丝裂原蛋白激酶两种抑制剂对弓形虫侵入宿主细胞影响的差异

目的观察两种丝裂原蛋白激酶(mitogen activated protein kinases,MAPK)抑制剂PD98059及U0126对刚地弓形虫侵入宿主细胞的影响,探讨其对弓形虫速殖子侵入宿主细胞信号转导途径的不同阻断效应。方法丝裂原蛋白激酶抑制剂PD98059或U0126分别在不同时间及不同剂量作用于速殖子-宿主细胞培养系统,用流式细胞仪(FCM)检测宿主细胞感染速殖子的差异。结果流式细胞仪检测加入U01261μmol/L、10μmol/L和100μmol/L的细胞培养孔的细胞感染弓形虫速殖子的量分别比对照组平均降低了26.10%(P<0.01),66.42%(P<0.01)和70.39%(P<0.01)。而加入PD980591μmol/L、10μmol/L和100μmol/L的分别比对照组平均降低了25.45%(P<0.01),53.01%(P<0.01)和64.70%(P<0.01)。实验中发现100μmol/L U0126作用培养细胞9h的时候,可导致HL-60细胞出现部分聚集成团、漂浮的中毒现象。结论U0126和PD98059均可明显抑制弓形虫速殖子侵入宿主细胞,但其差异无显著性,其机制有待进一步探索。     

丝裂原激活蛋白激酶抑制剂对弓形虫速殖子侵入宿主细胞信号转导途径的阻断效应

目的观察丝裂原激活蛋白激酶(mitogen activated protein kinases,MAPK)抑制剂对弓形虫速殖子侵入细胞的影响。方法 IFA 荧光标记检测在不同剂量抑制剂作用下及在不同时间其宿主细胞感染速殖子的差异;SDS-PAGE 和 Western blot 分析 MAPK 的表达及其相对活性。结果在 PD980591μM 和10μM 作用下其感染率仅分别为对照组的18.00%(u=9.03,P<0.01)和14.80%(u=7.93,P<0.001);在 PD98059 50μM 作用下宿主细胞感染率在24h 和48h 分别为对照组的55.45%(u=3.58,P<0.01)和49.15%(u=4.08,P<0.01);与对照比较,其 MAPK 的相对活性明显下降,10μM 和50μM 组分别降低了43.03%(u=2.72,P<0.01)和78.79%(u=5.74,P<0.001)。结论 PD98059通过作用于 MAPK 信号转导途径而抑制速殖子侵入宿主细胞的过程。     

ERK1/2信号途径与弓形虫侵入细胞及胞内增殖关系的研究

目的探讨阻断细胞外蛋白调节激酶(ERK1/2)信号途径对弓形虫速殖子侵入宿主细胞及在胞内增殖的影响。方法姬氏染色法检测ERK1/2途径不同时间及不同剂量的抑制剂U0126或PD98059作用下宿主细胞感染弓形虫速殖子的百分率,根据细胞感染率和感染细胞内虫荷量分析ERK1/2途径抑制剂对速殖子在细胞中增殖的影响。结果速殖子在细胞培养系统中以1、10和100μmol/L U0126或PD98059作用3 h、6 h和9 h后,前者细胞培养孔中的细胞感染率分别较对照组平均下降34.62%(P<0.01)、53.55%(P<0.01)和67.76%(P<0.01),后者分别较对照组平均下降22.67%(P<0.01)、52.21%(P<0.01)和58.99%(P<0.01);感染细胞感染速殖子率与对照组比较差异无统计学意义(P>0.05)。结论阻断ERK1/2途径的不同信号位点对弓形虫速殖子侵入宿主细胞影响不同,ERK1/2途径在速殖子侵入宿主细胞起主要作用,但对侵入后的增殖无明显影响。     

尖吻蝮蛇蛇毒对刚地弓形虫侵入宿主细胞的作用研究

目的　观察细胞培养系统中宿主细胞在不同剂量源自皖南地区的尖吻蝮蛇 (Agkistrodonacutus)蛇毒的作用下感染刚地弓形虫速殖子百分率的差异 ;探讨运用蝮蛇蛇毒作为辅助因子提高细胞培养用于弓形虫病病原学诊断的敏感性。方法　以尖吻蝮蛇蛇毒加入分孔培养的Hela细胞和THP - 1细胞中 ,分别用IFA和流式细胞仪进行检测 ,观察尖吻蝮蛇蛇毒对弓形虫速殖子侵入细胞的影响。结果　在相同的时间内 ,在尖吻蝮蛇蛇毒的作用下 ,Hela细胞和THP - 1细胞感染弓形虫速殖子的百分率在不同的时段均明显的提高。结论　尖吻蝮蛇蛇毒可以促进弓形虫速殖子侵入宿主细胞 ,在实际应中 ,可以作为一种辅助因子在检测标本时提高速殖子检出率 ,有利于缩短弓形虫的检出时间     

