运动对瘦素水平的影响

瘦素是一种主要由脂肪细胞分泌的蛋白质激素,由146个氨基酸组成,研究表明它对动物摄食、能量代谢、脂肪存储、生殖活动等方面都有调控作用.文章讨论了瘦素的结构和功能、影响瘦索水平的因素等,着重探讨了急性运动及长期运动训练对瘦索水平的不同影响,并对未来瘦素与运动的研究前景提出了展望.     

Sry基因的克隆及其在HeLa细胞中的表达

目的:构建真核表达重组质粒pCR3.1-Sry.并检验其在真核细胞内的表达.方法:用PCR法从小鼠基因组中扩增出Sty全长基因,重组入pMD18-T载体,酶切鉴定重组质粒,对酶切正确的重组质粒测序.双酶切测序验证的重组质粒.将切下的片段与真核表达质粒pCR3.1相连构建pCR3.1-Sty重组质粒.将其转染HeLa细胞后,RT-PCR法检测重组质粒在细胞中mRNA的转录;免疫荧光法检测重组质粒在细胞中蛋白质的表达.结果:PCR法扩增得到了1.2 kb的特异性Sry基因片段;成功地构建了真核表达重组质粒pCR3.1-Sry;重组质粒pCR3.1-Sry在HeLa细胞中mRNA及蛋白水平均可检测到表达.结论:重组质粒构建成功并可以在体外真核细胞中表达,为进一步研究其作为核酸疫茁的免疫作用提供了可控的实验材料.     

草鱼生长激素基因在COS7细胞中转染表达的研究

应用RT-PCR技术克隆草鱼生长激素(gcGH)的cDNA,将此cDNA定向插入真核表达载体VR1020中,构建成重组真核表达质粒VgcGH.利用脂质体法使质粒VgcGH转染哺乳动物细胞COS7,对转染后的COS7细胞进行RT-PCR、ELISA和免疫荧光检测,分别在转录和翻译水平证实gcGH基因在COS7细胞中得到了持续和正确的转染表达.     

柔嫩艾美耳球虫Serpin基因在293T细胞中的表达

为了检测柔嫩艾美耳球虫(Eimeria tenella,Et)丝氨酸蛋白酶抑制剂(Serine protease inhibitor,Serpin)基因(EtSerpin)在真核细胞293T中的表达情况,以Et孢子化卵囊cDNA为模板,经PCR扩增含完整Serpin开放阅读框的序列,将其克隆至pGEM-Teasy载体,构建pGEM-Teasy-EtSerpin质粒,双酶切回收目的片段后与相应酶切的真核表达载体pCAGGS连接,构建真核重组表达质粒pCAGGS-EtSerpin.该重组质粒经酶切和测序鉴定正确后转染293T细胞,用间接免疫荧光法和Western blot鉴定EtSerpin基因的表达情况,间接免疫荧光实验可以检测到红色荧光,Western blot结果显示出大小约45.5kDa的目的蛋白条带,结果表明构建的EtSerpin可以在293T细胞中获得表达.     

猪传染性胃肠炎病毒S基因B和C抗原位点片段在昆虫细胞中的胞内表达

通过基因重组的方法,在昆虫细胞胞内表达了猪传染性胃肠炎病毒（TGEV）S基因B和C抗原位点片段.表达蛋白经Dot-ELISA检测具有良好的抗原性.本研究为TGEV的血清学检测方法的建立提供了必要的物质基础.     

IL-21/4-IgG4融合基因的真核表达

利用新型细胞因子IL-21的变异体IL-21/4与IgG4构成融合基因IL-21/4-IgG4. 将构建好的融合基因载体poptivec- IL-21/4-IgG4转染到中华仓鼠卵巢细胞中,成功并稳定表达了IL-21/4-IgG4融合蛋白.     

IL-21/4—IgG4融合基因的真核表达

利用新型细胞因子IL-21的变异体IL-21/4与IgG4构成融合基因IL-21/4—IgG4。将构建好的融合基因载体poptivec- IL-21/4—IgG4转染到中华仓鼠卵巢细胞中，成功并稳定表达了IL-21/4—IgG4融合蛋白，证明了此融合蛋白真核表达的可行性，并为以后进一步研究，以及大量生产和应用IL-21/4打下了基础。     

人类8型疱疹病毒K1基因真核表达载体的构建

为构建人类8型疱疹病毒(Human herpesvirus 8,HHV-8)K1基因的真核表达载体,采集卡波氏肉瘤(Kaposi's sarcoma,KS)患者血清,采用柱式病毒DNA抽提试剂盒抽提血清中的病毒DNA,采用套式PCR技术检测HHV-8的特异性片断KS330233以判断是否得到了HHV-8 DNA,采用PCR技术扩增HHV8 K1基因并纯化回收,酶切、连接入真核表达载体pcDNA3.1,转化大肠杆菌DH5α,挑取阳性克隆pcDNA3.1/HHV8-K1扩增后,提取质粒,进行双酶切和DNA测序鉴定.结果显示采集了经典型KS血清3例,提取了血清HHV-8DNA,扩增了特异性片断KS330233,测序结果正确提示我们得到了HHV8 DNA,扩增了HHV8 K1基因,构建了pcDNA3.1/HHV8-K1真核表达载体,经限制性内切酶酶切鉴定及测序分析证实了其序列的正确性.由此可知:成功构建了pcDNA3.1/HHV8-K1真核表达载体,为进一步研究该基因的功能奠定良好的实验基础.     

