建立幽门螺杆菌感染BALB/c小鼠动物模型的实验研究

[目的]建立幽门螺杆菌(Hp)BALB/c小鼠感染动物模型。[方法]用cagA基因和vacA基因同时阳性的Hp小鼠适应株给BALB/c小鼠灌胃。[结果]经Hp小鼠适应株灌胃4周后,病理模型组10只感染的小鼠胃黏膜尿素酶试验、细菌学培养和组织病理学检查均为阳性,感染率为100%;正常对照组未见Hp生长,两者比较差异有统计学意义(P<0.05)。[结论]成功地建立了云南Hp小鼠适应株感染的BALB/c小鼠动物模型。这种ca gA基因和vacA基因同时阳性的Hp小鼠适应株毒力强容易定植;可在普通饲养条件下建立,即有利应用于云南地区Hp的相关研究,又可节约研究经费,易于推广使用。     

幽门螺杆菌相关性胃炎活动性与TNF-α,EGF的相关性

目的:研究肿瘤坏死因子(tumor necrosisfactor-α,TNF-α)和表皮生长因子(epidermal growth factor,EGF)水平与幽门螺杆菌(Helicobacterpylori,Hpylori)相关性胃炎炎症活动性的关系.方法:通过快速尿素酶试验和血清HpIgG抗体水平判断H pylori感染;组织学方法评定胃窦炎症活动性;放射免疫法测定胃窦黏膜中TNF-α,EGF的含量.结果:Hpylori阳性的慢性胃炎组胃窦黏膜炎性活动性显著重于Hpylori阴性组;胃窦黏膜中TNF-α水平在H pylori阳性组显著高于H pylori阴性组(0.103±0.034vs 0.062±0.022;P＜0.05),且随黏膜炎症活动加剧而增高;胃窦黏膜中EGF水平在Hpylori阳性组显著低于Hpylori阴性组(0.215±0.102 vs 0.319±0.187;P＜0.05),与黏膜炎症活动性无明显相关性.结论:TNF-α在Hpylori致炎过程中起重要作用,且与炎症活动性成正相关;EGF水平在Hpylori感染中下降,可能减弱了对组织细胞损伤的保护作用,与炎症活动性无关.     

幽门螺杆菌感染对胃上皮细胞增殖影响的实验研究

目的在幽门螺杆菌(H.pylori)长期感染蒙古沙土鼠腺胃模型基础上,探讨H.pylori感染诱致胃癌发生的可能机制.方法采用H.pylori国际标准菌株NCTC11637灌喂蒙古沙土鼠,建立H.pylori长期感染动物模型;用免疫组化及原位杂交方法检测H.pylori感染致胃上皮细胞增殖的改变.结果成功建立了H.pylori长期感染蒙古沙土鼠腺胃模型,其胃粘膜的组织学变化显示,H.pylori感染可致正常胃粘膜→慢性胃炎→萎缩→肠化生→异型增生的发展过程.H.pylori感染对胃上皮细胞增殖的影响:H.pylori感染能引起胃窦上皮细胞增殖增加(P<0.05);随着H.pylori感染时间的增加,EGF mRNA及EGFR mRNA的阳性信号表达呈逐渐增加的趋势(P<0.05) H.pylori定植致胃窦上皮细胞增殖的异常,对正常胃窦粘膜向不典型增生进展的过程起重要作用:EGF及EGFR在mRNA水平的异常表达可能是H.pylori定植致胃窦上皮细胞增殖异常的重要原因。     

