HBsAg真核表达质粒及其诱导的小鼠特异性免疫应答

目的 研究HBsAg真核表达质粒pCI-S和pcDNA3.1-S在真核细胞中的表达和质粒DNA的免疫效果。方法 应用基因重组技术构建HBsAg真核表达质粒pCI-S和pcDNA3.1-S；经酶切和测序鉴定无误后，用阳离子脂质体介导的方法将重组质粒转染HepG2和COS-7细胞，48h后，再ELISA的方法检测重组质粒在细胞中HBsAg的表达，同时同质粒DNA免疫小鼠，用ELISA检测免疫小鼠血清抗-HBs抗体水平；用乳酸脱氢酶释放法检测小鼠肿瘤细胞HBsAg特异性CTL反应。结果 重组质粒pCI-S和pcDNA3.1-S转染的HepG2和COS-7细胞培养上清液和sAg均为阳性。DNA免疫小鼠血清可检测到高滴度的抗-HBs抗体，免疫小鼠脾细胞可检测到校强的HBsAg特异性CTL反应。结论 HBsAg真核表达质粒pCI-S和pcDNA3.1-S可在HepG2和COS-7细胞中高效表达，DNA免疫小鼠成功地诱导出抗-HBs和HBsAg特异性CTL反应。     

乙型肝炎病毒复制调控元件对HBV DNA疫苗诱导的免疫应答

目的研究乙型肝炎病毒（HBV）复制调控元件增强子Ⅰ（ENHⅠ）及前S2（Pre-S2）抗原基因对HBV DNA疫苗诱导的免疫应答的影响。方法采用常规聚合酶链反应（PCR）从HBV adr亚型全基因DNA序列中分别扩增HBsAg、PreS2-HBsAg、HBsAg-ENHI和PreS2-HBsAg-ENHⅠ基因片段，重组到VR1012载体中，构建4种HBV DNA疫苗，转染HepG2细胞并免疫Balb／C小鼠。通过细胞免疫化学、酶联免疫分析（ELISA）、酶联免疫斑点试验（ELISPOT）等方法检测其在HepG2细胞内的表达及小鼠的体液及细胞免疫应答。结果转染的HepG2细胞表达相应的目的蛋白．ENHⅠ及Pre-S2抗原基因均可增强HBV DNA疫苗转染HepG2细胞表达HBsAg；免疫接种小鼠后第2周产生抗-HBs及HBsAg特异性细胞毒T淋巴细胞（CTL），Pre—S2抗原基因可增强HBV DNA疫苗免疫Balb／C小鼠诱导的抗-HBs及HBsAg特异性CTL的产生，ENHⅠ基因对免疫应答无影响。结论ENHI及Pre—s2抗原基因均可增强HBVDNA疫苗转染HepG2细胞表达HBsAg．Pre-S2抗原基因可增强HBVDNA疫苗免疫Balb／C小鼠引起的免疫应答。     

IL-18重组体联合HBV S基因核酸疫苗免疫小鼠的实验

目的：研究IL－18重组体对HBV S基因核酸疫苗诱导小鼠产生特异性体液免疫和细胞免疫反应的影响，以探求HBV核酸疫苗免疫预防和治疗的新策略。方法：将pcDNA3-S单独或联合pcDNA3-18免疫Balb/c小鼠，检测特异性体液免疫和细胞毒性T淋巴细胞（CTL）反应，并作HBsAg特异性淋巴细胞增殖试验和特异性细胞因子诱导试验。结果：与pcDNA3-S免疫组比较，pcDNA3-18和pcDNA3-S联合免疫组小鼠的抗－HBs水平略高，但差异无显著性（P＞0．05）。特异性CTL活性显著升高（P＜0．05）。免疫小鼠的脾细胞体外经特异性抗原HBsAg刺激后，联合免疫组脾细胞上清液中IFN－γ水平显著升高（P＜0．05），IL－4水平差异无显著性（P＞0．05），联合免疫组特异性HBsAg作用后脾细胞的刺激指数（SI）高于pcDNA3-S免疫组，但差异无显著性（P＞0．05）。结论：IL－18重组体联合HBV S基因核酸疫苗免疫小鼠，可促进机体诱导特异性TH1细胞和CTL细胞反应，增强机体的特异性细胞免疫功能，IL－18是具有较好应用前景的免疫佐剂。     

