电转联合热休克蛋白佐剂疫苗引发抗HBV的免疫应答

针对HBV感染的治疗性DNA疫苗虽然具有很好的应用前景,但目前抗病毒效果并不高,表明在病毒长期感染过程中存在免疫抑制机制。以HBV的表面蛋白(HBsAg)和核心蛋白(HBcAg)为DNA疫苗抗原,采用gp96和HSP70作为佐剂联合电转以提高疫苗的活性。将gp96为佐剂的HBsAg/HBcAg DNA疫苗免疫HBV转基因鼠后引发抗原特异性的细胞免疫和体液免疫应答。使用gp96和HSP70佐剂引起Treg下调20%。与没有免疫的小鼠相比,以gp96和HSP70为佐剂的DNA疫苗显著降低血清中病毒S抗原水平和DNA拷贝数,大幅降低小鼠肝脏中HBc的表达。该研究为设计以gp96为佐剂的乙肝治疗性DNA疫苗提供了依据。     

乙肝病毒标志物在胎儿和胎盘组织中的表达及其与 HBV母婴传播关系的研究

目的通过对乙肝阳性产妇外周血、胎儿及胎儿附属物进行乙肝病毒标志物的检测,探讨HBV宫内感染发生的机制。方法通过ELISA法及实时荧光定量PCR法检测血清标本中HBV标志物及HBV DNA水平;通过对组织标本的免疫组织化学染色检测组织中HBV标志物的表达。结果胎儿脐血HBV DNA水平与母血HBV DNA水平相关,母血HBV DNA高水平(≥107copy/mL)时脐血HBV DNA阳性率明显增高,P<0.05。胎儿脐血HBV DNA水平显著低于母血HBV DNA水平,P<0.05。胎盘组织可见HBsAg免疫组织化学染色阳性,但未发现HBcAg染色阳性。在引产胎儿胎肝和胎肾组织中发现HBsAg和/或HBcAg免疫组织化学染色阳性细胞。结论母亲HBV DNA高水平是发生HBV宫内感染的高危因素。脐血HBV DNA阳性是判断HBV宫内感染的相关指标;HBV可能通过胎盘感染的途径由母体进入胎儿体内,并可能在胎儿体内定位和复制,这可能是导致HBV宫内感染的原因。     

高复制HBV转基因小鼠模型对抗病毒药物拉米夫定评价的研究

[目的]探讨乙型肝炎病毒(HBV)转基因小鼠模型筛选抗HBV药物的可行性。[方法]用公认抗HBV复制药物拉米夫定对我们建立高复制HBV转基因鼠进行实验,选我们建立的1.3copy高复制HBV转基因小鼠20只,随机分成两组,每组10只。采用灌胃针灌胃法给药。对照组灌喂生理盐水,实验组灌喂拉米夫定,剂量为100mg/kg,每天2次,连续灌21d,每7d采血1次。荧光定量PCR检测血清中HBVDNA。[结果]实验组用拉米夫定前小鼠血清HBVDNA5.50±0.42(拷贝数log10数值),3周后HBVDNA已显著降低(4.63±0.57),4周后,小鼠血清HBVNDA为4.08±0.51,停药1周后,再次检测血清HBVDNA,小鼠血清HBVDNA又恢复正常水平(5.70±0.39)。[结论]我们建立的高复制HBV转基因小鼠模型验证了拉米夫定对HBV复制的抑制程度和持续时间,表明该模型可应用于抗HBV药物的筛选、评价研究。     

孕期大鼠暴露PFOS对胎鼠肝脏毒性的影响

目的:研究在孕期暴露PFOS对胎鼠的肝脏毒性的影响.方法:将孕期为12天的16只SD雌性大鼠,随机分为4组给予不同剂量的PFOS[0(对照),5,10,20 mg·kg-1],连续灌胃7天,在GD19天时对母鼠和胎鼠的体重、胎鼠肝脏的生化指标、母鼠血清的生化指标进行了相应的检测.结果:与对照组相比,母鼠体重在20 mg·kg-1组显著下降(P＜0.001);胎鼠的体重和体长在20mg·kg-1组显著下降(P＜0.001);胎鼠的肝脏重量降低,呈剂量依赖性,并伴有肝细胞浊肿、变性甚至坏死;10 mg· kg-1组胎鼠肝脏中的酶活性(ALT、AST、GGT和ALP等)显著升高(P＜0.001);母鼠血清的大部分生化指标未发生明显变化.结论:孕期大鼠暴露在PFOS的环境下会严重损伤胎鼠的肝脏功能.     

