恒河猴胰岛的半自动分离和纯化

目的探讨恒河猴胰岛分离和纯化的技术方法。方法采用自制的半自动胰腺消化分离装置,对3只成年恒河猴胰腺进行消化分离,用非连续密度梯度高渗枸橼酸盐嘌呤溶液（HCA液）-蔗聚糖液（Ficoll液）纯化恒河猴胰岛,通过双硫腙染色在倒置显微镜计数胰岛的数量和纯度,用胰岛素释放试验检测胰岛的分泌功能。分离和纯化结果采用配对t检验进行统计分析。结果消化后平均每个胰腺可获得（101420±12054）胰岛当量（IEQ）,纯化后平均每个胰腺可获得（71480±8054）IEQ,平均每克恒河猴胰腺组织可获得（2310±252）IEQ,纯化后胰岛平均纯度为（89．8±8．7）％,活率为（93．1±2．8）％。纯化后的胰岛对葡萄糖刺激反应良好,高糖（16．7mmol／L）时胰岛素的释放量为低糖（3．3mmol／L）时的5．9倍（t=45．2,P〈0．01）。分离胰岛在移植到糖尿病大鼠肾包膜下后能够影响降低血糖至支持水平。结论通过采用半自动胰腺消化分离装置,胶原酶P灌注消化及非连续密度梯度法纯化,能获取较高纯度及生物活性良好的恒河猴胰岛。     

糖尿病大鼠胰岛β细胞Fox01表达对细胞凋亡的影响

以高糖高脂饲料及链脲佐菌素诱导SD大鼠建立2型糖尿病模型,结果发现叉头转录因子(Fox01)[细胞核内(15.00+1.15 vs 6.45±0.62)%,P<0.05]、半胱天冬蛋白酶3(caspase-3)[(23.73±1.48vs 6.30±2.20)%,P<0.01]在糖尿病大鼠胰岛β细胞的表达和β细胞凋亡率[(22.29±1.84 vs 6.25±2.42)%,P<0.01]均高于正常大鼠;并且Fox01(核内)与caspase-3高表达的胰岛细胞正是发生凋亡的胰岛细胞.因此,Fox01可能参与2型糖尿病胰岛β细胞凋亡的调控.     

一种简便实用的大鼠Kupffer细胞的分离与鉴定方法

目的：研究一种简便实用的大鼠Kupffer细胞（KCs）的分离与鉴定方法.方法：原位两步灌流法对大鼠肝脏进行冲洗消化;利用Percoll液进行不连续密度梯度离心分离KCs;;选择性贴壁纯化KCs;台盼蓝染色法鉴定细胞存活率;吞噬实验鉴定细胞功能;ED1单克隆抗体免疫荧光细胞化学鉴定KCs;显微镜下观察KCs形态变化.结果：获取的KCs数量为（2.41±0.32）×107/只,其中活细胞数量占（92.3±2.12）％;吞噬实验显示（95.2±2.58）％的细胞内存在碳素颗粒;免疫荧光化学检测证明KCs纯度为（96.3±1.46）％;在显微镜下观察KCs形态,36h细胞形态变得不规则,3d后呈星形或多角形,体外培养可以存活7～10d.结论：此种KCs分离方法操作相对简便,获取的细胞数量、活性功能、纯度等方面均能达到进一步的实验要求,值得推广.     

五指山小型猪胰岛对糖尿病大鼠的治疗作用

目的:探讨小型猪胰岛治疗糖尿病大鼠的作用和功效.方法:成年健康♂五指山小型猪和普通家猪作为胰岛供体.胰腺获取后经胶原酶消化后进行胰岛提取纯化,纯化后的胰岛通过门静脉移植到糖尿病SPF大鼠的肝内,术后每天肌注环孢菌素(20mg/kg)作为免疫抑制剂,并通过检测糖尿病大鼠的血糖变化和肝脏组织学检查来判定胰岛移植后的存活情况和纠正胰岛移植的效果.结果:五指山小型猪胰腺消化后胰岛产量为4608 IEQ/g±593 IEQ/g,纯化后为3820 IEQ/g±718 IEQ/g,纯度为85%,取自屠宰场猪胰岛产量为纯化前为3500 IEQ/g±625 IEQ/g,纯化后为2720 IEQ/g±435 IEQ/g,纯度为80%.两组84.6%糖尿病大鼠在移植术后第1天,血糖降至正常.维持时间:小型猪胰岛术后存活时间为3-5d(中位存活时间:4.5d),普通家猪胰岛存活时间为2-4d(中位存活时间:3.7d).经Kaplan-Meier分析,两组胰岛存活时间无差别.结论:封闭群五指山小型猪分离纯化后的胰岛产量高,功能良好,可以纠正糖尿病大鼠的高血糖,适合作为异种胰岛移植的理想供体.     

