稳定表达乙型肝炎病毒X基因肝细胞株的构建

目的 构建稳定表达乙型肝炎病毒x基因（HBVx）的正常人肝细胞株。方法用PCR法扩增含EcoRI和HindⅢ酶切位点的x基因序列,构建HBVx基因真核表达载体pcDNA3．1（＋）-HBVx,转化大肠杆菌DHSct,筛选阳性克隆并对其进行双酶切和测序鉴定;用脂质体转染法将HBVx基因导入Chang细胞系,G418筛选,RT-PCR和Westernblot鉴定。结果x基因亚克隆入pcDNA3．1（＋）,含有完整的x基因片段,转染后Chang细胞有HBVxmRNA表达,Chang／HBVx有HBVx蛋白的表达。结论构建了稳定表达HBVx基因的肝细胞株Chang／HBVx。     

抗人VEGF发卡状核酶基因的构建及其对肝癌细胞VEGF蛋白表达的影响

利用计算机辅助设计合成针对人VEGF的发卡状核酶（RZ），定向亚克隆于真核表达载体pcDNA3．1^＋中，转染肝癌细胞SMMC-7721，RT—PCR鉴定。ELISA法和MTT法检测SMMC-7721细胞对照组、SMMC-7721／pcDNA3．1^＋空载体对照组和SMMC-7721／RZ基因转染组细胞VEGF表达和增殖情况，流式细胞术检测各组细胞周期和凋亡情况。结果为SMMC-7721／RZ基因转染组细胞VEGF表达水平和细胞增殖速率显著低于SMMC-7721细胞对照组和SMMC-7721／pcDNA3．1’空载体对照组。MC-7721／RZ基因转染组细胞出现凋亡峰，其凋亡率达11．O％，而细胞对照和空载体细胞对照均末出现。认为SMMC-7721／RZ转基因细胞株的成功构建和生物学特性的初步研究为进-步建立裸鼠人肝癌模型及评价RZ对体内肝癌生长的抑制作用奠定了-定的基础。     

乙型肝炎病毒X蛋白对MMPs及TIMPs的影响

目的全面分析乙型肝炎病毒X蛋白（Hepatitis Bvirus Xprotein，HBx）对基质金属蛋白酶（Matrix Metalloproteinases，MMPs）及组织金属蛋白酶抑制物（Tissue Inhibitors of Metalloproteinases，TIMPs）的影响，探讨其在肝细胞癌的侵袭转移中的可能作用。方法PCR扩增HBVX基因并克隆人真核表达载体pcDNA3．1／HisC，重组载体及空载体分别以Lipofectamine2000转染HepG2细胞并以800μg／mlG418筛选抗性细胞克隆。以Western blot检测抗性细胞HBx表达。抽提细胞总RNA，半定量RT-PCR检测MMPs及TIMPs。收集细胞培养液上清，以明胶酶谱检测MMP2及MMP9活性，反相明胶酶谱检测TIMPs活性。结果构建了HBx重组载体pcDNA3．1-XB。该重组载体及对照空载体转染HepG2细胞后，G418筛选，分别获得抗性细胞克隆HepG2-XB及HepG2-HIS，前者经Westernblot证实可表达HBx。半定量RT—PCR显示HBx可促进MMP2、7、13、14、16、17、19、23、24及TIMP1、4基因的转录，抑制MMP1、3、8、9、10、11、12、15、20及TIMP2、3基因的转录。明胶酶谱检测显示HBx可促进酶原MMP2（Pro—MMP2）及活性MMP9（Active—MMP9）表达，抑制酶原MMP9（Pro—MMP9）表达；反相明胶酶谱显示HBx可促进TIMP1、TIMP4的表达，同时抑制TIMP2及糖基化TIMP3的表达。结论HBx蛋白对MMPs、TIMPs转录表达的影响是多方面的，但这种影响在HBx促进肝癌细胞侵袭转移过程中的确切机制有待进一步阐明。     