磷酸铝佐剂对乙型肝炎DNA疫苗抗体应答的增强作用

目的 探讨磷酸铝佐剂对乙型肝炎DNA疫苗诱导体液免疫应答的增强作用。 方法 应用基因重组技术构建乙型肝炎表面抗原(HBsAg)真核表达质粒pcDNA 3.1-S,经酶切和测序鉴定无误后,作为乙型肝炎DNA疫苗,将不同浓度的磷酸铝悬液与之混合后免疫小鼠;用酶联免疫吸附法检测重组质粒在小鼠局部肌肉中HBsAg的表达和在血清中HBsAg的含量;并于免疫小鼠6周后检测小鼠血清抗-HBs水平。 结果 与单纯使用质粒pcDNA 3.1-S相比,将质粒pcDNA 3.1-S与磷酸铝悬液混合后免疫小鼠,重组质粒在小鼠局部肌肉中HBsAg表达差异无显著性;HBsAg在血清中的浓度均为阴性。将质粒pcDNA3.1-S与磷酸铝悬液混合后免疫小鼠,6周后检测小鼠血清抗-HBs,每1μl质粒中含1、10、50、100 μg磷酸铝组,小鼠血清抗-HBs抗体的P/N值分别为11.00±6.62、20.30±10.20、49.1 8±24.40和48.68±27.78,单纯使用质粒pcDNA3.1-S组P/N值为11.54±5.60。含1μg和10μg磷酸铝组,抗-HBs抗体P/N值与单纯用质粒pcDNA3.1-S组相比,差异无显著性;50μg和100μg磷酸铝组抗-HBs抗体P/N值高于单纯用质粒pcDNA3.1-S组,但50μg和100μg磷酸铝组二者差异无显著性。 结论 磷酸铝与质粒pcDNA3.1-S混合对pcDNA3.1-S在小鼠局部肌肉中HBsAg的表达无明显增强作用,但一定含量的磷酸铝能显     

小干扰RNA有效抑制乙型肝炎病毒的动物实验

目的 以乙型肝炎病毒（HBV）S区为靶位，观察小干扰RNA（siRNA）在动物体内抗HBV的效果。方法 以流体动力学法建立HBV感染的动物模型，将pcDNA3．1-HBV和细胞体外实验证明有效的siRNA尾静脉共注射Balb／c小鼠，用时间分辨免疫荧光分析法（IFMA）检测小鼠血清中HBsAg，用定量聚合酶链反应法（FQ-PCR）检测血清HBV DNA，用逆转录聚合酶链反应（RT-PCR）法检测HBV S-mRNA，用免疫组织化学法检测肝组织HBsAg和HBcAg。结果 在小鼠体内，siRNA能有效抑制HBsAg的分泌，降低HBVDNA的滴度，免疫组织化学结果也证实HBsAg、HBcAg刚性细胞数明显减少，干扰效果至少持续3d，而无关siRNA则无抑制作用。结论 在动物体内靶向HBV S区的siRNA能有效特异抑制HBV。     

HBsAg和HBcAg融合表达质粒对HBV转基因小鼠的体液免疫治疗效应

目的观察融合表达质粒pcDNA3．1-SC对HBV复制和表达的抑制效果。方法 构建融合表达质粒pcDNA3．1-SC，以100μg肌肉接种C57，BL／6 HBV DNA转基因小鼠，2、4周后各加强免疫1次。然后动态检测小鼠血清抗-HBs、抗-HBc的诱生，以及肝组织HBsAg、HBcAg和血清HBV DNA的消长情况。结果接种后4、8、12周，小鼠血清抗-HBs阳转率分别达到55％、67％和33％；而抗-HBc阳转率低于20％；同时，与接种前相比，肝组织内HBsAg、HBcAg表达和血清HBV DNA水平呈逐渐减弱的趋势，在接种后8周，有1／3的小鼠已不能检出。结论融合表达质粒pcD—NA3．1-SC能够在一定程度上抑制转基因小鼠体内的HBV DNA复制和抗原表达，提示了研制乙型肝炎治疗性DNA疫苗的可能性。     