牛磺酸对大鼠胰岛活性和功能的保护作用

目的:探讨牛磺酸(taurine,Tau)在大鼠胰岛培养过程中对胰岛的保护作用及其机制.方法:实验Wistar大鼠分为2组,RPMI 1640组和牛磺酸组(Tau组).流式细胞仪检测Caspase-9和磷酸化Akt阳性细胞比例,观察牛磺酸对Caspase-9及Akt的影响.RT-PCR检测单纯1640组和Tau组胰岛细胞的TNF-α、IL-1β、NF-κB和HO-1基因的表达.结果:胰岛单纯用RPMI 1640培养1 wk后激活的Caspase-9与加入牛磺酸后相比有统计学意义(41.03%±4.46% vs 23.85%±3.09%,P<0.05).胰岛分离纯化后有明显的TNF-αmRNA、IL-1βmRNA、NF-κB mRNA、HO-1mRNA表达,随着培养时间的延长TNF-αmRNA、IL-1β mRNA、NF-κB mRNA表达逐渐减弱,HO-1 mRNA表达逐渐增强.加入牛磺酸后胰岛TNF-α mRNA、IL-1β mRNA、NF-κB mRNA表达明显减弱,在6 h,72 h及1 wk时(TNF-α:0.34±0.02.0.24±0.01,0.19±0.02;IL-1β:0.24±0.09,0.09±0.01,0.05±0.01;NF-κB:1.76±0.30,0.93±0.15,0.37±0.02)和单纯培养组(TNF-α:0.57±0.1,0.39±0.02,0.29±0.02;IL-1β:0.34±0.02,0.24±0.01,0.19±0.02;NF-κB:2.52±0.24.1.21±0.14,0.76±0.07)比较差异具有显著性(均P<0.05).HO-1mRNA的表达较单纯培养组明显增强且随着时间的延长表达更明显(3.74±0.10,4.33±0.29.5.28±0.29 vs 2.46±0.30,3.13±0.07,3.59±0.22;均P<0.05).结论:牛磺酸能抑制TNF-α、IL-1β和NF-κB转录并活化Akt,抑制细胞凋亡.     

大鼠骨髓间充质干细胞向类肝细胞体外诱导分化

目的:观察不同条件下骨髓间充质干细胞(MSCs)体外诱导分化为类肝细胞的差异.方法:Wistar大鼠24只随机均分为3组,分别为正常对照组、肝纤维化模型组、自拟中药干预组.采用CCl4乳剂皮下注射建立肝纤维化模型.造模成功后中药干预组采用丹金舒肝胶囊药液灌胃治疗.治疗结束后剖杀大鼠,留取大鼠肝脏标本,HE染色观察各组病理改变.采用密度梯度离心法和贴壁法分离各组大鼠的MSCs,经培养传代获得纯化的MSCs.各组纯化的MSCs采用HGF、FGF-4进行诱导培养.留取15、21、27 d细胞培养液进行白蛋白(Alb)、甲胎蛋白(AFP)检测;于27 d收集细胞爬片,进行糖原染色和CK-18免疫细胞化学染色.结果:3组15、21和27 d各MSCs诱导组AFP水平均高于MSCs非诱导组(P<0.01),其中21 d AFP水平最高(肝纤维化模型组:48.94±0.08 vs 9.90±0.09;中药干预组:49.86±0.29vs 8.69±0.62;正常对照组:38.65±0.33 vs9.04±0.11,均P<0.01);3组15 d各MSCs诱导组与MSCs非诱导组白蛋白水平无统计学意义:21 d、27 d各MSCs诱导组白蛋白水平均高于MSCs非诱导组(1.11±0.08 vs 0.32±0.00,1.25±0.04 vs 0.32±0.00,1.06±0.03 vs 0.33±0.00;1.52±0.02 vs 0.33±0.00,1.79±0.01vs 0.31±0.03,1.63±0.04 vs 0.32±0.01,均P<0.01),27 d最高.27 d各MSCs诱导组糖原染色阳性,免疫细胞化学染色CK-18均阳性,而MSCs非诱导组糖原染色、CK-18均阴性.从AFP、Alb水平综合比较,3组诱导效果发现自拟中药干预组优于其他2组.结论:HGF、FGF-4可在体外诱导实验性大鼠的MsCs分化为具有肝细胞样细胞表型和功能的类肝细胞:自体MSCs可以作为治疗临床重症肝病的一种细胞来源,而临床联合中药复方治疗可能会使MSCs体外诱导的效果更为理想.     