金丝桃素体外抗刚地弓形虫速殖子效果的观察

目的观察金丝桃素对体外培养的刚地弓形虫(Toxoplasma gondii)RH株速殖子的杀伤作用,探讨金丝桃素抗弓形虫的效果。方法将生理盐水(A组)和终浓度为5μg/ml(B组)、50μg/ml(C组)、500μg/ml(D组)的金丝桃素分别加入含弓形虫速殖子1×106个/孔的24孔培养板中,于2、4、6 h后收集虫体,台盼蓝染色检测速殖子着色率,吉氏染色观察速殖子形态和结构变化,透射电镜观察速殖子超微结构的变化,流式细胞仪检测相同处理的携带黄色荧光蛋白的弓形虫的存活情况。结果弓形虫速殖子经不同浓度金丝桃素作用2 h后,B组、C组和D组虫体的台盼蓝着色率分别为(11.0±3.6)%、(25.0±6.3)%和(40.0±2.7)%,D组着色率高于C组和B组(P0.01),3组均高于A组(6.0±3.0)%,但B组和A组间差异无统计学意义(P0.05)。作用4 h后,D组着色率为(97.0±2.0)%,高于C组(30.0±7.2)%、B组(20.0±3.0)%和A组(10.0±1.0)%(P0.01);作用6 h后,D组着色率为(98.0±1.7)%,亦高于C组(42.7±5.5)%、B组(34.0±6.6)%和A组(19.3±4.9)%,两两比较,各组间差异均有统计学意义(P0.01)。吉氏染色观察发现,随时间的延长和剂量的增高,虫体两端逐渐变圆、肿胀、坏死。透射电镜观察结果显示,随金丝桃素作用时间延长,虫体逐渐肿胀,胞膜与基质间出现明显空隙,虫体内空泡增多、变大,胞膜破裂,内部结构溶解。流式细胞仪检测结果表明,弓形虫速殖子存活率在金丝桃素作用后的各时间段内与对照组间差异有统计学意义(P0.01);金丝桃素作用2 h后,D组无弓形虫存活,C组弓形虫存活率为(7.9±1.9)%,低于B组(38.1±5.5)%和A组(81.8±6.0)%(P0.01);作用4 h后,C组无弓形虫存活;B组在作用4 h和6 h后,弓形虫存活率分别为(14.3±7.9)%和(1.4±1.8)%,均低于A组的(73.8±11.3)%和(64.1±14.4)%(P0.01)。结论金丝桃素具有较强的体外抗弓形虫速殖子效果,且随剂量增高和作用时间延长抗虫效果更明显。     

刚地弓形虫RH株速殖子协同VP-16体外杀伤小鼠结肠癌细胞ct26的作用

目的探讨刚地弓形虫RH株速殖子对小鼠结肠癌ct26细胞周期的影响及其协同细胞周期特异性抗癌药依托泊甙(VP-16)对ct26细胞的杀伤作用。方法建立弓形虫RH株速殖子与小鼠结肠癌ct26细胞不同比例的共培养模型(虫/细胞比例分别为2:1、4:1、8:1、16:1),流式细胞仪检测24h细胞周期改变;以虫细胞比例2:1预先感染ct26细胞24h,CCK-8比色法观察不同浓度VP-16对细胞增殖抑制的改变;荧光显微镜观察VP-16(40μg/mL)对感染虫体细胞的形态学改变。台盼蓝染色观察不同浓度VP-16分别作用弓形虫RH株速殖子悬液(1×106/mL)2h、4h、12h、24h的虫体存活度改变。琼脂糖凝胶电泳观察VP-16(20μg/mL)对虫体DNA影响。结果流式细胞仪检测发现各浓度弓形虫速殖子均可明显改变ct26细胞24h周期分布,使G0/G1期细胞百分比下降,G2/M期百分比显著升高(P0.01)。2×106个速殖子感染细胞24h可使S期和G2期细胞百分比共同增长14.92%。CCK-8比色法检测结果显示虫细胞比例2:1预先感染ct26细胞24h可有效增强各浓度VP-16对ct26细胞的杀伤作用;Hochest33258染色结合荧光显微镜观察到虫体胞质寄生形态及VP-16诱导感染细胞凋亡典型形态;10μg/mL及更高浓度VP-16作用弓形虫4h以上均可使虫体全部死亡;琼脂糖凝胶电泳发现VP-16(20μg/mL)可使弓形虫速殖子DNA呈现典型的云梯状条带。结论弓形虫RH株速殖子可使小鼠结肠癌ct26细胞发生G2/M期,可有效增强细胞周期特异性抗癌药VP-16的抗癌作用,且弓形虫增殖亦明显受抑并发生凋亡。     

