乙型肝炎病毒-Alu-聚合酶链反应检测肝细胞癌中乙型肝炎病毒DNA的整合

目的 了解肝细胞癌(HCC)中HBV DNA的整合情况.方法 收集24例HBsAg阳性患者的HCC组织,提取其DNA,应用套式PCR原理,在设计的引物中引入U碱基,根据已知基因序列和人Alu重复序列分别设计引物,建立克隆整合的HBV DNA及其相邻的细胞基因序列的PCR技术.PCR产物测序所获结果经美国国家生物技术信息中心(NCBI)BLAST及Map Viewer检索确定HBV整合在染色体上的精确位置.结果 24份HCC组织中,14份存在HBV整合现象,整合的标本中11份正向插入宿主基因,8份反向插入宿主基因,其中5份标本既有正向插入也有反向插入,从病毒基因分析,整合可发生于X基因的任何长度,且均以截短形式插入宿主细胞DNA.另有8份标本未测到整合.结论 HBV在HCC细胞染色体上的整合呈不均衡分布.     

靶向HBx基因shRNA载体稳定表达人肝癌细胞的建立

本研究采用整合有HBV DNA而HBsAg表达阴性的人肝癌细胞株MHCC97-H,应用免疫荧光法初步研究HBx表达,并从基因组中克隆HBx基因,采用RNA干扰(RNAi)技术,构建靶向该HBx基因的短发夹RNA(shRNA)真核表达载体,通过转染和筛选,研究shRNA载体在MHCC97-H细胞中稳定表达对HBx基因表达的影响,最终构建稳定表达靶向整合HBx基因的shRNA载体的人肝癌细胞模型,为进一步研究构建平台.     

乙型肝炎与肝癌关系

目的乙型肝炎病毒(HBV)持续感染和宿主免疫反应可导致肝细胞癌(HCC)发生.研究HBV感染致癌机制和乙肝免疫预防HCC具有重要的理论和实际意义.①从分子生物学观点出发探索HBV感染与HCC关系与HCC相关的最危险因素HBV的XORF常整合于宿主DNA并高度表达.现有越来越多的证据表明反式激活剂HBx可能通过封闭抑癌基因p53的功能而致癌.结构与功能分析表明HBx的远C末端为其抑制p53诱导凋亡的必需区.最近发现HBx基因反式激活功能区发生天然"突然”,即可消除其诱导细胞生长停顿和凋亡的效应.②HBx是一多功能调节因子近来研究相对集中在HBx对肝细胞凋亡的作用上,促凋亡或抑凋亡.发现HBx可抑制cospase3酶活性阻断细胞凋亡.但另有报告HBx可致敏细胞引起凋亡.联系上述HBx可封闭由于p53过表达而诱导的凋亡,可见病毒根据不同细胞环境或外来刺激采取不同的策略.③乙肝免疫预防HCCHBVDNA,多在HBx基因整合于宿主DNA,可激活或抑制与生长增殖有关的细胞基因.如整合得到阻遏,细胞转化不至发生.台湾儿童广行乙肝免疫使HBV携带率10%降至0.9%,HCC发生率0.52%降至0.13%,证明控制乙肝是有效防癌措施.     

慢性乙型肝炎患者血清HBV增强子Ⅰ区分子特征分析

目的分析乙型肝炎病毒基因组中增强子Ⅰ区的特征,进一步探讨乙肝慢性化的机制。方法收集16份慢性乙型肝炎患者的血清标本HBV-DNA,扩增HBV EnhⅠ基因,PCR产物测序后对HBV进行分型,并分析HBVEnhⅠ突变情况。结果 10例(62.5%)标本HBV EnhⅠ基因PCR扩增阳性,测序标本经在线Genotyping比对后,均属于B型,与参考序列AF100309同源性最高,10份标本的HBV EnhⅠ区共发现12个突变位点。结论 HBVEnhⅠ基因存在多位点突变,此可能为促进病毒复制导致乙肝慢性化的机制。     

慢性乙型肝炎患者血清HBV增强子I区分子特征分析

目的分析乙型肝炎病毒基因组中增强子I区的特征,进一步探讨乙肝慢性化的机制。方法收集16份慢性乙型肝炎患者的血清标本HBV—DNA,扩增HBVEnhI基因,PCR产物测序后对HBV进行分型,并分析HBVEnhI突变情况。结果10例（62．5％）标本HBVEnhI基因PCR扩增阳性,测序标本经在线Genotyping比对后,均属于B型,与参考序列AFl00309同源性最高,10份标本的HBVEnhI区共发现12个突变位点。结论HBVEnhI基因存在多位点突变,此可能为促进病毒复制导致乙肝慢性化的机制。     

