧组相比,MSCs组及HIF-1α-MSCs组的凋亡率降低(P0.05),caspase-3的m RNA和蛋白质水平下调(P0.05),且HIF-1α-MSCs组更为明显(P0.05)。结果证明,HIF-1α修饰的MSCs能增强MSCs的抗PC12缺氧损伤作用,其机制可能与HIF-1α-MSCs降低PC12细胞caspase-3基因表达有关。     

低氧处理不同时间对人肺腺癌A549细胞增殖的影响 及相应缺氧模型探讨*

目的:观察低氧处理不同时间对人肺腺癌A549细胞增殖的影响,探讨合理的人肺腺癌细胞株A549体外模拟缺氧时间。方法:将人肺腺癌细胞A549细胞株在低氧环境下分别培养12 h、24 h、48 h、72 h,设置常氧对照组,通过CCK8法测定A549细胞存活率,RT-PCR和免疫印迹分别检测细胞缺氧诱导因子-1α(hypoxia-inducible factor-1α, HIF-1α)和血管内皮生长因子(vascularendothelial growth factor, VEGF)mRNA及蛋白的表达。结果:低氧24 h组A549细胞存活率最高,低氧48 h、72 h组A549细胞存活率呈时间依赖性明显下降(P0.001)。自低氧12 h起,A549细胞HIF-1αmRNA和VEGFmRNA的表达开始随低氧时间延长而显著增加(P均0.001);HIF-1α和VEGF蛋白表达自24 h开始随低氧时间延长而显著增加(P均0.001)。结论:低氧诱导的A549细胞存活率呈时间依赖性降低,而HIF-1α、VEGF表达呈时间依赖性增高,人肺癌细胞株A549缺氧模型最适时间为24 h。     

肺癌脑转移型A549／GFP-2细胞的筛选及其条件液对脑微血管内皮细胞的作用

目的：建立肺癌脑转移模型,筛选脑转移倾向细胞A549／GFP-2,探讨A549和A549／GFP-2条件液对脑微血管内皮细胞的作用,揭示肺癌脑转移的机制。方法：利用胸腔原位注射法筛选出A549脑转移细胞亚型A549／GFP-2,原代培养大鼠脑微血管内皮细胞,观察A549和A549／GFP-2细胞条件液对脑微血管内皮细胞增殖的影响和细胞内HIF-1α和VEGF表达的改变。结果：胸腔内原位种植较好地反应了临床肺癌脑转移的过程。不同浓度的A549和A549／GFP-2细胞条件液对脑微血管内皮细胞增殖的影响不同,低浓度（〈30％）对脑微血管内皮细胞有促进的作用;高浓度（〉60％）对脑微血管内皮细胞的增殖有不同程度的抑制作用,且有随浓度增加抑制作用增强的趋势。A549和A549／GFP-2细胞条件液能提高脑微血管内皮细胞内HIF-1α和VEGF的表达。结论：胸腔内原位种植是建立肺癌脑转移的稳定模型。肺癌脑转移与肺癌细胞在生长过程中分泌的HIF-1α和VEGF等细胞因子破坏了脑微血管内皮细胞的结构有关。     

低氧诱导因子-1α在低氧促成肌细胞增殖中的作用

目的:探讨低氧对大鼠骨骼肌成肌细胞(SkMs)增殖的影响及低氧诱导因子(HIF-1α)在低氧促成肌细胞增殖中的相关机制。方法:采用流式细胞仪观察了3、10%O2对SkMs细胞数量和增殖指数的影响;用RT-PCR方法检测了HIF-1αmRNA的表达,用Western blot方法检测了SkMs胞浆、胞核及总HIF-1α蛋白的水平。结果:低氧组较常氧组细胞数量和增殖指数增加(P0.05);HIF-1αmRNA、总蛋白水平在常氧组和低氧组中没有明显差异,常氧下胞浆中HIF-1α蛋白水平高于胞核内,低氧下HIF-1α蛋白水平在胞核内高于胞浆。结论:低氧能够促进SkMs增殖,HIF-1α可能是通过氧浓度调控的核转位的方式参与了低氧促SkMs的增殖。     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

国内外蝗害治理技术现状与展望

本文首先概述了国内外蝗虫发生与为害的态势,总结了现阶段我国蝗虫发生与为害的主要特点:即农田飞蝗暴发频繁而且严重,草原土蝗的发生时常造成严重的经济损失,而且侵入城市干扰市民生活,我国与周边国家之间蝗虫过境迁移频繁,使用化学农药污染环境和农产品;分析了国内外蝗虫防治对策与技术的发展现状,重点介绍了应急防治和可持续治理对策、...     

