胃癌和慢性胃炎患者幽门螺杆菌蛋白质组学分析

背景：胃癌的发生是一个多因素多步骤的过程,幽门螺杆菌（H．pylori）感染在此过程中发挥重要作用。目的：建立胃癌和慢性胃炎H．pylori的双向凝胶电泳（2-DE）图谱,识别鉴定差异表达的蛋白质．探讨细菌本身因素在胃癌发病过程中的作用。方法：取H.pylori而阳性的胃癌和慢性胃炎患者胃黏膜组织后分离培养H.pylori,收集菌体并采用BCA法行蛋白定量,以2-DE分离Hpylori蛋白．采用PDQuest软件分析差异表达的蛋白质点．应用电喷雾四极杆飞行时间串联质谱（Q—TOF）进行鉴定,并在Mascot数据库中进行检索。结果：胃癌和慢性胃炎H．pylori总蛋白均获得分辨率高、重复性好的2-DE图谱。共6个蛋白质点在胃癌和慢性胃炎中存在表达差异．其中超氧化物歧化酶、尿素酶仪亚单位、分子伴侣60kDa在胃癌Hpylori菌株中高表达,而巯基过氧化物酶、二磷酸核苷激酶、S．核糖基同型半胱氨酸裂解酶在慢性胃炎H．pylori菌株中高表达。结论：成功建立了分辨率高且重复性好的胃癌和慢性胃炎H．pylori蛋白的2-DE图谱,并鉴定出两种疾病相关菌株中差异表达的蛋白质点．这些蛋白质点可能在胃癌的发生中起重要作用。     

幽门螺杆菌26695及其不同克拉霉素耐药水平衍生株的比较蛋白质组学分析

背景：目前克拉霉素耐药机制的研究主要集中于对幽门螺杆菌（H．pylori）23S rRNM基因的分析．其他基因与克拉霉素耐药的关系在国内外尚未见报道。目的：应用蛋白质组学技术分析不同克拉霉素浓度下耐药H．pylori菌株的发生机制，以期发现新的克拉霉素耐药相关基因。方法：以H．pylori 26695为出发菌株，在含克拉霉素平皿上选择耐药突变体，提取全菌蛋白后通过双向凝胶电泳（2-DE）分离蛋白，银染后应用Image Master 2D Elite软件比较不同浓度克拉霉素耐药株和野生株的蛋白表达差异．应用基质辅助激光解吸电离飞行时间质谱（MALDI—TOF—MS）进行鉴定．并在NCBI的H．pylori蛋白数据库中搜索理论上酶解肽段能与之相匹配的蛋白。结果：共10个蛋白点在耐药株与野生株以及不同浓度耐药株间有明显差异，其中3个蛋白点为DNA指导的RNA多聚酶d亚单位，其表达量和等电点在耐药株与野生株间存在明显差异；1个蛋白点为黄素氧化还原蛋白，其在耐药株中的表达量上调；2个蛋白点为30S核糖体蛋白S1。其在耐药株中的表达量下调；3个蛋白点为过氧化氢酶，其在耐药株中的表达量上调；1个蛋白点为细胞分裂抑制剂，其在低浓度克拉霉素耐药株中的表达量较野生株上调，而在高浓度克拉霉素耐药株中的表达量较野生株下调。结论：不同浓度克拉霉素耐药株的耐药机制有不同的相关靶点，为研究H．pylori 23S rRNA以外克拉霉素的耐药机制提供了线索。     

