毛冠鹿胚胎有限细胞系的建立及生物学特性研究

建立了毛冠鹿胚胎肺、肾有限细胞系,并对细胞形态、生长速度及核型等生物学特性进行了观察,发现了毛冠鹿新核型,根据C－带分析结果,提出了毛冠鹿中存在B染色体,而核型多态的实质是B染色体的多态。     

大鼠肝细胞系的建立及其特性研究

从 Wistar 新生大鼠肝组织已成功获得正常肝细胞体外长期培养细胞系 NRL-84.细胞生长情况、分裂指数、染色体分析、软琼脂集落形成试验、相差显微镜及扫描电镜等多项指标观察.表明符合正常肝细胞生物学特征;用间接免疫荧光、对流免疫电泳、核酸原位杂交等检测甲胎蛋白,符合正常肝细胞的变动规律.正常新生大鼠的建立是国内首次报导,它将为进一步研究恶性肿瘤的发生发展机制提供了一个良好的实验模型和手段.     

聚苯乙烯薄膜三维培养肝细胞的实验研究

采用三维聚苯乙烯薄膜作为细胞培养支架,在体外进行大鼠肝细胞的三维培养,并与常规二维肝细胞培养进行比较.分析了细胞的生长状态及肝细胞功能,包括细胞存活率、白蛋白分泌功能、尿素合成功能及葡萄糖消耗功能.结果显示:三维细胞增殖活力均始终明显高于二维;在显微镜下观察,可看到三维支架表面黏附生长的肝细胞逐渐增多,有肝细胞黏附成团,且肝细胞保持着良好的形态学结构.研究表明,三维培养的肝细胞其增殖生长和代谢功能都优于同样条件下的二维培养,三维聚苯乙烯薄膜可以作为体外肝细胞培养支架,在生物人工肝培养体系中具有应用前景.     

细胞色素P450与农药相互作用及其机理研究

综述了细胞色素P450的种类和功能多样性,介绍了细胞色素P450参与除草剂代谢及其作用机理．同时,简述了细胞色素P450 14DM与杀菌剂特异性作用的机制．报道了我们从抗除草剂的瑞士黑麦草中克隆CYP8181同源基因及其功能的研究,以及柑橘绿霉菌细胞色素P450 14DM基因的克隆表达．为深入研究杀菌剂作用细胞色素P450 14DM的机理从而设计以P450 14DM为特异性靶标的新型农药、以及进一步研究细胞色素P450酶系与除草剂代谢的关系打下了良好的基础．     

昆虫细胞色素P450的多样性和功能

细胞色素P450是一类广泛存在的催化酶，能够在昆虫体内参与外源性物质的代谢和内源性物质的合成与降解，帮助昆虫进行正常的生理活动。尽管昆虫细胞色素P450的多样性和功能一直是研究的热点，但目前仍缺乏对这个领域的系统总结和归纳。本文综述了昆虫细胞色素P450数量、表达和反应类型的多样性以及细胞色素P450在昆虫中的功能和进化等方面的研究进展，为更好地研究和利用昆虫细胞色素P450的多样性和功能提供有力的参考依据，同时也能促进我们更好地理解昆虫的代谢和生理活动。     

用大鼠心肌条件培养基建立来源于C57BL/6J小鼠的ES细胞系

报道一种新的建立C57BL/6J小鼠ES细胞系的方法。采用大鼠心肌条件培养基,在不使用饲养层细胞和白血病抑制因子(LIF)的情况下,从C57BL/6J品系小鼠中建成1个ES细胞系即MESPU 41,成系率为1.0%。MESPU 41细胞为XX型,核型正常率高达89%,表现出XX型ES细胞系少有的稳定性。进行体内分化实验时MESPU 41细胞能发生广泛分化形成畸胎瘤。嵌合体制作实验证实MESPU 41细胞具有嵌合能力,能参与胚胎的发育。采用RT-PCR方法,检测出大鼠心肌细胞有LIF mRNA的表达,这可能与其条件培养基保持ES细胞未分化状态并使X染色体稳定有关。同时,还对大鼠心肌细胞进行了永生化的尝试,共得到了4个永生化克隆,这将进一步简化ES细胞建系和培养工作,为进一步研究ES细胞在体外培养过程中的稳定性开创了新的起点。     

