缬草波春诱导MKN-45胃癌细胞凋亡

目的:研究缬草波春诱导胃癌细胞凋亡,探讨其诱导凋亡与半胱氨酸酶(Caspase)及生存素(Survivin)mRNA、P53蛋白、Survivin蛋白表达的关系.方法:以100 mg/L的缬草波春作用于加Caspase-3抑制剂、Caspase-8抑制剂、Caspase-9抑制剂和未加Caspases抑制剂培养的MKN-45细胞24,48和72 h,用流式细胞仪分别检测细胞凋亡率;不同浓度的缬草波春(5,10,25,50,100 mg/L)作用MKN-45细胞不同时间(24,48,72 h)后,用tripure提取液提取细胞RNA,用RT-PCR法,检测Survivin mRNA的表达.不同浓度缬草波春(50和100 mg/L)作用MKN-45胃癌细胞株24 h后,用免疫组化的方法,检测P53蛋白和Survivin蛋白的表达.结果:单用Caspase抑制剂组,作用24,48和72 h对MKN-45细胞凋亡率无明显影响,与对照组比较差异无显著意义.Caspase-3抑制剂、Caspase-9抑制剂与缬草波春联合应用后24,48和72 h使MKN-45细胞凋亡率高于对照组(24 h:5.73%,5.41% vs 4.38%,P＜0.01;48 h:6.88%,6.32% vs 4.35%,P＜0.01;72 h:7.72%,8.62% vs 4.54%,P＜0.01),低于缬草波春组(24 h:5.73%,5.41% vs 8.14%,P＜0.01;48 h:6.88%,6.32% vs 12.31%,P＜0.01;72 h:7.72%,8.62% vs 26.41%,P＜0.01),与对照组及缬草波春组比较差异均有显著意义(P＜0.01).Caspase-8抑制剂与缬草波春联合应用后24,48和72 h MKN-45细胞凋亡率明显增加,与对照组比较差异有显著意义(8.02% vs 4.38%,P＜0.01;11.05% vs 4.35%,P＜0.01;24.86% vs 4.54%,P＜0.01),与单用缬草波春组比较差异无显著意义.缬草波春降低MKN-45胃癌细胞株Survivin mRNA的表达,并有浓度依赖性和时间依赖性,而且使MKN-45胃癌细胞株P53蛋白表达增加,Survivin蛋白表达降低,均有浓度依赖性.结论:缬草波春可诱导MKN-45细胞凋亡,其作用可部分被Caspase-3,Caspase-9抑制剂所抑制,但不能被Caspase-8抑制剂所抑制.缬草波春诱导MKN-45胃癌细胞株凋亡与P53蛋白表达提高及Survivin mRNA和Survivin蛋白低表达降低有关.     

表没食子儿茶素没食子酸酯诱导细胞凋亡蛋白酶途径依赖的人胃癌细胞株MKN45凋亡

背景：表没食子儿茶素没食于酸酯（EGCG）可诱导人胃癌细胞株MKN45凋亡，但其凋亡信号的传导途径尚不清楚。目的：研究EGCG诱导人胃癌细胞株MKN45凋亡的作用是否通过细胞凋亡蛋白酶（caspsse-3）依赖途径，为其临床应用提供进一步的理论依据。方法：采用四甲摹偶氮唑蓝（MTT）比色法检测EGCG和caspase-3抑制剂z-DEVD-fmk作用后MKN45细胞的存活率：采用Annexin V-FITC＋PI双染色法检测EGCG和caspase-3抑制剂作用后MKN45细胞的凋亡率：采用酶联免疫吸附测定（ELISA）检测EGCG和caspase-3抑制剂作用后MKN45细胞内caspase-3活性的改变。结果：EGCG可诱导MKN45细胞凋亡，且细胞内caspase-3活性显著升高。而caspase-3抑制剂干预后，EGCG抑制MKN45细胞生长的作用明显减弱，细胞凋亡率下降，caspase-3活性显著下降。结论：EGCG可诱导MKN45细胞凋亡，该作用可被caspase-3抑制剂显著抑制，提示EGCG诱导MKN45细胞凋亡的作用是通过caspase-3依赖途径的。     

缬草波春诱导胃癌细胞凋亡与信号分子表达的关系

缬草波春是从缬草中提取的化合物，体外实验表明缬草波春对Kreb Ⅱ腹水癌细胞、肝癌细胞、骨髓造血祖细胞和人T2淋巴细胞有抑制作用。新近发现生存素是凋亡抑制因子，而p53是重要的抗癌基因，参与化疗药物诱导肿瘤细胞凋亡过程。我们试图通过研究缬草波春诱导MKN-45胃癌细胞凋亡时P53蛋白、生存素mRNA及生存素蛋白的表达阐明其诱导凋亡的机制。     