不同疾病来源幽门螺杆菌菌株对人胃癌细胞系AGS基质金属蛋白酶表达影响的比较研究

背景：幽门螺杆菌（H.pylori）是胃癌的Ⅰ类致癌原，但H.pylori感染与胃癌发生的分子机制仍不明了。目的：在体外观察不同疾病来源的H.pylori菌株对人胃癌细胞系AGS基质金属蛋白酶（MMPs）表达的影响是否存在差异。方法：将AGS细胞分别与5株分离自胃癌和5株分离自轻度非萎缩性胃炎患者的H.pylori共培养，以逆转录聚合酶链反应（RT-PCR）和蛋白质印迹法（Western blot）检测MMP-2、MMP-7和MMP-9 mRNA和蛋白的表达，以肠道致病性大肠杆菌作为细菌对照。结果：胃炎H.pylori菌株和大肠杆菌基本不影响AGS细胞MMP-2、MMP-7和MMP-9 mRNA和蛋白的表达，而所有胃癌菌株均能上调MMP-2、MMP-7和MMP-9 mRNA和蛋白的表达。结论：分离自胃癌和胃炎患者的H.pylori菌株对AGS细胞MMP-2、MMP-7和MMP-9表达的影响有所不同，说明不同疾病来源的H.pylori菌株在促细胞恶变能力上存在差异。     

塞莱昔布对幽门螺杆菌蛋白质组的影响

目的:探讨选择性COX-2抑制剂塞莱昔布对幽门螺杆菌(H.pylori)蛋白质组的影响.方法:双向凝胶电泳技术(2-DE)分离塞莱昔布处理前后的H.pylori标准株26695全菌蛋白,银染法凝胶染色,图像扫描分析软件识别差异蛋白,基质辅助激光解吸电离飞行时间质谱(MALD-TOF-MS)、串联质谱(MALDI-TOF-MS/MS)分析差异蛋白;SYBRGreenⅠ实时定量PCR测定差异蛋白基因的表达变化.结果:塞莱昔布处理后,H.pylori标准株26695发现17个蛋白质斑点表达有差异.7个蛋白质斑点得到阳性鉴定,分别归为H.pylori的3种蛋白质:热休克蛋白60(HSP60),即groEL,延伸因子(EF-TU),谷氨酰转肽酶(GGT).与DMSO溶媒对照相比,塞莱昔布处理后的H.pyloriHSP60(groEL)、EF-TU、GGT表达下调.实时定量PCR测定显示,编码H.pyloriEF-TU、GGT基因的mRNA水平降低(0.07±0.06vs1.01±0.16;0.31±0.13vs0.98±0.01,均P<0.05),而编码H.pyloriHSP60(groEL)基因的mRNA水平增加(1...     

江苏不同地区胃疾病相关幽门螺杆菌差异蛋白质的比较

目的:鉴定、比较江苏地区胃癌高、低发区胃癌与慢性胃炎相关幽门螺旋杆菌(Helicobacter pylori,H.pylori)差异表达蛋白质.方法:选取江苏省泰州市(胃癌高发区)和苏州市(胃癌低发区)胃癌和慢性胃炎患者胃镜下胃黏膜活检组织分离培养出的H.pylori,提取蛋白质后通过双向凝胶电泳进行分离,使用PDQuest软件对蛋白质点进行分析和比较,通过四极杆飞行时间电喷雾串联质谱对差异蛋白质点进行鉴定,并通过Mascot数据库检索.结果:所选H.pylori菌株总蛋白进行双向凝胶电泳后获得优质2-DE图谱,经Q-TOP鉴定为1号(巯基过氧化物酶)、3号(超氧物歧化酶)、4号(尿素酶α)、5号(核苷二磷酸激酶)、8号(分子伴侣dnaK)、9号(S-核糖基同型半胱氨酸裂解酶)、13号(无机焦磷酸酶)、14号(DNA引导的RNA聚合酶α)和16号(分子伴侣60×103)9个差异蛋白质点,其中3号、4号、8号和14号蛋白质点在胃癌菌株中均为高表达,1号和5号蛋白质点在慢性胃炎菌株中均为高表达,9号、13号和16号蛋白质点仅在一个地区存在差异表达.结论:胃癌高、低发区胃癌及相应地区慢性胃炎H.pylori蛋白质比较存在一定差异,但其中2/3的差异蛋白质是一致的,胃癌高发区H.pylori差异蛋白质高表达多于低发区,表明H.pylori在不同胃癌发病区致病机制相似,但在胃癌高发区作用强于低发区.     