幽门螺杆菌长期感染诱发C57B L/6小鼠胃癌模型的建立及对血管新生的影响

目的:研究H.pylori长期感染C57BL/6小鼠致胃黏膜病变及其致癌性,并从血管新生的角度探讨H.pylori感染致胃癌发生的可能机制.方法:将80只SPF级C57BL/6小鼠随机分为正常组和模型组,每组40只.正常组正常饲养,模型组经口直接灌服H.pylori标准株SS1,距末次灌胃后10 wk、25 wk、45 wk、72 wk分4批处死,每批每组各处死10只小鼠.快速尿素酶试验、Giemsa染色法观察H.pylori在胃黏膜的定植情况,HE染色观察小鼠胃黏膜病理变化,免疫组织化学法观察微血管密度(microvessel density,MVD)的变化.结果:正常组动物的胃窦、胃体及十二指肠黏膜的尿素酶实验、Giemsa染色结果均呈阴性,在末次接种H.pylori后第10周、第25周、第45周、第72周,模型组H.pylori的定植率分别为88.9%、100%、100%、100%;72 wk时,模型组慢性胃炎、萎缩性胃炎、肠化生、异型增生和胃癌的发生率分别为100%,88.9%,77.8%,33.3%和22.2%,胃黏膜组织中MVD为18.56±2.62,较正常组(2.50±1.54)明显升高(P<0.01).结论:成功建立了H.pylori长期感染诱发C57BL/6小鼠胃癌模型,且H.pylori可增加小鼠胃黏膜MVD,可能在胃黏膜的癌变中发挥作用.     

幽门螺杆菌疫苗接种小鼠产生免疫后胃炎的影响因素

目的:研究幽门螺杆菌(H pylorl)疫苗接种小鼠产生免疫后胃炎的影响因素．方法:将H pylori疫苗免疫C57BL／6和BALB／c的小鼠,观察攻击后胃黏膜H pylori定植和炎症情况．将H pylori疫苗免疫C57BL／6小鼠,然后予不同菌量的H pylori攻击,观察胃黏膜H Pylori定植和炎症情况．将H pylori疫苗经口和经腹腔免疫C57BL／6小鼠,观察攻击后胃黏膜H pylori定植和炎症情况.对感染H pylori的C57BL／6小鼠予H pylori疫苗治疗,观察治疗免疫后胃黏膜H pylori定植和炎症情况．结果:不同品系的小鼠免疫保护程度无明显差异,但C57BL／6小鼠免疫后胃炎重于BALB／c小鼠．接受不同攻击茵量的小鼠保护程度无明显差异,但大的攻击茵量可诱导更严重的免疫后炎症．不同免疫途径诱导的免疫保护程度及攻击后不同时间点的炎症程度均无显著性差异．治疗性免疫导致H pylori定植明显降低,同时也引发更为严重的胃炎．结论:在不同的免疫宿主、免疫途径和治疗性免疫中均存在免疫后胃炎．免疫后胃炎的强弱程度受免疫宿主和攻击菌量的影响．     

单味中药治疗幽门螺杆菌的动物研究

目的研究单味中药治疗幽门螺杆菌(Helicobacter pylori,H.pylori)的疗效。方法建立感染H.pyloriSS1株BALB/c小鼠感染模型,将小鼠随机分成7组,每组25只,生理盐水(对照组)和黄连、黄芩、大黄、玄胡、紫珠水煎剂组及阿莫西林组(实验组),分别服药2周,停药4周后用胃黏膜组织Giemsa染色法、快速尿素酶试验及H.pylori分离培养方法判断H.pylori是否定植在小鼠胃黏膜中,从而判定H.pylori是否根除,最终评价中西药根除H.pylori的疗效。结果对照组根除率为20%;而实验组小鼠中,黄连、黄芩、大黄组根除率分别为76%、64%、68%,而玄胡、紫珠分别为28%、36%,阿莫西林组H.pylori根除率为80%,黄连、黄芩、大黄组及阿莫西林组与生理盐水有明显差异(P0.05),而玄胡、紫珠组与生理盐水组无明显差异(P0.05)。结论黄连、黄芩、大黄及阿莫西林对根除H.pylori效果好,玄胡、紫珠根除H.pylori的疗效差。     

外膜蛋白疫苗对幽门螺杆菌感染的免疫保护作用

研究幽门螺杆菌(H.pylori)外膜蛋白(OMP)加大肠杆菌不耐热内毒素(LT)经口免疫对小鼠H.pylori感染的保护作用。探讨定量细菌培养在疫苗免疫效果评价中的应用。方法从H.pyloriNCTC11637菌株制备OMP和全菌超声粉碎抗原,LT作为粘膜免疫佐剂。通过灌胃分别以H.pylori全菌超声粉碎抗原1mg加LT10μg(A组)、H.pyloriOMP0.5mg加LT10μg(B组)和生理盐水(C组)免疫小鼠(n=30),每周1次,共4次。免疫完成后4周以H.pyloriSydneyStrain1菌株攻击小鼠1次(1×107CFU/只),攻击后4周处死小鼠,取胃分别作快速尿素酶试验、病理检查及定量细菌培养,观察H.pylori定植情况。结果C组小鼠Hpylori定植密度为2.40×107CFU/g胃组织,A组和B组小鼠分别为2.03×105CFU/g和6.76×105CFU/g胃组织,免疫组的定植密度明显降低。结论口服HpyloriOMP加LT对小鼠H.pylori感染具有一定免疫保护作用,但不能完全预防感染,需进一步筛选更为有效的抗原和佐剂。定量细菌培养可以更敏感、更客观地反映胃粘膜H.pylori定植量和评价疫苗免疫效果。     