尿酸辅助树突细胞免疫对小鼠淋巴细胞增殖及CTL效应的影响

目的：观察尿酸辅助HBsAg蛋白负载的树突细胞免疫接种对小鼠免疫功能的影响。方法：将负载HB-sAg的小鼠骨髓来源树突细胞经尾静脉注射接种小鼠，1次／w，共2次。分DC（树突状细胞）对照组、联合尿酸组、尿酸对照组。MTT法检测体外经HBsAg或PBS重刺激的脾T淋巴细胞增殖反应；流式细胞仪法检测CTL（细胞毒性T淋巴细胞）活性。结果：免疫2周时，小鼠脾T细胞增殖反应及体内HBsAg特异性CTL的杀伤活性，联合尿酸组明显强于各对照组。结论：尿酸可促进负载HBsAg树突细胞免疫后小鼠脾T淋巴细胞的增殖及HBsAg特异性CTL效应。尿酸具有增强DC疫苗免疫效应的活性，可用作研制抗HBV的治疗性疫苗的免疫佐剂。     

淋病奈瑟球菌表面蛋白A基因疫苗的构建及其诱导小鼠的免疫应答

目的构建淋病奈瑟球菌表面蛋白A（NspA）基因疫苗，并接种小鼠，评价其诱导的体液和细胞免疫应答。方法将NspA基因插入到真核表达载体pcDNA3．1（＋）中，构建重组真核表达质粒pcDNA3．1（＋）／NspA，并经PCR、双酶切及测序鉴定。反转录一聚合酶链反应（RT-PCR）和免疫组织化学法分别检测NspA基因转染细胞后mRNA和蛋白的表达。以pcDNA3．1（＋）／NspA重组质粒免疫45只雄性BALB／c小鼠，试管凝集法检测免疫小鼠抗体滴度，ELISA检测IFN-γ水平，四甲基偶氮唑蓝（MTT）比色法检测脾淋巴细胞增殖。提取接种部位股四头肌总DNA，PCR检测BALB／c小鼠肌细胞内NspA基因。结果成功构建pcDNA3．1（＋）／NspA基因疫苗，能在真核细胞中转录和表达。pcDNA3．1（＋）／NspA免疫组的抗体滴度达1：640，pcDNA3．1（＋）和PBS免疫组均无特异性抗体检出。空载体pcDNA3．1（＋）组IFN-γ为（23．79±11．85）pg／mL，pcDNA3．1（＋）／NspA免疫组为（169．71±30．52）pg／mL（P〈0．01）；脾淋巴细胞增殖的刺激指数（SI）分别为1．05±0．30和1．94±0．74（P〈0．01）。并证实NspA基因可在小鼠肌细胞中稳定存在。结论构建淋病奈瑟球菌NspA基因疫苗，将其免疫BALB／c小鼠可诱导特异性体液和细胞免疫应答，为进一步用于淋病预防奠定了基础。     

248例慢性乙型肝炎患者血清HBV DNA水平与肝组织HBsAg／HBcAg表达的相关性研究

目的 探讨慢性乙型肝炎患者血清HBV DNA水平与肝组织病理学损害和HBsAg／HBcAg表达的关系。方法 对248例慢性乙型肝炎患者进行肝穿刺活检,采用免疫组织化学法检测HBsAg和HBcAg,同时检测患者血清HBVDNA水平。结果 在肝脏炎症程度G1～G4四组患者之间血清HBV DNA水平无显著性差异（P〉0．05）,肝纤维化程度S1～S4四组患者之间血清HBV DNA水平也无显著性差异（P〉0．05）;在肝组织HBsAg表达强度-～＋＋＋四组患者之间血清HBV DNA水平无显著性差异（P〉0．05）,而肝组织HBcAg随血清HBV DNA水平的增高而表达增强。结论 慢性乙型肝炎患者肝组织炎症活动度和纤维化程度与血清HBV DNA水平无相关性。     