不同疾病来源的幽门螺杆菌菌株对AGS细胞动力学的影响

体内外研究发现，幽门螺杆菌(H.pylori)感染可促进胃黏膜上皮细胞增殖或触发细胞凋亡，从而引起细胞动力学改变，而菌株的差异可导致不同的结果。目的：通过体外实验观察不同疾病来源的H.pylori菌株对人胃癌细胞株AGS的细胞活力、细胞凋亡和细胞周期的影响，以确定不同疾病来源H.pylori菌株的细胞毒性是否存在差异。方法：采用聚合酶链反应(PCR)鉴定分离自胃癌和胃炎患者的H.pylori菌株的毒力基因和相关亚型。将AGS细胞分别与H.pylori菌株共培养，采用四唑蓝(MTT)比色法检测细胞活力，流式细胞仪检测细胞凋亡和细胞周期。结果：cagA、uacA、iceA和babA2基因和相关亚型在胃癌和胃炎H.pylori菌株中呈均匀分布。不同菌株抑制AGS细胞活力和促进细胞凋亡的能力虽有强弱差别，但胃癌菌株与胃炎菌株之间不存在整体上的差别。多数H.pylori菌株能抑制AGS细胞周期的G1/S期转换。结论：不同H．pytori菌株对共培养的AGS细胞动力学的影响存在差异，但胃癌菌株与胃炎菌株的细胞毒性无整体上的差别。     

幽门螺杆菌感染个体中菌株基因多态性的研究

幽门螺杆菌(H.pylori)感染的临床结局与宿主易感性、环境因素和菌株的基因多态性相关。了解感染个体中菌株的基因多态性，有助于阐明H.pylori感染的影响因素、致病机制和治疗后复发等问题。目的：研究同-H.pylori感染个体中的菌株是否存在基因多态性。方法：分别从14例临床H.pylori感染者的胃窦活检标本中分离、培养H.pylori,从每例菌株中分离出10个单克隆菌株。从分离自另7例患者胃窦和胃体活检标本的配对菌株中各分离出1个单克隆菌株。采用随机扩增多态性DNA(RAPD)指纹分析方法检测H.pylori菌株的基因多态性。结果：分离自不同感染个体的H.pylori菌株的RAPD指纹图有显著差异。大多数来自同一胃活检部位或同一感染个体的单克隆菌株的RAPD指纹图相同或十分相似，仅少数单克隆菌株的指纹图存在差异。结论：不同H.pylori感染个体所携带菌株的基因型存在显著多态性，同一个体携带的H.pylori菌株基因型相对均一。     

Cdx2蛋白在不同亚型肠上皮化生中的表达及其与H.pylori感染的关系

目的探讨胃黏膜肠上皮化生（intestinal地metaplasia,IM）中尾型同源异型盒基因2（caudal type homeobox genes2,Cdx2）蛋白的表达及其与幽门螺杆菌（helicobacter pylori,H.pylori）感染的关系。方法选取内镜下活检的18例正常胃黏膜和53例IM胃黏膜。采用高铁二铵-爱先蓝-过碘酸雪夫反应（HID-AB-PAS）将胃黏膜IM分为Ⅰ、Ⅱ、Ⅲ型,然后用Cdx2单克隆抗体进行免疫组织化学染色,检测Cdx2在正常胃黏膜及不同亚型IM中的表达。采用一分钟快速尿素酶实验及甲苯胺蓝染色检测H．pylori感染。结果Cdx2蛋白在正常胃黏膜中不表达,IM中Cdx2阳性表达率为83．02％（44／53）,其中Ⅰ型IM阳性率为95．45％（21／22）。Ⅱ型为83．33％（15／18）,Ⅲ型为61．53％（8／13）,三者间Cdx2蛋白表达有显著性差异（P=0．036）。IM中H．pylori感染阳性率为47．17％（25／53）,其中Ⅰ型IM中H．pylori感染阳性率为27．27％（6／22）,Ⅱ型为55．56％（10／18）,Ⅲ型为69．23％（9／13）,三者间Mpylori感染率亦有显著性差异（P=0．038）。Cdx2蛋白表达主要集中于H．pylori感染阴性者,但H．pylori感染阳性者和阴性者Cdx2表达无统计学差异（P〉0．05）。结论Cdx2蛋白异常表达可能参与了胃黏膜IM向胃癌的转变,检测其表达有助于预测胃黏膜IM向胃癌转变的可能性。     