乙型肝炎病毒表面抗原基因的克隆及序列分析

目的克隆并鉴定乙型肝炎病毒表面抗原基因,为下一步构建该重组腺病毒载体以及进一步研究腺病毒载体的包装及在乙型肝炎病毒基因治疗中的作用。方法参照人 HBV adr 亚型序列,设计和合成 S 基因特异引物。应用 PCR 技术,从含有 HBsAg 的 HBV DNA 中扩增目的 DNA,通过 TA 连接将其克隆人 pGEM-T easy 载体,经限制性内切酶 BglⅡ/SalⅠ鉴定后,进一步测序鉴定。结果从乙肝表面抗原阳性(HBsAg )血清中成功提取 HBV DNA,并以此 DNA 为模板,成功扩增出 S 基因,测序结果与 GenBank 中注册的相应序列比对,核苷酸序列的同源性高达92％～99％,预测氨基酸序列同源性亦达82％。结论从 HBsAg 阳性血清中成功克隆出 S 基因序列,为进一步构建重组腺病毒及后续实验奠定了基础。     

HBsAg/HBsAb双阳性慢性乙型肝炎患者HBV S基因免疫逃逸相关变异分析

目的分析HBsAg/HBsAb双阳性慢性乙型肝炎患者HBV S基因主要亲水区免疫逃逸相关位点变异特点。方法收集89例HBsAg/HBsAb双阳性和148例HBsAg单阳性的慢性乙型肝炎患者血清及临床资料,提取患者血清HBV DNA,扩增患者HBV S基因并进行测序,应用DNASTAR Lasergene Meg Align软件对主要亲水区已有文献报道的46个免疫逃逸相关位点及新增N-糖基化变异进行比对分析。结果 2组患者在年龄、ALT、TBIL、HBV DNA载量和HBe Ag阳性率方面差异均无统计学意义(P均0.05)。HBsAg/HBsAb双阳性患者HBV S基因主要亲水区变异的总检出率为31.46%,明显高于HBsAg单阳性患者的18.92%(P0.05),其中s L110I/S、s T113N/S、s T131I/N/P和s S143L/M/T的变异检出率明显高于单阳性组;双阳性患者多位点联合变异检出率亦明显高于单阳性患者(20.22%vs.6.08%,P0.05)。双阳性患者新增N-糖基化变异检出率高于单阳性患者(7.87%vs.2.03%),差异具有统计学意义(P0.05)。结论 HBsAg/HBsAb双阳性患者比HBsAg单阳性患者的HBV S基因免疫逃逸相关变异种类更多,单位点和多位点联合变异检出率更高,并且新增N-糖基化变异检出率也更高,这些变异可能是引起慢性乙型肝炎患者HBsAg/HBsAb双阳性共存的驱动因素之一。     

一种新的乙型肝炎病毒X基因变异方式

目的:证实乙型肝炎病毒(hepatitis B virus,HBV)X基因一种新的变异方式.方法:从HBV慢性感染患者血清中提取HBVDNA,扩增X基因序列,克隆入pMD19 T载体,选择阳性克隆进行DNA测序,与已知HBV基因相应序列比较该患者体内HBV基因变异位点以及变异形式.结果:从21例患者中共挑选74个克隆测序,测序结果提示54个克隆X基因下游大段缺失突变,长度达234 nt,位于1601-1834 nt处,另有1个克隆发生245 nt缺失突变.发生缺失变异的病毒株同时存在G/A1515C、G1518C和A1585T替换突变,这两种突变具有联动特征.缺失突变株HBx仅编码76 aa其第44和45位编码为LL,具有特异性.结论:观察到一种X蛋白变异方式,这种大段缺失突变导致X蛋白下游编码序列丢失,其为X因子还是X蛋白以及这种变异是否为常态形式尚需进一步研究.     

HBsAg与抗-HBs同时阳性慢性乙肝患者HBV基因全序列分析

目的 报告HBsAg与抗-HBs同时阳性慢性乙肝患者基因序列变化特点.方法 对三例HBsAg与抗-HBs同时阳性慢性乙肝患者血清中HBV DNA基因全序列进行PCR扩增、测序,并对测序结果及S区氨基酸进行分析.结果 测序结果发现三例患者血清中HBV S区氨基酸均发生替换突变,并且,三例患者中HBV DNA发生碱基替换的位点大部分相同,且替换后碱基基本相同.结论 HBsAg与抗-HBs同时阳性慢性乙型肝炎患者常发生S区氨基酸序列突变,双阳现象的出现亦可能与S区外其他位点碱基突变有关.     

长效干扰素治疗慢性乙型肝炎表面抗原转阴可能与HBV序列本身相关

目的明确peg-IFN抗HBV治疗后HBsAg转阴是否与HBV序列相关。方法对一对夫妻慢性乙型肝炎患者在peg-IFNα2a抗病毒治疗后均获得HBsAg阴转的血清HBVDNA,进行HBV全序列基因组扩增、克隆,各挑选3个克隆进行病毒基因测序。分析比对两者HBV序列的异同。结果例1男,45。岁在治疗24周达到HBsAg和HBVDNA阴转;例2女,47。岁在治疗36周获得HBVDNA阴转,在48周时获得HBsAg阴转。两者HBV均为C型。两者感染的HBV序列完全一致。结论 Peg-IFNα抗病毒治疗后出现HBsAg转阴可能与病毒本身相关。     

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.     

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.     

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.