重组转化生长因子β3基因对大鼠肝星状细胞Ⅰ型胶原表达的影响

目的观察转化生长因子D3基因（TGFβ3）对大鼠肝星状细胞株（HSC—T6）Ⅰ型胶原合成的影响。方法TGFβ3表达质粒[pcDNA3．1（＋）-TGFβ31和TGFβ1表达质粒[pcDNA3．1（＋）-TGFD11的构建。通过脂质体介导方法，将pcDNA3．1（＋）-TGFβ1、pcDNA3．1（＋）-TGFβ3分别及共同转染体外培养的HSC—T6细胞，荧光定量PCR法及Westernblot法分别检测转染后TGFβ1、TGFD3、Ⅰ型胶原mRNA及蛋白质的表达。将pcDNA3．1（＋）-TGFD1转染HSC—T6细胞，经G418筛选建立高表达TGFD1的HSC～T6细胞克隆，pcDNA3．1（＋）-TGFD3转染克隆细胞，荧光定量PCR法检测转染后TGFβ3、TGFβ1及Ⅰ型胶原mRNA的表达，Westernblot法检测TGFβ1、Ⅰ型胶原蛋白的表达情况。结果构建的pcDNA3．1（＋）TGFD3、pcDNA3．1（＋）-TGFD1质粒可转染HSC—T6细胞，转染率28．2％。pcDNA3．1（＋）TGF侈3转染细胞后，Ⅰ型胶原mRNA及蛋白的表达较空白组及对照组增加，以72h增高最为明显（P〈0．05）；共转染组Ⅰ型胶原mRNA及蛋白质的表达较pcDNA3．1（＋）-TGFβ1转染组明显降低（P〈0．05）。TGF侈3转染克隆细胞后，TGFD1mRNA表达较克隆组无明显改变（P〉0．05），而蛋白质表达明显下降（P〈0．05），Ⅰ型胶原mRNA及蛋白质表达均较克隆组明显降低（P〈0．05）。结论TGFD3基因转染正常培养的HSC—T6细胞，增加Ⅰ型胶原的表达；转染高表达TGFβ1的克隆组HSC—T6细胞，Ⅰ型胶原表达明显降低，提示TGFβ3对肝纤维化的发生有抑制作用。     

HBV X基因转染对HepG2肝癌细胞凋亡的影响及其机制

目的:研究HBV X基因转染对HepG2肝癌细胞凋亡及凋亡相关因子表达的影响.方法:用脂质体转染法将HBx真核表达载体pcDNA3/HBx瞬时转入HepG2细胞,以未转染的HepG2细胞及转染空载体pcDNA3的细胞为对照.RT-PCR法检测HBx基因的表达;MTT法检测各组细胞的增殖活性;TUNEL法检测各组的凋亡情况.β-actin为内参,半定量RT-PCR法检测凋亡相关基因Bax、Bcl-xL、c-myc的表达量变化.结果:pcDNA3-X转染HepG2细胞后,RT-PCR扩增出HBV X片段,空质粒组及正常对照组均未扩增出相应片段.HepG2细胞转染HBx基因后,相对于对照组细胞增殖能力明显下降,凋亡增多,差异有显著性意义(P＜0.01);转染HBx的细胞Bax、Bcl-xL、c-myc mRNA相对表达量较转染空质粒组和未转染质粒组明显增高,差异有显著性意义(P＜0.05).结论:成功将HBx基因转染入HepG2细胞,并在细胞中表达,转染HBx基因可同时上调Bax、Bcl-xL、c-myc mRNA表达,促进HepG2细胞凋亡,抑制增殖.     

一种高效表达HBx的肝癌细胞模型的建立

目的构建稳定、高效表达HBx的转基因细胞模型BEL-7404／HBx。方法通过脂质体转染将亚克隆有HBVX基因的重组质粒pcDNA3．1（＋）-V5-HisB—HBx导人肝癌细胞系BEL-7404，并用G418筛选获取阳性克隆，分别用逆转录聚合酶链反应（RT-PCR）和免疫印迹（Westernblot）检测不同生长时期阳性克隆中HBxmRNA和蛋白的表达情况。结果不同生长时期阳性克隆均能检测到HBxmRNA和蛋白的高效表达。结论成功构建稳定、高效表达HBx的转基因肝癌细胞模型BEL-7404／HBx。     