肺癌与正常人血清蛋白质双向电泳差异分析

利用双向电泳(2-DE)分离肺癌患者和正常人的血清蛋白质,银染显色,Umax扫描仪获取图像,Melanie4软件分析图像,寻找肺癌相关差异蛋白质.结果建立了较稳定的肺癌和正常血清蛋白质2-DE图谱,平均蛋白质点数约400个.软件分析显示,两组共有11个差异蛋白质点,其中肺癌患者量上调的差异点9个,量下调的差异点2个.提示差异蛋白质分离为进一步肺癌相关血清蛋白质的鉴定和其他肿瘤血清蛋白质组学研究奠定了良好基础.     

枸橼酸铋钾对幽门螺杆菌耐药菌株体外抗菌活性研究

目的铋制剂被广泛应用于幽门螺杆菌（H．pylori）感染的根除治疗，目的在于了解枸橼酸铋钾在体外对H．pylori耐药菌株是否存在抗菌活性及枸橼酸铋钾在体外对甲硝唑和克拉霉素抗H．pylori耐药菌株活性的影响。方法选取H．pylori标准菌株NCTC11637（对照）和8株l临床分离H．pylori耐药菌株作为实验菌株：抑菌研究采用琼脂稀释法测定枸橼酸铋钾对H．pylori菌株的最小抑菌浓度（MIC），通过E试验法在含枸橼酸铋钾培养基和普通培养基上分别测定甲硝唑或克托霉素对H．pylori菌株的MIC值。杀菌研究采用时间一杀菌曲线法，分别在含有枸橼酸铋钾、甲硝唑、克拉霉素及枸橼酸铋钾与甲硝唑或克拉霉素混合物的液体培养基中对H．pylori标准菌株NCTC11637及l临床分离的H．pylori耐药菌株进行培养，分别计算空白对照组和各实验组0、4、8、24h的细菌数量，采用半对数法绘制时间-杀菌曲线：结果枸橼酸铋钾对H．pylori标准菌株及临床分离的H．pylori耐药菌株平均MIC值为2．6mg／L（铋）：枸橼酸铋钾降低甲硝哗和克拉霉素对H．pylori耐药菌株的MIC值。枸橼酸铋钾1mg／L（铋）对标准菌株H．pylori11637具有明显的体外杀菌作用，4mg／L（铋）对临床分离耐药菌株也具有明显的体外杀菌作用，其杀菌效果随剂量增加而增强：、枸橼酸铋钾与抗生素混合物对H．pylori标准菌株及临床耐药菌株的体外杀菌作用增强。结论枸橼酸铋钾对H．pylori标准菌株及临床分离的H．pylori耐药菌株均有体外抑菌和杀菌作用，并且与甲硝唑或克拉霉素联用对标准菌株及临床分离的H．pylori耐药菌株具有体外协同抑菌或杀菌作用。     