幽门螺杆菌感染对胃上皮细胞凋亡影响的实验研究

目的在H.pylori长期感染蒙古沙土鼠腺胃模型基础上,探讨H.pylori感染诱致胃癌发生的可能机制.方法采用H.pylori国际标准菌株NCTCli637灌喂蒙古沙土鼠,建立H.pylori长期感染动物模型:用末端脱氧核苷酸转移酶介导的dUTP切口末端标记法(TUNEL)及原位杂交方法检测H.pyiori感染致胃粘膜上皮细胞凋亡的变化。结果成功建立了H.pylori长期感染蒙古沙土鼠腺胃模型,其胃粘膜的组织学变化显示,H.pylori感染可致正常胃粘膜→慢性胃炎→萎缩→肠化生→异型增生的发展过程。H.pylori感染对胃上皮细胞凋亡的影响:接种H.pylori后25周内能引起胃窦粘膜上皮细胞凋亡增加(P<0.05),H.pylori感染45周时,与25周时相比,胃窦粘膜上皮细胞凋亡指数呈逐渐递减的趋势,65周时甚至明显低于正常值(P<0.05):FAS及FASL原位杂交检测结果显示随着H.pylori感染时间的延长,炎症及病变程度的加重,FAS及FASL mRNA的阳性信号表达有逐渐增加的趋势(R<0.05)。结论 H.pylori走植致胃窦粘膜上皮细胞凋亡的异常,对正常胃窦粘膜向不典型增生进展的过程起重要作用;FAS和FASL在mtLNA水平的异常表达可能是H.pylori定植致胃窦粘膜上皮细胞凋亡异常的重要原因.     

抗幽门螺杆菌中性粒细胞激活蛋白的单克隆抗体的制备和鉴定

目的:从抗幽门螺杆菌(Hpylori)全菌蛋白的单克隆抗体(mAb)中,应用重组中性粒细胞激活蛋白(NAP),筛选出抗NAP单抗并进行鉴定.方法:临床分离H pylori DY01,DY04株.免疫BALB/c小鼠后,应用杂交瘤技术制备mAb.再用ELISA方法以重组表达的NAP蛋白筛选相应的单抗,对NAP单抗进行亚类鉴定和效价检测,并用Western blot和免疫组化方法鉴定其特异性.结果:获得3株针对H pylori-NAP蛋白的特异性mAb,抗体亚类为IgG1,轻链为k型.单抗细胞培养液的抗体效价为1/16-1/32,腹水的抗体效价是1/32000-1/64000.Western blot鉴定表明,抗NAPmAb针对NAP蛋白产生特异性条带,具有高度的特异性;免疫组化分析显示:3株NAP mAb能与H pylori临床菌株发生特异性结合反应,菌体染成深棕色.结论:获得抗Hpylori-NAP蛋白的特异性mAb,为幽门螺杆菌感染的诊断、预后判断及表位疫苗的研究提供基础.     

胃食管反流病与幽门螺杆菌感染的关系探讨

为了探讨胃食管反流病(GERD)与幽门螺杆菌(Helicobacter pylori, H.pylori)感染的关系,我们将经过电子胃镜确诊的GERD患者115例及对照组轻度慢性浅表性胃炎患者90例予活检胃窦组织快速尿素酶法及14C呼气试验法进行H.pylori检测,对比两组H.pylori感染情况.结果 显示:115例GERD组H.pylori感染率为37.39%,90例对照组H.pylori感染率为62.22%,GERD组H.pylori感染率明显低于对照组,有显著性差异(P＜0.01).     

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.     

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.     

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.     

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.     

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

Abstract: The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M_r range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [¹²⁵I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (K_a of 4.7 ± 0.2 × 10⁹ M^-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M_r 24,000. The functional status of PRL-and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.