弓形虫入侵的宿主细胞骨架重塑触发线粒体重新分布北大核心CSCD

目的探讨宿主细胞骨架重塑在弓形虫侵入HFF细胞以及触发细胞线粒体重新分布中的作用。方法体外培养HFF细胞,预先用1μg/mL细胞松驰素D(CD)处理30min,接种弓形虫速殖子分别培养1h和20h,Western blotting检测弓形虫表面抗原(surface antigen,SAG)1蛋白表达。同时用MitoTrackerRed CMXRos荧光探针标记细胞线粒体,激光共聚焦显微镜下观察HFF细胞线粒体在弓形虫侵入前后和CD处理前后聚集和分布情况。结果感染后1h,CD处理组HFF细胞内虫体量与CD未处理组差异不大,20h时CD未处理组细胞内虫体量显著多于CD处理组。此时可见HFF细胞线粒体明显聚集成明亮点状且分布于纳虫泡周围,而未感染组细胞线粒体未见明显聚集分布。CD可以显著抑制弓形虫侵入HFF细胞后引起的线粒体聚集。结论触发宿主细胞骨架重塑是弓形虫侵入宿主细胞并引起细胞线粒体向纳虫泡聚集所必须的。     

刚地弓形虫速殖子与鼠星形胶质细胞体外共培养的实验观察

原代培养星形胶质细胞,待其长至培养皿的80%时,将星形胶质细胞与弓形虫速殖子(细胞与虫体的比例为1∶10)共培养0～72h,吉氏染色观察星形胶质细胞和弓形虫速殖子的变化。共培养1h,此时定为0点,再培养0～48h,单磺酰戊二胺染色在荧光显微镜下观察星形胶质细胞内自噬囊泡的变化。结果发现共培养1h时,弓形虫速殖子开始进入星形胶质细胞,并出现荧光颗粒;8h星形胶质细胞内荧光颗粒最多;12h后显著减少,星形胶质细胞开始被破坏;48h星形胶质细胞内荧光颗粒消失;72h大部分星形胶质细胞已被涨破,留下大量增殖的弓形虫速殖子。说明自噬在弓形虫速殖子体外感染星形胶质细胞的过程中是受到抑制的。     

《热带病与寄生虫学》杂志2006年第4卷总目次

述评食源性寄生虫病不容忽视.····...··.....……卢思奇(125)论著弓形虫微线体分泌蛋白飞Mlclo基因的克隆和序列分析.................……，……张松涛汪学龙沈继龙(l)丝裂原蛋白激酶抑制剂U0126对刚地弓形虫侵人宿主细胞的影响杨小迪.............……，.陈兴智孙新等(     

刚地弓形虫细胞培养的研究Ⅰ．磷脂酶A2对成囊株速殖子侵入细胞的影响

已知多种酶和细胞因子对弓形虫侵入细胞有促进或抑制作用。本研究旨在探讨利用对弓形虫侵入细胞有促进作用的因子提高以细胞培养作病原诊断的敏感性。以激素弱化的小鼠接种成囊株包囊获得速殖子，以单核细胞ＴＨＰ－１细胞系的培养系统定量接种速殖子并加入源自蛇毒的磷脂酶Ａ２（ＰＬＡ２），以免疫荧光法和流式细胞仪检测细胞感染率。结果表明ＰＬＡ２对弱毒株速殖子侵入细胞有明显促进作用，在４株成囊株培养不同时间的检测，其感染细胞比率均比对照有显著提高。     

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.