Three layer functional model and energy exchange concept of aging process

Relying on a certain degree of abstraction, we can propose that no particular distinction exists between animate or living matter and inanimate matter. While focusing attention on some specifics, the dividing line between the two can be drawn. The most apparent distinction is in the level of structural and functional organization with the dissimilar streams of ‘energy flow’ between the observed entity and the surrounding environment. In essence, living matter is created from inanimate matter which is organized to contain internal intense energy processes and maintain lower intensity energy exchange processes with the environment. Taking internal and external energy processes into account, we contend in this paper that living matter can be referred to as matter of dissipative structure, with this structure assumed to be a common quality of all living creatures and living matter in general. Interruption of internal energy conversion processes and terminating the controlled energy exchange with the environment leads to degeneration of dissipative structure and reduction of the same to inanimate matter, (gas, liquid and/or solid inanimate substances), and ultimately what can be called ‘death.’ This concept of what we call dissipative nature can be extended from living organisms to social groups of animals, to mankind. An analogy based on the organization of matter provides a basis for a functional model of living entities. The models relies on the parallels among the three central structures of any cell (nucleus, cytoplasm and outer membrane) and the human body (central organs, body fluids along with the connective tissues, and external skin integument). This three-part structural organization may be observed almost universally in nature. It can be observed from the atomic structure to the planetary and intergalactic organizations. This similarity is corroborated by the membrane theory applied to living organisms. According to the energy nature of living matter and the proposed functional model, the decreased integrity of a human body's external envelope membrane is a first cause of the structural degradation and aging of the entire organism. The aging process than progresses externally to internally, as in single cell organisms, suggesting that much of the efforts towards the restoration and maintenance of the mechanisms responsible for structural development should be focused accordingly, on the membrane, i.e., the skin. Numerous reports indicate that all parts of the human body, like: bones, blood with blood vessels, muscles, skin, and so on, have some ability for restoration. Therefore, actual revival of not only aging tissue of the human body's membrane, but the entire human body enclosed within, with all internal organs, might be expected. We assess several aging theories within the context of our model and provide suggestions on how to activate the body's own anti-aging mechanisms and increase longevity. This paper presents some analogies and some distinctions that exist between the living dissipative structure matter and inanimate matter, discusses the aging process and proposes certain aging reversal solutions.     

Unusual responses of nocturnal pineal melatonin synthesis and secretion to swimming: Attempts to define mechanisms

Abstract: The effect of swimming at night on rat pineal melatonin synthesis was compared with that of light exposure at night. Rats were forced to swim at 0030 hr (lights out at 2000 hr) and sacrificed by decapitation 15 and 30 min later, immediately after swimming. Other groups of animals were exposed to white light (650μW/cm²) for 15 and 30 min at same time. Swimming caused a rapid and highly significant drop in the melatonin content in the pineal gland; however, the activity of N-acetyltransferase (NAT), the supposed rate limiting enzyme in the melatonin production, was not changed. Despite the drop in pineal melatonin levels, serum concentrations of the indole remained elevated in the rats that swam. In contrast, melatonin levels in the pineal and serum of light exposed rats fell precipitously, accompanied by a significant suppression of NAT activity. Since we anticipated that the strenuous exercise associated with swimming may induce release of artrial natriuretic peptide (ANP) from the heart, which in turn could cause the release of pineal melatonin, in a second study we injected physiological saline intravenously to stretch the cardiac muscle and release ANP. Three milliliters of normal saline was injected during the day into the jugular vein of anesthetized rats that were pretreated with isoproterenol to stimulate pineal melatonin production. Animals were killed 15 min after the saline injection, and pineal NAT activity and pineal melatonin levels were measured. The saline injections caused no alteration in the elevated levels of either NAT or melatonin. These data suggest that the disparity in pineal NAT activity (which was high) and pineal melatonin (which was low), in animals swum at night, may not be caused by ANP which is released during strenuous exercise such as swimming.     