复制型HBV转基因小鼠遗传稳定性研究

目的：提高复制型HBV转基因小鼠的遗传稳定性。方法：应用回交传代及双杂交育种法,经荧光定量PCR、ELISA和化学发光法研究HBV基因在小鼠体内的复制与表达。结果：HBV转基因小鼠已稳定传至第23代,血清HBsAg达4122.31±2044.74IU/ml,93.93%的转基因小鼠血清HBV DNA达104-106copies/ml,表达复制水平较早期有显著提高并稳定传代;雌雄小鼠之间表达水平无显著性差异。结论：该转基因小鼠经过培育传代,已成为一个高表达且遗传稳定的复制型HBV小鼠模型。     

用重组8型腺相关病毒载体介导的乙型肝炎病毒持续感染小鼠模型评价核苷类似物的抗病毒效果

探索用重组8型腺相关病毒载体携带1.3拷贝乙型肝炎病毒(rAAV8-1.3HBV)介导的HBV持续感染小鼠模型评价核苷酸类似物抗病毒药物的抗病毒效果.首先,通过将rAAV8-1.3HBV经尾静脉注射到30只C57BL/6小鼠体内,建立HBV持续感染模型并对模型成功率进行检测,将建模成功的27只小鼠随机分成6组.然后采取灌胃的方式给予不同剂量的抗病毒药物恩替卡韦(ETV)及拉米夫定(LAM),每日1次,连续l0d,后停药15d,同时设置生理盐水及空白对照组.其中ETV分为高剂量(1.0 mg/(kg·d))和低剂量(0.1 mg/(kg·d) 两组;LAM分为高剂量(500 mg/(kg·d)) 和低剂量(100 mg/(kg·d)) 两组.检测给药前后和停药前后小鼠模型血清中HBV DNA、HBeAg和HBsAg表达水平并比较变化情况.结果发现连续给药10d后,各给药组与生理盐水组相比,血清中HBV DNA水平均显著下降,具有统计学差异(P＜0.05).停药15d后,低剂量的ETV与LAM两组血清HBV DNA水平出现反弹,差异存在统计学意义(P＜0.05).在整个实验过程中,各组小鼠血清中HBeAg和HBsAg表达水平均未出现明显变化.上述结果表明,ETV和LAM能有效抑制模型小鼠中HBV病毒的复制,而对HBeAg和HBsAg表达水平无明显影响;提示AAV8-1.3HBV介导的HBV持续感染小鼠模型制备简单,成模率高,可有效体现出ETV和LAM抗HBV的作用效果,从而用于核苷酸类似物抗HBV药物的筛查.     

应用慢病毒载体携带HBV基因组DNA制备乙型肝炎病毒感染小鼠模型的影响因素分析

应用慢病毒载体构建不同HBV转导质粒,通过高压水动力法尾静脉注射小鼠,比较不同HBV转导质粒、剂量(5μg和10μg)、小鼠品系(Balb/c和C57BL/6)及鼠龄(6周龄和18周龄)对建立HBV感染模型的影响。不同的时间点尾静脉采血,ELISA检测血清HBsAg、HBeAg的表达水平及动力变化,Real-time PCR检测血清及肝组织病毒载量;免疫组织化学法检测肝组织HBcAg的定位与表达。1.3倍HBV基因组慢病毒载体转导质粒(pCSHBV1.3)优于1.1倍与1.2倍HBV基因组转导质粒(pCS-HBV1.1or pCS-HBV1.2);pCS-HBV1.3注射Balb/c小鼠后抗原表达维持时间短,抗体出现早;pCS-HBV1.3注射C57BL/6小鼠后,HBsAg、HBeAg抗原表达及血清HBV DNA水平维持时间长;且注射5μg质粒相对于10μg质粒注射小鼠后抗原表达维持时间更长;而6周龄和18周龄小鼠血清均可在较长时间内检测到HBsAg、HBeAg及HBV DNA的表达,但在注射后35周内,前者的表达量均高于后者;所有注射质粒的小鼠肝组织中均可检测到HBcAg的表达,且在血清HBV感染标志转阴时均可检测到肝内HBV DNA的存在。注射质粒的HBV基因组长度、剂量以及宿主的遗传背景均对建立乙肝成体转基因小鼠模型有影响,且发现以5μg含1.3倍HBV基因组的转导质粒pCS-HBV1.3注射6周龄C57BL/6小鼠,HBV抗原表达和HBV DNA水平维持时间长,更适合建立HBV持续感染模型。     

大剂量HBsAg 对HBV 转基因小鼠T 细胞免疫调节的研究

目的:研究大剂量HBsAg对HBV转基因小鼠其T细胞免疫效果的影响。方法:用大剂量血源性HBsAg免疫HBV转基因小鼠,采用ELISA方法观察转基因小鼠所诱生的HBsAg特异性Th1类细胞因子的水平,ELISPOT方法检测不同免疫方案对小鼠HBsAg特异性分泌IFN-γT细胞数量的影响,同时检测对小鼠淋巴细胞增殖的影响。结果:HBsAg组免疫后脾细胞产生的Th1类细胞因子(IFN-γ、IL-2)、HBsAg特异性分泌IFN-γT细胞及T细胞增殖水平较对照组显著增加(P<0.05)。结论:大剂量的HBs-Ag可以诱导乙肝转基因小鼠产生高水平Th1类细胞因子并打破免疫耐受。     