人IGFBP-1基因真核表达载体的构建及其对胃癌细胞BGC-823增殖的影响

目的构建IGFBP-1真核表达载体,观察IGFBP-1基因真核表达载体转染胃癌细胞株BGC-823后基因的表达及其对BGC-823细胞增殖的影响.方法以GES-1细胞基因组为模板,PCR技术扩增人IGFBP-1的基因片段,克隆人pc DNA3-His-Flag真核表达载体,重组载体pc DNA3-IGFBP-1-His-Flag,并通过PCR、双酶切和测序鉴定.重组质粒经阳离子脂质体法转染BGC-823细胞,RT-PCR和Western blot检测细胞IGFBP-1的表达情况,并通过CCK8法观察IGFBP-1过表达对BGC-823细胞增殖的影响.结果经PCR、双酶切和测序鉴定证明IGFBP-1真核表达载体构建成功,并在胃癌细胞株BGC-823高效表达.CCK8实验显示:IGFBP-1过表达对BGC-823细胞增殖无影响.结论成功构建IGFBP-1真核表达载体,IGFBP-1过表达对BGC-823细胞的增殖无影响,可为进一步研究IGFBP-1生物学功能奠定基础.     

PRV闽A株gE基因抗原编码区片段在昆虫细胞中的表达研究

伪狂犬病病毒(PRV)糖蛋白E是一种在伪狂犬病的根除计划中具有重要作用的糖蛋白.将伪狂犬病病毒闽A株gE基因抗原编码区片段克隆到杆状病毒Bac-to-Bac系统表达载体pFastBacHTa中,获得的重组表达载体pFastBacHTa-FS转化大肠杆菌DH10BAc感受态细胞后,得到的重组Bacmid DNA在脂质体介导下转染粉蚊夜蛾Hi5细胞,通过细胞内同源重组,获得了含目的片段的重组病毒.此重组病毒感染Hi5细胞后72 h,细胞总蛋白的SDS-PAGE与Western-Blot结果表明,gE基因抗原编码区片段在Hi5细胞中得到了正确表达,表达产物约35000,占细胞总蛋白的9.31%.溶解性分析显示该片段在Hi5细胞中为不可溶性表达.     

Cotransfection of ICAM-1 and HLA-DR reconstitutes human antigen-presenting cell function in mouse L cells

The initiation of a specific immune response is believed to require not only activation through antigen-specific receptors on T cells and B cells but also antigen-independent interactions between accessory molecules. One such molecule is LFA-1, which enhances the avidity of interactions between T cells and antigen-presenting cells, and is possibly involved in signal transduction across the T-cell membrane. Intercellular adhesion molecule-1 (ICAM-1), a surface glycoprotein of relative molecular mass (Mr) 80,000-110,000, has been defined as a ligand for LFA-1, and has been shown to participate in the interaction between T cells and monocytes. The determination of the precise contribution of such accessory molecules to antigen presentation, however, is complicated by the need to analyse against a background of multiple molecular interactions. We have investigated the role of LFA-1/ICAM-1 interactions in antigen presentation directly by quantifying the contribution of ICAM-1 expression to T-cell stimulation using L-cell transfectants that co-express ICAM-1 and HLA-DR. In the case of transfectants expressing modest levels of HLA-DR, co-expression of ICAM-1 is critical for effective HLA class II-restricted and allospecific T-cell activation, pointing to an important role for ICAM-1 in the induction of T-cell responses.     

鼠胚肝和全胚细胞悬液对荷瘤小鼠的影响

目的：试验鼠细胞悬液和全胚细胞悬液对荷瘤小鼠的影响。方法：本试验用TA2系小鼠孕中期和孕末期胚肝细胞,悬液及全胚细胞悬液,经尾静脉注射于荷瘤小鼠（乳腺癌、纤维肉瘤、艾氏腹水痛）。观测移植瘤生长情况和支持机体存活时间的作用。结果：经实验观察及统计学处理、全胚细胞悬液及胚肝细胞悬液输注荷瘤小鼠均未见到抑瘤作用及支持机体延长寿命的作用。结论：我们的试验结果可能预示着细胞疗法对肿瘤的治疗是有限的,需进一步研究以便做出客观的评估。     

Introduction of new genetic material into pluripotent haematopoietic stem cells of the mouse

An infectious retrovirus vector has been used to transfer a bacterial gene encoding resistance to the neomycin analogue G418 into pluripotent haematopoietic stem cells present in explanted murine bone marrow tissue. Subsequent transplantation of the cells into lethally irradiated mice results in engraftment of the animals with donor haematopoietic tissue containing the bacterial gene. This approach affords an efficient and rapid means of re-introducing genetically modified tissue into intact organisms and provides a system whereby the expression and regulation of cloned genes can be followed within the context of a well characterized developmental programme.     

