零下非结冰保存L-02细胞的效果及与细胞凋亡的关系

目的 采用UW液零下非结冰(-0.8℃)保存生物人工肝用L-02细胞,与常规低温(4℃及0℃)比较,探索零下非结冰保存肝细胞的效果以及同细胞凋亡的关系.方法 制备好的UW液保存的L-02细胞悬液分为3组:-0.8℃组(零下非结冰组),0℃组(0℃非结冰组),4℃组(对照组).低温保存24 h、48 h及72 h后,分别测定细胞存活率及凋亡率(流式细胞术)、LDH释放、尿素合成功能、白蛋白分泌功能.结果 零下非结冰(-0.8℃)较常规低温(0℃及4℃)明显提高了低温保存L-02细胞的存活率[72 h:-0.8℃(70.17±2.82)% vs 4℃(60.05±3.17)%],降低了细胞凋亡率[72 h:-0.8℃(5.73±1.68)% vs 4℃(9.20±2.35)%].零下非结冰明显抑制了LDH的释放[72 h:-0.8℃(113.88±5.64)U/L vs 4℃(170.47±11.80)U/L],更好地维持了L-02细胞的尿素合成功能[72 h:-0.8℃(1.01±0.14)mmol/L vs 4℃(0.66±0.09)mmol/L]及白蛋白分泌功能[72 h:-0.8℃(9.04±0.53)μg/ml vs 4℃(7.70±0.52)μg/ml].结论 与0℃及4℃相比,零下非结冰(-0.8℃)可明显提高L-02细胞存活率,降低低温损伤引起的细胞凋亡,有效的保护肝细胞尿素合成功能和白蛋白分泌功能.     

零下非结冰UW液、Celsior液和HTK液保存C3A细胞的效果比较

目的 比较UW液、Celsior液和HTK液零下非结冰(-0.8℃)保存生物人工肝用C3A细胞的效果.方法 制备好的C3A细胞悬液分以下3组:UW液保存组(UW液组);Celsior液保存组(CS液组);HTK液保存组(HTK液组).各组细胞于-0.8℃低温保存72 h后,分别测定细胞存活率及死亡率(流式细胞术)、LDH释放、尿素合成功能及白蛋白分泌功能.结果 UW液及Celsior液比HTK液显著提高了零下非结冰保存72 h的C3A细胞的存活率[(84.34±4.25)%,(83.53±3.73)% vs (75.65±3.01)%,P<0.001]和细胞内ATP含量[(5.54±1.44)μg/106 个细胞,(5.51±1.31)μg/106个细胞 vs (2.75±1.03)μg/106 个细胞,P<0.001];抑制了LDH释放(P<0.001);更好地维持了细胞尿素合成功能[(0.63±0.10)mmol/L,(0.62±0.06)mmol/L vs (0.39±0.04)mmol/L,P<0.001]和白蛋白分泌功能[(1.99±0.38)g/L,(1.96±0.24)g/L vs (1.50±0.18)g/L,P<0.05].UW液与Celsior液零下非结冰保存C3A细胞的效果无差异.结论 同HTK液相比,使用UW液或者Celsior液零下非结冰(-0.8℃)保存C3A细胞可以明显地提高复温后细胞存活率和细胞内ATP含量,降低低温损伤引起的LDH释放,有效地保护肝细胞尿素合成功能和白蛋白分泌功能.UW液同Celsior液零下非结冰保存C3A细胞的效果无差异,保存时间不宜超过72 h.     

