流体剪切力对5-氮杂胞苷诱导大鼠骨髓间充质干细胞向心肌样细胞分化的影响

目的观察5-氮杂胞苷诱导大鼠骨髓间充质干细胞向心肌样细胞分化,并探讨流体剪切力对诱导过程的影响。方法贴壁法从大鼠骨髓中分离骨髓间充质干细胞,进行纯化传代培养,取第4代骨髓间充质干细胞以3、5、10、15及20μmol/L 5-氮杂胞苷分别作用12、24及48 h,用免疫荧光法鉴定分化的心肌样细胞α-肌动蛋白表达率,10μmol/L作用24 h为最佳诱导浓度。通过建立流体剪切力模型,设立以下四组:不加载流体剪切力组、加载5 dyn/cm2流体剪切力组、加载15 dyn/cm2流体剪切力组和加载25 dyn/cm2流体剪切力组,作用24 h,倒置显微镜观察诱导后骨髓间充质干细胞形态的变化,4周后逆转录聚合酶链反应测定分化的心肌样细胞心肌肌钙蛋白ImRNA的表达。结果骨髓间充质干细胞随着传代逐渐变成梭形,5-氮杂胞苷诱导后骨髓间充质干细胞胞体逐渐增大并伸出细长突起,部分相邻细胞的突起连接成网,形态学上表现出向心肌细胞方向转化的特征。免疫荧光显示α-肌动蛋白表达阳性。经过流体剪切力作用细胞后,心肌肌钙蛋白I的表达增高,阳性条带均比未进行力学刺激的细胞表达明显,以15 dyn/cm2剪切力最明显。但是25 dyn/cm2剪切力作用结果并没有随着力的增大而增大。结论 5-氮杂胞苷可以诱导大鼠骨髓间充质干细胞向心肌样细胞分化,5-氮杂胞苷可联合剪应力诱导大鼠骨髓间充质干细胞向心肌样细胞分化,且诱导效果优于单独使用5-氮杂胞苷。     

大鼠骨髓间充质干细胞体外定向诱导分化为心肌样细胞的实验研究

目的研究大鼠骨髓间充质干细胞的体外培养及向心肌样细胞转化的条件。方法获取成年大鼠胫骨干骨髓,采用贴壁法进行间充质干细胞的培养、传代,观察经5-氮胞苷诱导后骨髓间充质干细胞的生长和分化。结果大鼠骨髓基质细胞贴壁呈集落生长,5-氮胞苷诱导骨髓基质细胞转化为心肌样细胞。结论骨髓基质细胞能够在体外被诱导分化为心肌样细胞,为自体心肌细胞移植提供了一种良好的来源。     

骨髓间充质干细胞体外定向诱导分化为心肌样细胞的实验研究

目的 研究大鼠骨髓间充质干细胞的体外培养及向心肌样细胞转化的条件。方法 获取成年大鼠胫骨干骨髓，采用贴壁法进行间充质干细胞的培养、传代，观察经5-氮胞苷诱导后骨髓间充质干细胞的生长和分化。结果 大鼠骨髓基质细胞贴壁旱集落生长，5-氮胞苷诱导骨髓基质细胞转化为心肌样细胞，结论 骨髓基质细胞能够在体外被诱导分化为心肌样细胞，为自体心肌细胞移植提供了一种良好的来源。     

骨髓间(充)质干细胞体外定向诱导心肌样细胞超微结构特征研究

目的：研究兔骨髓间（充）质干细胞（mesenchymal stem cell，MSC）经5～氮杂胞苷（5-azacytidine，5-aza）诱导在体外定向分化的心肌样细胞超微结构特征。方法：取兔髂骨骨髓，分离并培养骨髓MSC，用5～aza定向诱导向心肌样细胞分化。以相差显微镜、透射电镜观察心肌样钏胞形态学变化及超微结构特征。结果：5-aza诱导后，部分细胞体积增大，呈“捧状”或“珠状”结构，有肌管样结构形成，透射电镜下见有肌丝、心房颗粒及线粒体等心肌样细胞超微等结构。结论：经5-氮杂胞苷诱导分化的骨髓间（充）质干细胞具有心肌样细胞超微结构特征．     

犬骨髓间充质干细胞体外分离培养、诱导分化为心肌细胞实验研究

目的 观察犬骨髓间充质干细胞(BMSCs)分离体外培养的生长特性及诱导分化为心肌细胞.方法 利用骨髓穿刺,Percoll分离液密度梯度离心法分离穿刺骨髓细胞继而通过贴壁筛选纯化骨髓间充质干细胞进行接种培养、体外扩增经5-氮胞苷(5-azacytidine)诱导分化为心肌样细胞.倒置显微镜观察诱导前后细胞形态变化,流式细胞分析及免疫化学进行相关免疫抗原检测.结果 分离细胞流式细胞分析细胞表面分子抗原CD105(间充质干细胞抗原)阳性,CD34(造血干细胞表面抗原)阴性,CD31(内皮主细胞表面抗原)阴性.利用5-溴-2-尿嘧啶脱氧核苷(Brdu)进行细胞增殖标记,结果增殖细胞核标记率为(77.2±6.1)%.传4代后,经5-aza诱导,细胞形态发生改变,由梭形变为"心肌样",同时有自发性细胞搏动和"肌管"样结构.免疫化学检测分化细胞肌球蛋白重链(MHC)、心肌特异性肌钙蛋白I(cTnI)、连接蛋白-43(Cx-43)等心肌细胞特异蛋白表达阳性.结论 BMSCs分离纯化在体外培养条件下可以实现大量扩增,同时维持其未分化状态.与5-氮杂苷(5-azacytidine)共培养,可以诱导其分化为心肌样细胞.BMSCs是细胞移植"心肌再生"治疗缺血性心脏病的理想种子细胞来源.     