用rAAV8-1.3HBV制备两种品系小鼠乙型肝炎病毒感染模型的比较研究

我们先前用rAAV8-1.3HBV静脉注射C57BL/6小鼠成功地制备了慢性乙型肝炎病毒(Hepatitis B virus,HBV)感染模型。为了探讨不同品系的小鼠对rAAV8-1.3HBV静脉注射是否具有不同反应,本研究比较了C57BL/6和BALB/c小鼠静脉注射重组病毒后外周血中HBV抗原和抗体水平、病毒载量和肝脏组织HBcAg表达情况,以及不同剂量重组病毒注射与这些指标的关系。将低(4×109 Viral genome,vg)、中(4×1010vg)和高(4×1011vg)三种剂量的rAAV8-1.3HBV通过尾静脉注射至C57BL/6和BALB/c小鼠,分别利用ELISA和荧光定量PCR方法检测血清中的HBV抗原、抗体水平以及HBV DNA,利用免疫组化检测肝脏组织HBcAg的表达。结果发现,对于C57BL/6小鼠,三种不同剂量rAAV8-1.3HBV注射均可造成100%小鼠出现HBV持续感染;血清HBsAg、HBeAg和HBV DNA以及肝组织HBcAg稳定表达超过8个月,其表达水平随重组病毒注射剂量的增加而升高,高剂量注射时可造成超过40%的肝细胞感染HBV,血清中HBV DNA可达105 IU/mL以上;未检测到针对HBV的抗体。对于BALB/c小鼠,三种不同剂量rAAV8-1.3HBV注射也可造成100%小鼠出现HBV持续感染;血清HBeAg和HBV DNA以及肝组织HBcAg稳定表达超过8个月,但是血清HBsAg在重组病毒注射2周之后显著下降甚至消失;在中剂量注射组的BALB/c小鼠血清中检测到低水平的Anti-HBs;血清HBeAg和肝组织HBcAg的表达水平随重组病毒注射剂量的增加而增高,并且各剂量组表达水平均高于C57BL/6小鼠,高剂量注射时可造成超过50%的肝细胞感染HBV。本研究表明,低至4×109 vg剂量的rAAV8-1.3HBV注射即可造成C57BL/6和BALB/c两种品系小鼠出现HBV持续感染,并且HBV复制水平随重组病毒注射剂量增加而增高;BALB/c小鼠对HBV的免疫反应强于C57BL/6小鼠,可以产生针对HBsAg的体液免疫反应而使血清HBsAg转阴,但无法清除携带HBV的肝细胞。     

大剂量HBsAg对HBV转基因小鼠T细胞免疫调节的研究

目的：研究大剂量HBsAg对HBV转基因小鼠其T细胞免疫效果的影响。方法：用大剂量血源性HBsAg免疫HBV转基因小鼠,采用ELISA方法观察转基因小鼠所诱生的HBsAg特异性Th1类细胞因子的水平,ELISPOT方法检测不同免疫方案对小鼠HBsAg特异性分泌IFN-γT细胞数量的影响,同时检测对小鼠淋巴细胞增殖的影响。结果：HBsAg组免疫后脾细胞产生的Th1类细胞因子（IFN-γ、IL-2）、HBsAg特异性分泌IFN-γT细胞及T细胞增殖水平较对照组显著增加（P〈0.05）。结论：大剂量的HBs-Ag可以诱导乙肝转基因小鼠产生高水平Th1类细胞因子并打破免疫耐受。     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

龙胆科药用植物化学成分的研究现状

龙胆科植物在我国的分布范围很广,且多数为药用植物,其多数种属的药用植物,至今其化学成分尚未被系统研究。综述了目前龙胆科药用植物的化学成分的研究现状及一般提取方法,对近年来发现的环烯醚萜及裂环烯醚萜类化合物进行了总结,为本科药用植物的更深入研究提供了参考。     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

Specificity of action of piperidylcholine derivatives as substrates of cholinesterases of various origin

The review deals with study of enzymologic properties of a novel highly specific acetylcholinesterase substrate, N-(β-acetoxyethyl) piperidinium iodomethylate (“piperidylcholine”), and its 30 derivatives that were tested as effectors of cholinesterases of mammals and various species of Pacific squids. It was proven for the first time that responsible for specificity of action was structure of cyclic ammonium grouping of the alcohol part of molecule of the ester substrate. Analysis of specificity is performed based on enzymatic hydrolysis parameters—activity of catalytic center of cholinesterases and bimolecular constant of the reaction rate that are determined at optimal and low substrate concentrations. Among the specially synthesized group of thioester compounds there is revealed one more highly specific acetylcholinesterase substrate—N-(β-acetoxyethyl) piperidinium.     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.     

医疗机构合理用药评价模型的构建研究

目的 针对医疗机构的合理用药水平进行评价研究。方法 根据医疗机构合理用药的具体要求,构建医疗机构合理用药评价指标体系,采用基于模糊群决策的方法和多指标评价分析法构建医疗机构合理用药评价模型。结果 构建了基于模糊群决策的医疗机构合理用药评价模型,并通过实例分析证明了评价模型的可行性。结论 建立的基于模糊群决策的医疗机构合理用药评价模型能够对医疗机构的合理用药水平进行科学评价,为提高医疗机构合理用药水平奠定基础。