大鼠梗阻性胆管炎细胞免疫功能降低及中药锦红片的影响

目的:探讨梗阻性胆管炎大鼠细胞免疫功能降低的发生机制及清热通下中药锦红片的影响.方法:♂SD大鼠24只建立急性梗阻性胆管炎模型,随机分为模型组(n=8)、锦红片治疗组(n=8)和单纯胆管梗阻组(n=8),检测血浆IL-2,CD_3~ ,CD_4~ ,CD_8~ ,内毒素,胸腺指数,胸腺细胞凋亡指数及电镜下观察胸腺的超微结构及凋亡.结果:模型组IL-2,CD_3~ ,CD_4~ 和胸腺指数显著低于治疗组和单纯胆管梗阻组(IL-2:28.5±3.0 ng/L vs 33.9±3.6 ng/L,39.6±2.2 ng/L,P<0.05,P<0.01;CD_3~ :54.5%±5.5% vs 70.7%±4.8%,66.3%±7.1%,均P<0.01;CD_4~ :34.5%±8.3% vs 44.2%±3.3%,44.5%±4.2%,均P<0.01:胸腺指数:0.89±0.18 vs 1.10±0.13.1.12±0.24,均P<0.05),CD_8~ 3组间没有统计学差异,血浆内毒素和凋亡指数明显高于治疗组和梗阻组(内毒素:0.85±0.14 Eu/mL vs 0.53±0.10 EU/mL,0.49±0.11 EU/mL,均P<0.01;凋亡指数:25.7±5.1 vs 15.8±5.5.9.0±3.1.P<0.05,P<0.01),模型组胸腺可见较多典型的凋亡细胞,结果显示经中药干预治疗后,免疫功能、内毒素血症和胸腺细胞凋亡有所改善,接近单纯胆管梗阻组水平.结论:梗阻性胆管炎大鼠存在免疫功能降低,胸腺细胞异常凋亡.锦红片对维持免疫机能的稳定有积极的意义.     

高游离脂肪酸血症通过氧化应激导致胰岛β细胞功能受损

目的 研究高游离脂肪酸(FFA)血症对胰岛β细胞功能的影响.方法 正常雄性SD大鼠随机分为3组:对照组输注生理盐水,FFA组输注脂肪乳,氮乙酰半胱氨酸(NAC)干预组在输注脂肪乳的同时加用NAC.持续输液2～4天,行静脉葡萄糖耐最试验(IVGTY)和胰腺组织表面灌注试验评价在体及离体情况下胰岛β细胞胰岛素分泌功能.结果 胰腺组织表面灌注结果显示,FFA组脂肪乳输注2天,基础状态下的胰岛素分泌较对照组增加[(55.5±19.4 vs 27.4±6.7)mIU/L,P<0.01],而葡萄糖刺激的胰岛素分泌(GSIS)与对照组并无差别;延长脂肪乳输注到4天,高FFA刺激基础胰岛素分泌的作用消失,且GSIS明显抑制[(46.8±33.0 vs 214.7±27.4)mIU/L,P<0.05].IVG3T结果亦显示脂肪乳输注4天后胰岛素分泌功能受到抑制.但在脂肪乳输注的同时加用抗氧化剂NAC可使β细胞GSIS峰值部分恢复[(165.4±14.8)mIU/L,P<0.01].结论 循环中FFA水平增高可能通过激活氧化应激导致胰岛β细胞功能受损.     

内源性大麻素受体1在肥胖大鼠胰腺表达的实验研究

高脂饲料喂养20周的SD肥胖大鼠呈胰岛素抵抗特征,免疫荧光双标记显示内源性大麻素受体1(CBIR)主要表达于胰岛p细胞,免疫组化和实时定量PCR结果显示,高脂喂养组胰腺CBIR蛋白(6 002.8±855.5 vs 3 712.8±769.6,P<0.01)及mRNA(对照组的16.7倍,P<0.05)表达均高于对照组,提示肥胖时胰岛D细胞CB11R表达量上调可能导致胰岛素抵抗.     

针刺对结肠吻合术后Cajal间质细胞修复与再生的影响

目的:探讨针刺促进术后肠道动力恢复的机制.方法:30只SD大鼠随机分为空白组、模型组(行结肠吻合术)、针刺组.针刺组予每日针刺双侧足三针(足三里、三阴交、太冲),连续3d.观察大鼠排便情况,测量小肠推进率,观察结肠组织Cajal间质细胞(interstitial cells of Cajal,ICC)超微结构和胆碱能神经-ICC-平滑肌细胞网络结构.结果:针刺组术后首次排便时间较模型组提前,小肠推进率提高(2.00d±0.47d vs 2.50d±0.53d,66.30%±4.21% vs 46.33%±5.56%,P<0.05).与空白组比较,模型组ICC超微结构损伤明显,胆碱能神经-ICC-平滑肌细胞网络结构紊乱,ICC和小泡乙酰胆碱转移体(VAChT)阳性神经纤维数量明显减少(18.67±6.11 vs 32.33±5.51,18.67±3.79 vs 20.67±3.21,P<0.05)荧光强度减弱(35.00±9.54 vs 58.67±10.21,20.33±5.13 vs 34.67±6.81,P<0.05).而针刺组ICC超微结构损伤较模型组轻,网络样结构维持,ICC和VAChT阳性神经纤维的...     

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.