沙塘鳢不同组织同工酶生化表现型及遗传基础

采用垂直板聚丙烯酰胺凝胶电泳方法，对沙塘鳢（Odontobutis abscura）的消化道、心脏、肌肉、脾脏、肾脏、脑、肝脏、生殖腺和晶体9种组织的4种同工酶(EST、LDH、ADH、MDH)进行了研究，讨论了这几种同工酶在沙塘鳢不同组织中的分布以及遗传基础。EST有5个基因位点，单体酶；LDH只有1个基因位点，为四聚体酶；MDH为由3个基因位点控制的二聚体酶；ADH亦为二聚体酶，但仅在肝脏中得到表达。结果表明，同工酶的表达具有组织特异性。     

氯化汞染毒小鼠肝脏的病理形态与超微结构变化研究

采用氯化汞染毒的方法,对昆明种小白鼠的肝脏进行了显微及亚显微结构研究,结果表明,氯化汞对小鼠肝脏有明显的毒性作用,主要表现为线粒体肿胀,凝聚;仙质网脱核糖颗粒;次级溶酶体,脂滴数量增多,糖元颗粒减少等一系列的病理学变化,其病变程度呈剂量－反应关系。     

Attenuating effect of daidzein on polychlorinated biphenyls-induced oxidative toxicity in mouse testicular cells

The attenuating effect of daidzein （DAI） on oxidative toxicity induced by Aroclor 1254 （A1254） was investigated in mouse testicular cells. Cells were exposed to A1254 alone or with DAI. The oxidative damage was estimated by measuring malondialdehyde （MDA） formation, superoxide dismutase （SOD） activity and glutathione （GSH） content. Results show that A1254 induced a decrease of germ cell number, an elevation in thiobarbituric acid reactive substances （TBARS） but a decrease in SOD activity and GSH content. However, simultaneous supplementation with DAI decreased TBARS level and increased SOD activity and GSH content. Consequently, dietary DAI may restore the intracellular antioxidant system to attenuate the oxidative toxicity of A 1254 in testicular cells.     

Expression of dopaminergic phenotypes in the mouse olfactory bulb induced by the calcitonin gene-related peptide

In the olfactory bulb, tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines, is expressed after birth when the axons of olfactory epithelial neurons have made synapses in the bulb. It has been suggested that expression of TH is regulated trans-synaptically because on deafferentation of the bulb there is a marked decrease in the contents of TH, dopamine and 3,4-dihydroxyphenylacetic acid, which, however, return to normal levels after regeneration of the primary afferents. To date the molecular signalling involved in this trans-synaptic induction has not yet been characterized; I have therefore studied the expression of dopaminergic properties (presence of TH and dopamine uptake) in dissociated cell cultures from embryonic mouse olfactory bulb. I report that the number of dopaminergic cells increases fivefold when olfactory bulb neurons are co-cultured with olfactory epithelial neurons and that soluble factors, rather than cell interactions, mediate this effect. The dopaminergic-inducing factor is the calcitonin gene-related peptide (CGRP) which is present in chemosensory neurons of the olfactory epithelium and when added at nanomolar concentrations to olfactory bulb cultures mimics the effect of olfactory epithelial neurons. Significantly the induction of dopaminergic phenotypes brought about by olfactory epithelial neurons is abolished by an antiserum to CGRP. These observations show that CGRP is involved in the differentiation of dopaminergic olfactory bulb neurons.     

小鼠胎儿成纤维细胞的冷冻保存研究

为了摸索小鼠胎儿成纤维细胞冷冻保存的简易方法,采用常规冷冻和玻璃化冷冻2种模式,探讨了不同的冷冻方法、不同的冷冻前平衡时间、不同的冷冻保护液的配比等因素对成纤维细胞冷冻效果的影响。实验结果表明:常规冷冻采用含10%(体积分数)甘油的DMEM液作保护剂,可将0~5℃下平衡时间缩短至45~60 min,细胞活力可达0.87。玻璃化冷冻采用10%(体积分数)NBS+1.8 mol/L乙二醇+0.1 mol/L蔗糖的PBS冷冻小鼠胎儿成纤维细胞效果好于其他组合,细胞活力可达到0.86。玻璃化冷冻法与常规冷冻法的冷冻效果差异不显著。