体内外检测乙型肝炎DNA疫苗诱生小鼠特异性CTL活性的比较研究

目的：了解体内外两种方法检测乙型肝炎DNA疫苗诱生BALB／c小鼠特异性细胞毒性T淋巴细胞（CTL）活性的特点，比较其异同，建立与体外法互为补充，且更加直观、简便的活体CTL活性检测模型。方法：用HBV-S真核表达质粒pVAX1／S对SP2／0细胞进行转染，经鉴定后作为目的靶细胞（稳定转染并表达HBV主蛋白，命名为SP2／0-S细胞）；对照靶细胞为稳定转染pVAX1空载质粒的细胞（命名为SP2／0-P细胞）备用。活体诱生实验：用目的或对照靶细胞分别使小鼠荷瘤，观察免疫及非免疫小鼠在各靶细胞负荷下的生存时间与肿瘤的生长情况，设置不同组合做同期对照观察，小鼠生存率的差异可以提示体内特异性CTL是否存在。体外诱生实验：采用经典CTL活性检测方法，LDH释放法成品药盒。SP2／0-S细胞与SP2／0-P细胞作为靶细胞，免疫鼠与非免疫鼠的脾细胞为效应细胞，按照不同效／靶比共孵育后以酶标仪检测LDH的释放活性。结果：活体诱生实验：免疫小鼠存活时间明显长于对照小鼠（P〈0．05）。体外诱生实验：免疫鼠CTL活性在彬靶比为50／1时出现最大杀伤活性，为36．9％，明显优于对照组（P〈0．05）。结论：体内外两种方法均能够检测到乙型肝炎DNA疫苗诱生BALB／c小鼠特异性CTL活性，实验中发现体外LDH释放法CTL活性并不高，但是总体上仍然能够反映CTL活性。而活体诱生实验操作相对简便，表现更为直观，可望成为与经典方法相互补充的新型实验模型。     

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.     

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.     

Serological survey of HIV and syphilis in pregnant women in Madagascar

Objectives Peripartal transmission of human immunodeficiency virus (HIV) and Treponema pallidum, the causative agent of syphilis, leads to severe consequences for newborns. Preventive measures require awareness of the maternal infection. Although HIV and syphilis testing in Madagascar could be theoretically carried out within the framework of the national pregnancy follow‐up scheme, the required test kits are rarely available at peripheral health centres. In this study, we screened blood samples of pregnant Madagascan women for HIV and syphilis seroprevalence to estimate the demand for systemic screening in pregnancy. Methods Retrospective anonymous serological analysis for HIV and syphilis was performed in plasma samples from 1232 pregnant women that were taken between May and July 2010 in Ambositra, Ifanadiana, Manakara, Mananjary, Moramanga and Tsiroanomandidy (Madagascar) during pregnancy follow‐up. Screening was based on Treponema pallidum haemagglutination tests for syphilis and rapid tests for HIV, with confirmation of positive screening results on line assays. Results Out of 1232 pregnant women, none were seropositive for HIV and 37 (3%) were seropositive for Treponema pallidum. Conclusions Our findings are in line with previous studies that describe considerable syphilis prevalence in the rural Madagascan population. The results suggest a need for screening to prevent peripartal Treponema pallidum transmission, while HIV is still rare. If they are known, Treponema pallidum infections can be easily, safely and inexpensively treated even in pregnancy to reduce the risk of transmission.     

Variabilité des concentrations plasmatiques de l’angiotensinogène et risque d’hypertension artérielle chez le rat transgénique

Aim

Genetic polymorphisms of the human angiotensinogen gene are frequent and may induce up to 30% increase of plasma angiotensinogen concentrations with a blood pressure increase of up to 5 mmHg. Their role for the pathogenesis of human arterial hypertension remains unclear. High plasma angiotensinogen levels could increase the sensitivity to other blood pressure stressors.

Methods

Male transgenic rats with a 9-fold increase of plasma angiotensinogen concentrations and male non-transgenic rats aged 10 weeks were treated or not with NG-Nitro-L-arginine-methyl ester for 3 weeks in their drinking water (n = 3/group). Systolic blood pressure and body weight were measured at baseline and at the end of the study when left ventricular weight and ventricular expression of angiotensin I-converting enzyme and procollagen Iα1 were determined (polymerase chain reaction).