表没食子儿茶素没食子酸酯对人胃癌细胞株MKN45诱导凋亡作用的研究

目的了解表没食子儿茶素没食子酸酯（EGCG）是否诱导人胃癌细胞株MKN45凋亡及其凋亡信号传导途径，为其临床应用提供进一步的理论依据。方法用四甲基偶氮唑蓝（MTT）比色法检测EGCG对MKN45细胞生长的抑制作用；膜连蛋白V-异硫氰基荧光素（Annexin V-FITE）＋碘化丙啶（PI）方法测定EGCG作用后MKN45细胞的凋亡率；酶联免疫吸附测定（ELISA）检测EGCG对MKN45细胞内castmse-3活性的影响；Rhodamine123染色检测EGCG作用后MKN45细胞内线粒体膜电位的改变情况。结果EGCG作用后MKN45细胞发生凋亡，随着EGCG作用时间的延长及浓度的增加．凋亡率增加。在EGCG作用8h后MKN45细胞内的caspase-3的活性开始升高，并于作用12h后活性明显升高。线粒体的膜电位于EGCG作用4h后就开始明显下降，并与时间和浓度正相关。结论EGCG可以诱导人胃癌细胞株MKN45细胞凋亡，且与作用时间及浓度正相关。EGCG对MKN45细胞凋亡的诱导是通过线粒体途径发生的．     

西咪替丁对人胃癌细胞MKN45的生长抑制和促凋亡的研究

目的 探讨西咪替丁对胃癌细胞MKN45生长的抑制和凋亡作用。方法 采用MTT比色法测定不同浓度西咪替丁作用下胃癌细胞MKN45培养液的OD值，并用流式细胞仪测定西咪替丁对MKN45的细胞周期和细胞凋亡的影响。结果 西咪替丁作用72小时，抑制胃癌细胞。MKN45的生长并呈剂量依赖趋势。胃癌细胞MKN45细胞周期发生改变，G1期百分比减少，S期百分比增加，并出现明显的细胞凋亡峰。结论 西咪替丁可能通过诱导胃癌细胞。MKN45凋亡从而抑制其细胞增殖。     

凋亡相关蛋白在胃癌细胞凋亡中的作用研究

目的　研究核转录因子 (NF) κB、生存素 (survivin)、Bcl 2和Caspase3在肿瘤坏死因子相关凋亡诱导配体 (TRAIL)诱导的胃癌细胞凋亡中的作用。方法　应用细胞培养和流式细胞仪检测的方法 ,观察胃癌细胞SGC 790 1、MKN2 8、AGS、MKN45经TRAIL作用后的细胞凋亡率 ,Westernblot方法分析 4种胃癌细胞中NF κB、生存素、Bcl 2和Caspase3的表达。结果　TRAIL 50ng/ml作用于胃癌细胞 2 4h后 ,MKN2 8、MKN 45、AGS和SGC 790 1细胞的凋亡率分别为 2 4.0 5% ,7.83 % ,8.0 5%和3 .17%。TRAIL 3 0 0ng/ml作用于胃癌细胞 2 4h后 ,MKN2 8、MKN 45、AGS和SGC 790 1细胞的凋亡率分别为 3 6.0 5% ,2 0 .2 7% ,16.50 %和 11.80 %。Westernblot结果显示 ,SGC 790 1细胞NF κB、生存素的表达高于MKN2 8细胞 (P <0 .0 5) ,与Bcl 2的表达无明显差异。MKN2 8细胞Caspase3的表达高于SGC 790 1细胞 (P <0 .0 5)。结论　TRAIL具有选择性诱导肿瘤细胞凋亡的特性 ,肿瘤细胞对TRAIL的耐药现象可能与凋亡抑制因子生存素和NF κB的表达增强以及Caspase3 的表达抑制有关     

羟基喜树碱诱导胃癌细胞凋亡的作用机制初步研究

目的：研究羟基喜树碱（HCPT）诱导胃癌细胞的凋亡作用及对凋亡相关基因p53,c-myc,bcl-2,bcl-xl和bcl-xs表达的影响,探讨其诱导胃癌细胞凋亡的作用机制。方法：应用TUNEL染色、流式仪、免疫组化和RT－PCR技术等研究HCPT对胃细胞SGC－7901和MKN－45的诱导凋亡作用和对凋亡相关有达的影响。结果：HCPT作用于细胞后,可看到较为典型的细胞凋亡的形态学变化;细胞核固缩,染色质凝集,呈新月型紧核膜周边,核碎裂,染色质片段化,凋亡小体形成等。流式细胞仪DNA直方图上出现典型的亚二倍体的“凋亡峰”。流式细胞仪计数显示,10μg/ml的HCPT诱导胃癌细胞SGC－7901和MKN－45的凋亡率为21．88％和12．34％。TUNEL染色法显示,细胞凋亡指数在1．865－9．54％之间。免疫组化和RT－PCR结果显示：HCPT能够明显下调SGC－7901细胞的P53和bcl-2基因的蛋白和mRNA表达,对SGC－7901细胞的c-myc,bcl-xl和bcl-xs基因的蛋白表达无影响。HCPT作用后MKN－45细胞的p53蛋白和mRNA的表达增加,对MKN－45细胞的bcl-2,c-myc,bcl-xl和bcl-xs基因的表达无影响。结论：HCPT能够诱导胃癌细胞凋亡,可能是通过调控胃癌细胞的P53和bcl-2的表达而诱导胃癌细胞凋亡。     