Molecular characterisation of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic transmission

The molecular characterisation of species and genotypes of Cryptosporidium and Giardia is essential for accurately identifying organisms and assessing zoonotic transmission. Results of recent molecular epidemiological studies strongly suggest that zoonotic transmission plays an important role in cryptosporidiosis epidemiology. In such cases the most prevalent zoonotic species is Cryptosporidium parvum. Genotyping and subtyping data suggest that zoonotic transmission is not as prevalent in the epidemiology of giardiasis. Molecular characterisation of Cryptosporidium and Giardia is a relatively recent application that is evolving as new genes are found that increase the accuracy of identification while discovering a greater diversity of species and yet unnamed taxa within these two important genera. As molecular data accumulate, our understanding of the role of zoonotic transmission in epidemiology and clinical manifestations is becoming clearer.     

Synthesis and turnover of cerebrosides and phosphatidylserine of myelin and microsomal fractions of adult and developing rat brain.

The synthesis and turnover of cerebrosides and phospholipids was followed in microsomal and myelin fractions of developing and adult rat brains after an intracerebral injection of [U-14C]serine. The kinetics of incorporation of radioactivity into microsomal and myelin cerebrosides indicate the possibility of a precursor-product relationship between cerebrosides of these membranes. The specific radioactivity of myelin cerebrosides was corrected for the deposition of newly formed cerebrosides in myelin. Multiphasic curves were obtained for the decline in specific radioactivity of myelin and microsomal cerebrosides, suggesting different cerebroside pools in these membranes. The half-life of the fast turning-over pool of cerebrosides of myelin was 7 and 22 days for the developing and adult rat brain respectively. The half-life of the slowly turning-over pool of myelin cerebrosides was about 145 days for both groups of animals. The half-life of the rapidly turning-over microsomal cerebrosides was calculated to be 20 and 40 h for the developing and adult animals respectively. The half-life of the intermediate and slowly turning-over microsomal cerebrosides was 11 and 60 days respectively, for both groups of animals. The amount of incorporation of radioactivity into microsomal cerebrosides from L-serine was greatly decreased in the adult animals, and greater amounts of the precursor were directed towards the synthesis of phosphatidylserine. In the developing animals, considerable amounts of cerebrosides were synthesized from L-serine, besides phosphatidylserine. The time-course of incorporation indicated that a precursor-product relationship exists between microsomal and myelin phosphatidylserine. The half-life of microsomal phosphatidylserine was calculated to be about 8 h for the fast turning-over pool in both groups of animals.     

白术同源四倍体的诱导和鉴定及其与二倍体过氧化物酶的比较

以白术（Atractylodes macrooephala Koidz.）二倍体组培苗为材料,对其四倍体诱导方法进行研究,共获得45个白术同源四倍体株系,为优良株系的选育提供了材料。此外,还分析比较了其中8个白术四倍体株系与二倍体的过氧化物酶同工酶（POD）的酶谱差异,发现四倍体各株系过氧化物酶同工酶谱比二倍体的均多了Rf0.310的谱带,且总过氧化物酶比活力也发生了很大改变,对探讨白术四倍体优良株系的生理生化机理具有一定的参考价值。     

Identification and composition of the tonsillar and anal enterococcal and streptococcal flora of dogs and cats

Enterococcus faecalis was the most frequently isolated enterococcal species from anal swabs and tonsils of dogs and cats, although in the anal samples from dogs Ent. hirae was found almost as often as Ent. faecalis. Most Ent.faecium strains from dog tonsils differed from those associated with humans and other animals in that they fermented sorbitol. Typical Ent. avium as well as atypical Ent. avium -like strains were seen in dogs, while the related species Ent. raffinosus was associated with cat tonsils. Enterococcus cecorum also occurred mainly in cats. Certain atypical strains, presumptively identified as Ent. cecorum , shared characteristics with Ent. columbae.
The most frequent streptococcal species in tonsils of cats and dogs were Streptococcus suis and Strep. canis. Streptococcus canis and Strep. bovis predominated in anal swabs. The canine Strep. suis differed from the common porcine strains in fermenting mannitol.
Forty-seven of the 288 isolates examined could not be identified or related to known species. The characteristics of two groups of these bacteria, provisionally called 'Ton 31 group' and 'O7 group' are described.