Results

At baseline, transgenic rats had +18 mmHg higher bood pressure and –8% lower body weight compared to non-transgenic rats (P < 0.05) without significant changes for the vehicle groups throughout the study (P > 0.05). NG-Nitro-L-arginine-methyl ester increased blood pressure, left ventricular weight and left ventricular weight indexed for body weight by +41%, +17.6% and +18.6% (P < 0.05) in transgenic and +25%, +5.3% and +6.7% (P > 0.05) in non-transgenic rats compared to untreated animals, respectively. Cardiac gene expression showed no differences between groups (P > 0.05).

Conclusion

Increased plasma angiotensinogen levels may sensitize to additional blood pressure stressors. Our preliminary results point towards an independent role of angiotensinogen in the pathogenesis of human hypertension and associated end-organ damage.     

Morphological and immunocytochemical heterogeneity of cultured pinealocytes from one-week-and two-month-old rats: Planimetric and densitometric investigations

Abstract: In vitro preparations of rat pinealocytes are widely used for biochemical analyses of signal transduction processes. This paper deals with morphological and immunocytochemical features of such preparations. Special attention was paid to the problems of whether pinealocytes represent a heterogeneous cell population and how such heterogeneity may develop during ontogeny. The investigations were performed with cells which were obtained from the pineal organ of one-week-and two-month-old rats, attached to synthetic peptide-coated coverslips or tissue culture chamber slides, and maintained under in vitro conditions overnight. The attached cells were then fixed with paraformaldehyde. These preparations yielded monolayers of spherical cells of different sizes; most cells were isolated, but some of them were aggregated and formed small clusters. On the average, the cells from the one-week-old animals were smaller than the cells from the two-month-old animals. Immunocytochemical demonstration of S-antigen, a pinealocyte-specific marker, showed that the majority of the cells from two-month-old animals were intensely or moderately labelled. Pinealocytes from one-week-old animals were less S-antigen immunoreactive. Only very few cells (less than 1% displayed glial fibrillary acidic protein (GFAP)-immunoreactivity. Planimetric investigations of the cell size and semiquantitative densitometric investigations of the intensity of the S-antigen immunoreaction revealed that (i) pinealocytes kept in vitro form a heterogeneous cell population, and that (ii) this heterogeneity increases during postnatal development from one-week-old to two-month-old animals. Two groups of pinealocytes can be distinguished based on their developmental fate: pinealocytes of one group grow dramatically, but show only a moderate increase in S-antigen immunoreactivity, and pinealocytes of the other group retain their size, but display a distinct increment in S-antigen immunoreacti vitv.     

Effects of pinealectomy and melatonin administration on certain indices of ovarian hyperplasia and/or hypertrophy in rats with both ovaries intact or after unilateral ovariectomy

Abstract: In earlier studies from other laboratories it was shown that melatonin decreased ovarian weight in rats and inhibited compensatory hypertrophy of the remaining ovary after unilateral ovariectomy. This study was designed to examine the influence of melatonin on certain indices of ovarian hyperplasia and/or hypertrophy in adult female rats with both ovaries preserved and with either an intact pineal gland or with the pineal gland removed (pinealectomy, PX) or, finally, in sham-PX animals. Similar studies were conducted on rats after unilateral ovariectomy, referring the examined parameters to the remaining intact ovary. The studies included mitotic activity of granulosa layer cells and corpus luteum cells, ovarian weight, ovarian cross-sectional area, cross-sectional area of the granulosa layer of all the Graafian follicles and the cross-sectional areas of the corpora lutea, visible on the ovarian cross-section. On the basis of results, we conclude that: 1) the effect of PX on the processes of ovarian hyperplasia and hypertrophy may vary; analogously, exogenous melatonin administration may influence ovarian hyperplasia and hypertrophy in different ways; 2) PX and exogenous melatonin may, under certain conditions, exert similar biological effects, even synergistic effects; 3) melatonin inhibits ovarian growth processes, while the effects of PX are variable; 4) the results indicate that in experiments performed on rats, with the use of two control groups, i.e., intact and sham-PX, melatonin effects on these two groups may differ.