内皮祖细胞移植对糖尿病兔下肢缺血的病理及血管内皮生长因子的影响

目的探讨内皮祖细胞(EPC)移植治疗糖尿病兔下肢缺血的病理及缺血肌肉中血管内皮生长因子(VEGF)的变化。方法2006年1月至9月在哈尔滨医科大学附属第二医院内分泌科将30只健康日本大耳白兔随机分为3组,即糖尿病磷酸盐缓冲液(PBS)对照组(A组)8只、糖尿病内皮祖细胞移植治疗组(B组)14只、正常血糖内皮祖细胞移植治疗组(C组)8只。骨髓来源的内皮祖细胞经体外扩增培养7d后,通过肌内注射进行细胞移植,用病理改变及肌浆中VEGF的变化评价治疗效果。结果EPC移植后14d,病理示3组毛细血管密度分别为(9.29±1.63)个/视野、(12.60±2.16)个/视野、(12.51±1.56)个/视野;血管数/肌束数分别为0.66±0.05,0.83±0.11,0.90±0.13;肌浆中VEGF质量分数[每克肌肉中VEGF因子的质量(ng)]分别为0.22±0.05,0.30±0.07,0.31±0.08;糖尿病内皮祖细胞移植治疗组与糖尿病PBS对照组比较,毛细血管密度、血管数/肌束数、VEGF值差异均有显著性意义,P<0.05。结论EPC移植能有效治疗糖尿病兔下肢缺血。     

内皮祖细胞移植治疗糖尿病兔下肢缺血的影像学改变

目的探讨内皮祖细胞（EPC）移植治疗糖尿病兔模型下肢缺血的疗效。方法将日本大耳白兔随机分3组：糖尿病PBS对照组（A组）、糖尿病内皮祖细胞移植治疗组（B组）、正常血糖内皮祖细胞移植治疗组（C组）。骨髓来源的内皮祖细胞经体外扩增培养7d后,通过肌内注射进行细胞移植。结果EPC移植后14d,彩超示B组兔缺血侧／正常侧下肢胫前动脉收缩期峰值流速比值明显增加,动脉造影示该组缺血侧下肢动脉显影血管数多于对照组（P〈0．05）。结论EPC移植能有效治疗糖尿病兔模型下肢缺血。     

内皮祖细胞和单个核细胞移植对糖尿病大鼠血管病变疗效的比较

目的比较内皮祖细胞（EPC）和单个核细胞（MNC）移植改善糖尿病大鼠后肢血管病变的疗效。方法将大鼠EPC和外周血MNC标记后分别植入糖尿病大鼠缺血后肢。对照组仅植入PBS溶液。ELISA法检测VEGF的含量，免疫组化法计数毛细血管数目。结果EPC和MNC均能在缺血后肢分化为毛细血管。移植后EPC组和MNC组大鼠缺血肢体局部VEGF含量高于对照组，毛细血管数目也高于对照组。但EPC组和MNC组VEGF含量和毛细血管数目无明显差异。结论移植EPC及含有相当数量EPC的MNC对糖尿病大鼠后肢血管病变的改善作用相似。EPC可能在外周血单个核细胞移植中发挥主要作用。     

脂蛋白（a）对内皮祖细胞向缺血组织归巢的影响

目的　脂蛋白（a）　［Lp（a）］水平增高是冠状动脉和周围血管疾病的独立危险因素。本研究拟在下肢缺血小鼠模型中观察Lp（a）对内皮祖细胞（EPC）向缺血组织归巢的影响并初步探讨其机制。方法　从小鼠骨髓中分离诱导培养EPC。EPC经磷酸缓冲盐溶液（PBS）或Lp（a）　（20　mg/L）处理12　h后,分别移植入下肢缺血小鼠,以荧光显微镜检测EPC归巢到缺血组织和血管发生能力。同时,EPC经PBS或Lp（a）　处理后,以荧光显微镜、Western　blot检测Lp（a）对EPC黏附能力及相关基因表达的影响。结果　整体实验发现,植入对照的EPC（PBS-EPC）后,下肢缺血区显示有大量归巢的EPC及血管腔样结构。与此相反,植入Lp（a）处理的EPC［Lp（a）-EPC］后,下肢缺血区仅显示有极少量归巢的EPC及毛细血管腔样结构。体外实验显示,对照组EPC黏附性能强,P-选择素糖蛋白配体1（PSGL-1）、CXCR4蛋白表达丰富。与对照组相比,Lp（a）减弱EPC的黏附能力及下调PSGL-1和CXCR4蛋白表达。结论　Lp（a）抑制EPC归巢到下肢缺血组织,其机制可能与下调EPC的PSGL-1、CXCR4表达及降低EPC黏附性能有关。     