舒林酸对HBV X基因转染的肝癌细胞Wnt信号通路的影响

目的探讨非甾体类抗炎药舒林酸(Sulindac)对HBV X基因转染的人肝癌SMMC7721细胞系Wnt信号通路的影响。方法用脂质体转染方法将HBx真核表达载体pcDNA3-X瞬时转染SMMC7721细胞(SMMC7721/HBx),以转染空载体pcDNA3的SMMC7721细胞组(SMMC7721/pcDNA3)及未转染质粒的SMMC7721细胞组(SMMC7721)为对照,于转染后72 h用RT-PCR和Western blot检测HBx的表达;并以各种浓度的舒林酸于瞬时转染后24 h干预SMMC7721/HBx组和SMMC7721/pcDNA3组至72 h,Western blot和免疫细胞化学检测β-catenin的表达,Western blot检测cyclinD1的表达。结果RT-PCR和Western blot显示,SMMC7721/HBx组在RNA及蛋白水平均有HBx的表达,而SMMC7721/pcDNA3组及SMMC7721组均无表达。与SMMC7721/pcD-NA3组及SMMC7721组相比,Western blot示SMMC7721/HBx组β-catenin、cyclinD1的表达水平较高,免疫细胞化学显示SMMC7721/HBx组β-catenin的表达水平较高,核染色较深,而SMMC7721/pcDNA3组及SMMC7721组之间则无明显差异。Westernblot和免疫细胞化学示一定浓度的舒林酸干预SMMC7721/HBx组和SMMC7721/pcDNA3组细胞48 h后可下调β-catenin和cy-clinD1的表达,而对SMMC7721/HBx组的下调较SMMC7721/pcDNA3组明显。结论HBx基因成功瞬时转染入SMMC7721细胞,激活Wnt信号通路,并同时上调cyclinD1的表达。舒林酸可通过下调β-catenin抑制肝癌细胞的Wnt信号通路,下调cyclinD1的表达,且HBx阳性的肝癌细胞对舒林酸较敏感。     

丙型肝炎病毒1b基因型核心蛋白对HepG2细胞Bax与Bcl-2基因表达的影响

目的研究丙型肝炎病毒（HCV）1b基因型核心蛋白（C）对HepG2细胞B细胞淋巴瘤-2基因（Bcl-2）与Bcl-2相关X蛋白（Bax）表达的影响,以探索1b型HCV C蛋白与HepG2细胞凋亡的关系。方法利用RT-PCR扩增出HCV-1b-C基因,经双酶切后连接pcDNA3.1（-）,成功构建真核表达载体pcDNA3.1（-）/HCV-1b-C。利用脂质体转染HepG2细胞,RT-PCR及Western Blot检测其mRNA及蛋白的表达,RT-PCR及Western Blot检测转染成功后HCV-1b-C对HepG2细胞Bax与Bcl-2表达的影响,并设转染空质粒组及未处理组作对照。结果成功构建真核表达载体pcDNA3.1（-）/HCV-1b-C;瞬时转染HepG2细胞,成功表达HCV C mRNA及蛋白;转染C基因组的Bax的mRNA及蛋白相对表达量减少,与转染空质粒组及未处理组比较差异均有统计学意义（P〈0.01）;转染C基因组的Bcl-2的mRNA及蛋白相对表达量增多,与转染空质粒组及未处理组比较差异均有统计学意义（P〈0.01）。结论 1b基因型HCV C蛋白转染HepG2细胞会导致Bax表达减少及Bcl-2表达增多,降低Bax/Bcl-2比值,可能是抑制HepG2细胞凋亡的机制之一。     

肝癌组织中HBx基因的多态性及其突变体对QSG7701细胞生物学行为的影响

目的研究中国南部部分地区肝细胞癌患者中是否存在特征性HBx基因突变，以及基因变异在肝癌发病中的作用。方法采用巢式PCR，单链构象多态分析（SSCP）、异源性双链分析（HA）和DNA测序等方法对51份肝细胞癌组织石蜡包埋切片标本和25份HBV携带者血清HBx基因多态性进行分析，对有明显缺失变异的HBx基因进行克隆，转染肝细胞，通过体外和体内实验比较变异和野生型HBx基因转染对肝细胞QSG7701生物学行为的影响。结果肝癌组织中HBx基因存在点突变和缺失型突变，B和C基因型之间突变类型和数量有一定差异，C基因型点突变频率更高（t=-2．522，P〈0．05），而B基因型存在缺失变异（HBx—d382和HBx—d431）。获得了含HBx缺失变异的基因的稳定转染细胞株。该细胞株细胞大小形态不一、体积增大、核浆比例增大，生长速度更快，克隆形成率高（P〈0．05），在裸鼠体内成瘤机会和速度更快。结论HBx基因在肝细胞癌组织中频繁地发生突变，nt382～400位置上的缺失型突变体转染QSG7701后能促进肝细胞的生长速度，细胞凋亡减少，使之获得肿瘤性细胞特性，因此HBx—d382缺失型突变体可能在中国南部部分地区肝癌发生发展中发挥重要作用。     

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.     

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

Abstract: The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M_r range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [¹²⁵I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (K_a of 4.7 ± 0.2 × 10⁹ M^-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M_r 24,000. The functional status of PRL-and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.