生物人工肝用C3A细胞在零下非结冰时的保存

目的:探讨零下非结冰保存肝细胞的效果及其与细胞凋亡的关系.方法:UW液保存的C3A细胞悬液分为3组:-0.8℃组(零下非结冰组),0℃组(0℃非结冰组),4℃组(对照组).低温保存24、48及72 h后,采用流式细胞术分别测定细胞存活率及凋亡率,同时测定LDH、乳酸释放,细胞内ATP含量、尿素合成功能及白蛋白分泌功能,同时观察细胞形态.结果:零下非结冰组较0℃非结冰组及对照组明显提高了低温保存72 h的C3A细胞的存活率(86.49%±2.80%vs 81.50%±2.83%.77.83%±3.40%,均P<0.05),降低了细胞凋亡率(1.26%±0.84%vs 5.34%±1.20%,9.16%±1.99%,均P<0.05);明显抑制了低温保存72 h乳酸以及LDH释放(乳酸:10.38μg/106 Cells±1.40 μg/106 cells vs 12.02 μg/106cells±1.64μg/106 cells,17.41μg/10°cells±2.40μg/106cells:LDH:80.10 U/L±11.10 U/L vs 12004U/L±14.32 U/L,148.98 U/L±15.37 U/L,均P<0.05);更好地维持了低温保存72 h C3A细胞内ATP含量、尿素合成功能、及白蛋白分泌功能(均P<0.05).形态上,零下非结冰组保存细胞死亡比例少,细胞接触良好,未见对照组及0℃非结冰组细胞膜周围出现"毛刺"样改变及细胞内的"空泡样"改变.结论:在零下非结冰条件下保存肝细胞,建立一个"血库样"(ready to use)肝细胞库,可以有效促进生物人工肝的发展.     

抑制内质网应激关键信号通路蛋白eIF2α去磷酸化与肝细胞增殖及c-Met表达的关系

目的 探讨抑制内质网应激（ERS）关键信号通路蛋白真核细胞起始因子2α(eIF2α)去磷酸化与肝细胞增殖及促肝细胞生长因子受体（c-Met）表达的关系。方法 将人肝细胞株L02分为对照组和胡萝卜素（TG）组，TG组加入含内质网钙平衡抑制剂毒胡萝卜素（TG）的RPMI1640培养基，诱导构建细胞ERS模型；对照组仅加入RPMI1640培养基，培养48 h。采用CCK-8检测细胞活力，Western blotting法检测细胞ESR相关蛋白p-eIF2α、ERAD通路相关蛋白HRD1、XBP1以及c-Met蛋白表达。另取L02细胞，分为TG组和Salubrinal+TG组，Salubrinal+TG组给予eIF2α去磷酸化抑制剂Salubrinal预处理2 h，然后加入1μmol/L TG培养48 h;TG组加入1μmol/L TG培养48 h。采用CCK-8检测细胞活力，Western blotting法检测细胞eIF2α、p-eIF2α、HRD1、XBP1、c-Met蛋白表达。结果 与对照组相比，TG组p-eIF2α、HRD1表达水平升高，TG组ERS相关蛋白p-eIF2α表达上调，E...     

SMO保存液对犬肾低温保存期间氧自由基变化的研究

建立犬离体肾脏单纯低温保存模型，对照组供肾灌注和保存用HTK保存液和UW保存液，实验组用自制SMO保存液，分别在犬肾保存24、48、72h切取肾皮质标本，测定皮质丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性。结果显示，实验组SOD活性48h明显高于对照组，MDA含量各时点均显著低于对照组（P均〈0．05）。实验组各时点SOD活性和MDA含量与UW液组比较均无统计学差异。提示SMO液在减轻氧自由基损伤方面优于HTK液，与UW液相当。     

肝移植大鼠肝窦内皮细胞细胞凋亡与移植肝肝细胞损害的关系

目的探讨冷保存肝移植大鼠肝窦内皮细胞（SEC）细胞凋亡与移植肝肝细胞损害的关系。方法雄性SD大鼠随机分为假手术组（n=6）、UW1h肝移植组（11=48）、UW12h肝移植组（n=48）。参照Kamada的方法行原位肝移植（OLT）。观察大鼠I68h存活率。分别于术后不同时相点采取血液及组织标本，检测血清丙氨酸氨基转移酶（ALT）及透明质酸（HA）水平；TUNEL法检测SEC凋亡，透射电镜观察细胞凋亡的形态学改变。结果UW12h组168h存活率为50％，显著低于UW1h组（F=6．39，P〈0．05）。UW12h组肝移植后血清ALT、HA水平明显高于UW1h组（F=3．99，P〈0．05；F=12．43，P〈0．05），两组大鼠ALT水平均于术后6h达高峰。UW12h组SEC凋亡指数（AI）明显高于UW1h组和假手术组（F值分别为63．58和86．58，P值均〈0．01），两组大鼠SEC的AI也于术后6h达高峰，与血中丙氨酸氨基转移酶（ALT）的高峰时相点一致。且两组大鼠SEC的AI均与ALT水平呈显著正相关（，值分别为1．0和0．962，P〈0．05）。结论SEC凋亡程度与移植肝肝细胞损害呈显著正相关，SEC凋亡是冷保存再灌注损伤的关锋环节。     