丹红注射液改善糖尿病小鼠下肢缺血血管新生的研究

目的观察丹红注射液对糖尿病小鼠下肢缺血后血管新生的影响。方法 40只Balb/c小鼠,其中10只为正常对照组,30只采用链脲霉素腹腔注射联合左下肢股动脉结扎分离术制备糖尿病下肢缺血模型。模型小鼠中随机选取10只腹腔注射PBS作为Control组,10只腹腔注射丹红注射液作为DH组(2μL/g,连续注射7d)。采用激光多普勒血流灌注扫描仪观察血流灌注情况、动脉X线造影观察血管管样新生情况,及采集下肢肌肉组织匀浆,通过ELISA检测血管内皮生长因子(VEGF)表达量。结果与PBS对照组相比,丹红注射液显著增加糖尿病小鼠下肢缺血血流灌注、管样新生,并提高硫化氢浓度,VEGF表达量显著增高(P0.05)。结论丹红注射液能改善糖尿病小鼠下肢缺血的血管新生,其可能机制是通过VEGF信号通路发挥作用。     

猪心肌缺血再灌注损伤区自体骨髓单个核细胞和外周血内皮祖细胞移植后细胞分化探讨

目的探讨猪心肌缺血再灌注损伤区自体骨髓单个核细胞（BM-MNC）和外周血内皮祖细胞（EPC）移植后细胞存活和分化情况。方法23头中国小型猪随机分为BM-MNC组（n=9）、EPC组（n=7）和对照组（n=7）。BM-MNC组,将PKH26标记的BM-MNC经冠状动脉移植于心肌缺血再灌注损伤区。EPC组,将CM-DiI标记的EPC经冠状动脉移植于心肌缺血再灌注损伤区。对照组,磷酸盐缓冲液对照。4周后观察荧光标记的移植细胞,并在缺血再灌注区内取材进行病理染色,用兔抗Ⅷ因子相关抗原多克隆抗体,鼠抗兔α-肌小节-肌动蛋白抗体和兔抗人纤连蛋白多克隆抗体免疫组化染色,比较各组缺血再灌注损伤区血管再生情况和反应强度。选取免疫组化可疑分化的组织观察可疑分化细胞的超微结构。结果缺血再灌注区的小血管数目BM—MNC组为（13．39±6．96）个／高倍视野,EPC组为（12．39±4．72）个／高倍视野,两者间差异无统计学意义,而高于对照组的（3．50±1．90）个／高倍视野（P〈0．05）。BM—MNC和EPC组兔抗人纤连蛋白多克隆抗体阳性反应强度显著低于对照组（P〈0．05）,而鼠抗兔α-肌小节-肌动蛋白抗体阳性反应强度三组间差异无统计学意义。BM—MNC组1头猪的密集细胞团形态结构上结合免疫组化染色鉴定,可能是干细胞的原位生长的表现。电镜检查发现了比较幼稚的内皮细胞和心肌样细胞。结论EPC和BM-MNC移植于梗死区具有刺激小血管形成、抑制纤维化的作用,还可能向心肌细胞方向分化。     

脐血内皮祖细胞治疗糖尿病大鼠下肢缺血的实验研究

目的研究脐血内皮祖细胞(EPC)治疗糖尿病下肢缺血的有效性及机制,为临床治疗糖尿病足病(DF)提供理论依据。方法取产妇足月产脐血分离单核细胞,培养7d,流式细胞仪鉴定细胞,并计数活细胞数。将Wistar雄性大鼠分成3组:(1)糖尿病大鼠射线照射后结扎双后肢股动脉,尾静脉注射血管EPC(DE组)。(2)糖尿病大鼠结扎双后肢股动脉,尾静脉注射缓冲液(DP组)。(3)正常大鼠射线照射后结扎双后肢,尾静脉注射EPC(NC组)。用绿色荧光(GFP)示踪EPC,Ⅷ因子免疫组化检测肌纤维间毛细血管数,RT-PCR检测双后肢肌肉血管内皮生长因子(VEGF)mRNA水平。结果 DE、NC组右后肢腓肠肌溃疡及缺血明显好转,有明显荧光,肌纤维间毛细血管数多,VEGF表达高,DP组好转不明显,无荧光,毛细血管数少,VEGF表达少,其差异有统计学意义(P<0.05)。结论脐血EPC治疗糖尿病大鼠下肢缺血有效。     