骨髓间充质干细胞与心肌修复

骨髓间充质干细胞 (MSC)具有多向分化潜能 ,在一定的诱导条件下可以分化为多种细胞。 5 氮胞苷可以诱导离体MSC出现心肌表型。MSC移植研究表明 ,在心肌组织微环境中 ,MSC可向心肌分化并与宿主心肌功能整合 ,从而修复损伤坏死心肌、改善心脏功能     

诱导骨髓间充质干细胞向心肌细胞分化及相关信号通路的研究进展

骨髓间充质干细胞具有自我更新、增殖及多向分化的特点,在体外可通过5-氮胞苷、共培养等多种途径诱导分化为心肌样细胞。相关研究表明,Notch、W nt等信号通路在这一过程中发挥着重要作用。     

骨髓间质干细胞心肌分化过程中细胞内钙离子浓度的变化

目的研究骨髓间质干细胞心肌分化前、后钙离子浓度变化。方法用5-杂氮胞苷(5-azacytidine)体外诱导猪骨髓间质干细胞使之向心肌分化；ELASA法测定分化前、后细胞内心肌肌钙蛋白Ⅰ(cTnⅠ)变化；应用离子图像分析系统测定分化前、后细胞内钙离子浓度变化。结果(1)经5-杂氮胞苷诱导后细胞形态发生变化；(2)诱导后第3周起细胞内cTnⅠ明显增高；(3)诱导组细胞内钙离子浓度较对照组高，且诱导前后细胞内钙释放机制不同。结论骨髓间质干细胞体外经5-杂氮胞苷诱导后可具有心肌细胞的某些特性，这一过程与钙信号有关。     

人脐血间充质干细胞诱导分化成心肌细胞的研究

目的探讨体外脐血间充质干细胞诱导分化成心肌细胞的可行性和最佳方法。方法收集获知情同意的健康产妇脐血细胞,分离单个核细胞,从中进一步分离间充质干细胞,传代培养至第3代,应用免疫荧光流式细胞仪标记间充质干细胞特异性抗原CD34、CD44和CD90。5-氮胞苷诱导分化4周后,免疫组织化学染色和RT-PCR法分别检测心肌细胞标记物肌钙蛋白Ⅰ、转录因子GATA 4和β-肌球蛋白重链。结果在第3代细胞中,可检测到CD44、CD90的表达,未检测到CD34的表达。脐血间充质干细胞经5-氮胞苷诱导分化后,呈现成纤维细胞样形态和克隆增殖特点。免疫组织化学染色和RT-PCR可检测到肌钙蛋白Ⅰ,GATA 4和β-肌球蛋白重链的表达。结论脐血间充质干细胞能够被诱导分化成心肌样细胞,可成为干细胞移植的细胞来源。     

5-氮胞苷体外诱导骨髓间充质干细胞向心肌样细胞的分化

目的: 研究5-氮胞苷（5-Aza）对培养人骨髓间充质干细胞（MSC）的作用,并对分化后的心肌样细胞进行鉴定。方法: 采用密度梯度离心法分离到骨髓单个核细胞（MB-MNC）,用含200 ml/L胎牛血清的低糖型DMEM培养液进行培养。采用差速贴壁法纯化MSC,用流式细胞仪检测细胞表面抗原。以5-Aza诱导第3代MSC 24 h后继续培养。培养4周,用免疫细胞化学染色法检测肌系标记抗原:α-肌动蛋白（α-actin）及心肌细胞特异性标记抗原:肌钙蛋白T（cTnT）;在透射电镜下观察细胞的超微结构。结果: MSC经5-Aza诱导分化后,可表达α-actin和cTnT,未经诱导的同培养天数的MSC中均未见表达。透射电镜可观察到肌丝等心肌细胞的特异性结构。结论: 5-Aza可诱导MSC分化为心肌样细胞。     

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.     

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.     

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.     

Language of CTO interventions – Focus on hardware

The knowledge of variety of chronic total occlusion (CTO) hardware and the ability to use them represents the key to success of any CTO interventions. However, the multiplicity of CTO hardware and their physical character and the terminology used by experts create confusion in the mind of an average interventional cardiologist, particularly a beginner in this field. This knowledge is available but is scattered. We aim to classify and compare the currently used devices based on their properties focusing on how physical character of each device can be utilized in a specific situation, thus clarifying and simplifying the technical discourse.