维生素C诱导人胃癌细胞株MKN45凋亡的实验研究

目的 探讨维生素C对胃癌细胞株MKN45增殖、凋亡的影响及其相关机制。方法在体外培养的胃癌细胞株MKN45中加入不同浓度（0．05、0．1、0．5、1mg／m1）的维生素C,通过MTT比色法检测维生素C对细胞生长活力的影响;应用荧光显微镜、流式细胞术、DNALadder分析法检测细胞凋亡情况;应用分光光度法检测Caspase-3活性;应用化学显色法测定维生素C作用MKN4524h后细胞内超氧化物歧化酶（SOD）活性、丙二醛（MDA）含量。结果维生素C对胃癌细胞株MKN45有显著的生长抑制作用;与对照组比较,1mg／ml维生素C处理后,细胞凋亡率升高（P〈0．01）,并呈现凋亡的形态学改变,DNALadder分析呈典型“梯状”条带;Caspase-3活性升高（P〈0．05）。与对照组相比,维生素c处理组细胞内的SOD活性下降,MDA含量增加（P〈0．05）。结论1mg／ml浓度的维生素c作用24h后,可诱导胃癌细胞MKN45凋亡,其机制可能是通过提高Caspase-3的活性以及通过自身自氧化的氧化应激效应而诱导肿瘤细胞凋亡。     

5-氮杂-2’-脱氧胞苷诱导CDX2基因表达对人胃癌细胞株MKN45增殖和凋亡的影响

背景：DNA甲基化导致基因表达沉默是胃癌发生的重要步骤。近年发现肠表型调节因子CDX2在胃癌中具有抑癌基因的作用。目的：探讨5-氮杂-2’-脱氧胞苷（5-aza—CdR）对人胃癌细胞株MKN45中CDX2基因表达的影响及其对细胞增殖和凋亡的作用。方法：以不同浓度5-aza—CdR（2．5、5、10μmo]／L）处理胃癌细胞株MKN45,甲基化特异性PCR（MSP）检测CDX2启动子区甲基化状态;实时荧光定量RT-PCR和蛋白质印迹法分别检测CDX2mRNA和蛋白表达：CCK8法检测MKN45细胞增殖活性,AnnexinV．FITC／PI检测细胞凋亡和Hoechst33258染色观察其凋亡形态：比色法检测caspase-3活性。结果：MKN45细胞中CDX2基因启动子区高甲基化．CDX2mRNA表达极低且蛋白不表达：经3种浓度5-aza-CdR处理72h后．MKN45细胞CDX2基因启动子区高甲基化发生逆转．CDX2mRNA和蛋白表达均上调。与对照组相比,5-aza-CdR呈剂量依赖性地抑制MKN45细胞增殖和促进凋亡（P〈0．05）：Hoechst33258染色示细胞核致密浓染、细胞核碎裂;caspase-3活性随着5．aza．CdR浓度的增加而升高（P〈0．05）。结论：MKN45细胞中CDX2基因表达缺失可能与其启动子区CpG岛高甲基化有关;5-aza—CdR能有效逆转CDX2基因的异常甲基化．诱导其再表达．并抑制MKN45细胞增殖和促进凋亡．该作用可能与caspase-3活性有关．     

YM155对人胃癌细胞株MKN28的作用及其机制研究

背景:研究发现生存素(survivin)在胃癌组织中高表达,YM155是survivin的特异性抑制剂。目的:探讨YM155对人胃癌细胞株MKN28的作用及其机制。方法:以不同浓度YM155作用于人胃癌细胞株MKN28。采用甲基噻唑基四唑(MTT)法检测细胞增殖抑制率;以原位末端标记(TUNEL法)检测细胞凋亡率;以逆转录聚合酶链反应(RTPCR)、蛋白质印迹法分别检测survivin mRNA和survivin、多聚ADP核糖聚合酶(PARP)、caspase-3蛋白表达。结果:YM155作用后,MKN28细胞增殖抑制,凋亡增加。随着YM155浓度升高,survivin mRNA和蛋白表达水平明显降低,并伴随PARP、caspase-3蛋白裂解。结论:YM155可抑制人胃癌细胞株MKN28增殖,并诱导其凋亡,此机制可能与抑制survivin表达,继而激活caspase凋亡信号通路有关。     

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.