川芎嗪对糖尿病大鼠视网膜的保护作用

目的探讨川芎嗪（TMP）治疗糖尿病视网膜病变（DR）的效果及其机制。方法将链脲佐菌素诱发糖尿病（DM）模型的36只大鼠随机分为DM组和TMP治疗组（每组18只），同时设同批次同种属健康大鼠10只作为对照组，TMP治疗组每天用100mg／kg的TMP灌胃1次，DM组和对照组每天用100mg／kg的生理盐水灌胃1次，4个月结束实验。剥离各组视网膜，以NADPH组织化学方法显示微血管，并测量微血管面积密度；制作视网膜组织匀浆，ELISA法测定血管内皮生长因子（VEGF）浓度。结果DM组视网膜微血管面积密度和VEGF浓度均明显高于对照组及TMP治疗组（P均〈0．01）；TMP治疗组视网膜微血管面积密度和VEGF浓度均明显高于对照组（P〈0．05）． 结论 TMP对DR有治疗作用，其机制可能是抑制VEGF生成及微血管增生。     

氨氯地平对糖尿病大鼠心肌梗死后骨髓内皮祖细胞动员及血管新生的改善作用

目的：观察氨氯地平对糖尿病大鼠心肌梗死（心梗）后骨髓内皮祖细胞(EPC)动员和血管新生障碍的改善作用以及对心功能的影响,并探讨其可能的分子机制。
　　方法：180~200g SPF级雄性Sprague-Dawley大鼠60只随机分为两部分：糖尿病大鼠（n=40）给予高脂饲料饲养四周后,腹腔注射30 mg/kg链脲佐菌素;非糖尿病大鼠(n=20)为正常饮食饲养。40只糖尿病大鼠结扎冠状动脉左前降支造成急性心梗模型。术后将大鼠随机分成对照组(n=20),每日予以0.5%羧甲基纤维素钠溶剂1ml灌胃,治疗组(n=20)每日予以氨氯地平2 mg/kg灌胃,继续高脂喂养四周。流式细胞术检测术前及术后不同时间点（第1、3、5、7、14和28 d）外周血CD45-/low+/CD133+/KDR+早期EPC数量,酶联免疫吸附法检测血浆血管内皮生长因子(VEGF)水平。CD31免疫荧光染色法评估心梗周围区血管新生情况。超声心动图评估心功能。免疫印迹法测定骨髓细胞中EPC动员相关信号通路蛋白的表达。
　　结果：外周血CD45-/low+/CD133+/KDR+ EPC水平术后峰值治疗组（第7 d,112±30/106单核细胞）较对照组（第5 d,55±10/106单核细胞）升高;血浆VEGF水平在心梗后峰值治疗组[第7 d ,（5.63±1.33）ng/L]较对照组[第5 d,(3.68±0.98) ng/L]升高;骨髓细胞蛋白激酶B与内皮型一氧化氮合酶的活化水平及基质金属蛋白酶-9的表达增加;心梗周围区新生毛细血管密度治疗组大鼠较对照组显著增加,左心室射血分数及左心室短轴缩短率明显提高。上述比较差异均有统计学意义（P<0.05~0.01）。
　　结论：氨氯地平治疗改善糖尿病大鼠缺血诱导的骨髓EPC动员障碍,缺血区血管新生以及心梗后心功能。这种作用可能通过改善VEGF/内皮型一氧化氮合酶信号通路活化而介导。     

2型糖尿病外周血内皮祖细胞的诱导分化及影响因素的研究

目的观察2型糖尿病（T2DM）外周血内皮祖细胞（endothelial progenitor cell，EPC）在体外的分化增殖能力及影响因素。方法取20例T2DM无并发症患者、19例T2DM合并血管并发症患者和21例对照人群外周血EPC培养，观察细胞形态学变化并计数，用流式细胞仪检测贴壁细胞的特异性标志。结果糖尿病血管并发症组培养获得的EPC和EPC集落低于糖尿病无并发症组（P〈0．05）和对照组（P〈0．01）。EPC数量与SBP及FPG呈负相关（R=-0.266，P〈0.05；R=-0.619，P〈0．01），糖尿病人EPC集落生成数量与HbA1C水平呈负相关（R=-0．749，P〈0.05），与糖尿病病程年呈负相关（R=-0.406，P〈0．01）。结论FPG和SBP与EPC的增殖分化有关，T2DM外周血EPC数目减少，与HbA1c、SBP及病程相关。     

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.     

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.