新型肾脏保存液低温保存后犬肾移植效果评价

目的建立犬自体肾移植模型，评价经新型肾脏保存液（HCA-Ⅱ）低温保存后的犬肾移植效果。方法60只杂种犬随机分为A（H-CA液，保存48h）、B（HCA—I液，保存48h）、C（UW液，保存48h）、D（H-CA液，保存72h）、E（HCA-Ⅱ液，保存72h）、F（UW液，保存72h）等六组。切取左肾，灌洗并放入该保存液保存。左肾动脉与右侧髂外动脉端端吻合，静脉与右侧髂外静脉端侧吻合，输尿管与膀胱抗反流吻合，同时切除右侧肾脏。检测移植前后肾功能指标。结果手术成功率95％，HCA-Ⅱ液各组术后血肌酐的峰值均低于H-CA液组，且血肌酐恢复正常的时间也较H—CA液组早（P〈0．05）；以上各指标均与UW液组无明显差别（P〈0．05）。结论HCA-Ⅱ肾脏保存液对供肾的保存效果明显好于H—CA保存液，可安全保存犬肾72h。     

改良HCA肾保存液对肾脏低温保存效果的动物实验研究

目的:探讨改良高渗枸橼酸盐嘌呤肾保存液(HCA)对离体肾脏的低温保存效果。方法:将36只雄性草犬随机分为6组，分别用改良HCA(mHCA)液、Uw液(uNIVERSITY wISCOSIN器官保存液)、HCA液对其离体肾脏进行保存，并于24、48小时后观察肾脏皮质线粒体呼吸控制率(RCR)、细胞内ATP含量、pH值以及丙二醛(MDA)含量；比较肾脏移植前后血肌酐变化。结果肾脏保存24小时后，各项检测指标与移植前比较无明显差异，48小时后，mHCA液组与UW液组血肌酐差异无显著性，但均明显低于HCA液组；肾脏细胞RCR、ATP含量，mHCA液组与UW液组差异无显著性，但均高于HCA液组，mHCA液组与UW液组MDA明显降低，mHCA液组pH最高。结论:mHCA液能明显提高肾脏的低温保存效果，延长肾脏保存时间。     

树肝细胞的分离培养及其意义

本文采用体外简易两步灌流法和激素限定RPMI1640培养液对树鼯肝细胞进行分离与培养。结果显示：分离所获肝细胞总数约为1．8×108个,细胞存活率达94％,肝细胞贴壁伸展能力强,培养时间较长,2周内保持良好的形态特征。认为分离培养的树鼯肝细胞可用作肝病研究的体外细胞模型,有助于人肝炎病毒感染的进一步研究。     

左旋泊尼汀对离体大鼠心脏保存效果的影响

将25只成年雄性Wistar大鼠（体重350～400g）随机分为A组（对照组）及B、C、D、E组，每组5只，经麻醉和抗凝后，快速取下心脏。A组心脏悬挂在Langendorff灌注模型上K-H液恒压灌注，37℃下测定血流动力学指标；B、C、D、E组分别经主动脉灌注4℃ HTK液、UW液、HTK液＋左旋泊尼汀、UW液＋左旋泊尼汀心肌保存液，置于4℃相应的心肌保存液中保存6h；重新复温复灌。再次测定血流动力学指标，留取标本分别测定心肌含水量、心肌酶漏出量、ATP含量变化。结果 D组及E组平均左室发展压（LVDP）与B、C组比较显著升高，磷酸肌酸激酶同工酶（CK—MB）漏出量明显减少，心肌ATP含量明显增多。认为HTK液和UW液中加入左旋泊尼汀能改善离体鼠心脏的低温保存效果。     

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.