Melatonin and photoperiodic time measurement: Seasonal breeding in the sheep

Abstract: Well-established circadian physiology supports the view that photoperiodic time measurement utilizes the coincidence between the presence of light and a photosensitive phase of a 'biological clock' to alter reproductive status—the so-called external coincidence model of seasonal breeding. In this review, we examine the mechanism whereby photoperiod interacts with presumed suprachiasmatic nuclei activity to allow endogenous melatonin to normally synchronize reproductive activity to the optimal time of year. The Romney Marsh sheep is particularly explored as an experimental model. It is suggested that the on/off activity of seasonal reproduction may be a robust mechanism able to be predictably manipulated by the judicious use of the light/dark cycle and exogenous melatonin, but firmly based on circadian principles.     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Serological survey of HIV and syphilis in pregnant women in Madagascar

Objectives Peripartal transmission of human immunodeficiency virus (HIV) and Treponema pallidum, the causative agent of syphilis, leads to severe consequences for newborns. Preventive measures require awareness of the maternal infection. Although HIV and syphilis testing in Madagascar could be theoretically carried out within the framework of the national pregnancy follow‐up scheme, the required test kits are rarely available at peripheral health centres. In this study, we screened blood samples of pregnant Madagascan women for HIV and syphilis seroprevalence to estimate the demand for systemic screening in pregnancy. Methods Retrospective anonymous serological analysis for HIV and syphilis was performed in plasma samples from 1232 pregnant women that were taken between May and July 2010 in Ambositra, Ifanadiana, Manakara, Mananjary, Moramanga and Tsiroanomandidy (Madagascar) during pregnancy follow‐up. Screening was based on Treponema pallidum haemagglutination tests for syphilis and rapid tests for HIV, with confirmation of positive screening results on line assays. Results Out of 1232 pregnant women, none were seropositive for HIV and 37 (3%) were seropositive for Treponema pallidum. Conclusions Our findings are in line with previous studies that describe considerable syphilis prevalence in the rural Madagascan population. The results suggest a need for screening to prevent peripartal Treponema pallidum transmission, while HIV is still rare. If they are known, Treponema pallidum infections can be easily, safely and inexpensively treated even in pregnancy to reduce the risk of transmission.     

Response of rat pineal melatonin to calcium, magnesium, and lithium is circadian stage dependent

Abstract: Herein we documented the response of pineal melatonin production to electrolytes known to be effective on pineal function in view of a possible circadian stage dependence. We studied the release of melatonin by perifused rat pineal glands at 2 different circadian stages corresponding to the middle of the light and dark periods, i.e., respectively, 7 and 19 HALO (Hours After Light Onset, L:D = 12:12). The initial efflux rates were, as expected, much higher in the perifusates of glands removed from rats sacrificed during the dark phase than of those removed during the light phase. After 3 hr of perifusion, melatonin release reached similar levels which were found constant up to the 8th hr of perifusion, whatever the circadian stage. Perifusion of the glands with physiological concentrations for the rat of calcium (5.2 mmol/1) and magnesium (1.34 mmol/1) resulted in a stimulatory effect on the pineal glands removed from rats sacrificed in the middle of the dark period (19 HALO), whereas no effects were observed on the pineal glands removed from rats sacrificed during the light (7 HALO). Lithium (0.28 and 0.55 mmol/1) was ineffective on melatonin release in pineal glands removed 7 and 19 HALO. Our results show differences in the initial efflux rates of melatonin and in the response of perifused pineal glands to calcium and magnesium according to the circadian stage.     

Conservative management of perforated duodenal diverticulum： A case report and review of the literature

Duodenal diverticula are a relatively common condition. They are asymptomatic, unless they become complicated, with perforation being the rarest but most severe complication. Surgical treatment is the most frequently performed approach. We report the case of a patient with a perforated duodenal diverticulum, which was diagnosed early and treated conservatively with antibiotics and percutaneous drainage of secondary retroperitoneal abscesses. We suggest this method could be an acceptable option for the management of similar cases, provided that the patient is in good general condition and without septic signs.     

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.