腺病毒介导的SPINK6表达在肝癌细胞QGY-7703中功能的初步研究

为了探讨SPINK6对肝癌细胞的抑制作用,使用缺陷型腺病毒载体构建载有SPINK6基因的重组腺病毒,感染肝脏肿瘤细胞QGY-7703,上调SPINK6蛋白的表达.通过CCK8细胞增殖实验和软琼脂克隆形成实验证实SPINK6对QGY-7703细胞生长的抑制,通过Transwell实验和细胞划痕试验证实SPINK6对QGY-7703细胞的体外细胞迁移与侵袭能力的抑制,为我们进一步揭示SPINK6的作用机制奠定了基础.     

人脂肪来源的干细胞体外培养特性及分化为肝细胞样细胞的研究

肝脏组织工程的兴起和不断发展使各种急慢性肝病得到彻底的治愈成为可能.而肝脏组织工程的发展很大程度上受制于种子细胞的来源不足.为了寻找理想的肝脏组织工程种子细胞，本实验从吸脂术所得的脂肪抽吸液中分离出干细胞，利用流式细胞仪、染色体核型分析等方法对其生长特性、表面标志及生长的稳定性作了分析；用含有肝细胞生长因子（HGF）和成纤维细胞生长因子（FGF-4）的条件培养基将其向肝细胞样细胞诱导分化，并利用RT-PCR及免疫荧光的方法检测了分化后细胞的标志物CK18，甲胎蛋白（AFP）和白蛋白以及分化后细胞的功能.结果显示，脂肪来源的干细胞不仅有良好的生物学稳定性，而且分化后的细胞表达了肝细胞的标志物且具有一定的肝细胞的功能.这为解决肝脏组织工程种子细胞来源的问题提供了可能.     

人肝癌细胞系HepG2生物学特性分析

人肝癌细胞系HepG2是进行抗癌药物检测及肝细胞生理性状研究的重要细胞系.充分了解该细胞系的生长增殖和遗传特性,对于细胞系的应用具有重要意义.本研究对体外连续传代培养的HepG2细胞,进行了生长曲线、分裂指数、染色体核型等生物学特性分析.结果表明:HepG2细胞在体外培养条件下,生长曲线呈典型的S形,群体倍增时间为24 h;分裂指数最高达4.5%;染色体数目为41～114,染色体众数为54～69(80%).     

人细胞色素P450氧化还原酶的克隆表达和抗体的制备

细胞色素P450还原酶(CPR)是人细胞色素P450酶系的电子供给者,对后者活性的发挥起到重要作用.本研究将从人胚胎cDNA文库中得到的人CPR的编码基因,连接入pT7450表达载体中,在大肠杆菌BL21(DE3)中高效表达并纯化.每升发酵液可得到纯化蛋白49 mg,占总蛋白含量的10%,以细胞色素C为底物检测酶活性,比活为65 u/mg.利用纯化的CPR作为抗原,常规免疫纯系大耳家兔,获得高效价、高特异性的抗血清,抗体滴度为1∶200 000(ELISA法),纯化后抗体应用于不同组织中CPR含量的评价,证明重组CPR及其抗体可用于细胞色素P450的体外药物代谢及细胞色素P450与CPR作用机理的研究.     

Agar coating in multicellular spheroids culture of rat hepatocytes

Aggregation of freshly isolated adult rat hepatocytesin vitro is important for the construction of artificial liver support system. In the experiment, agar has been used as an extracellular matrix substrate and results demonstrate that hepatocytes in serum-free culture medium can effectively form floating spheroids in tissue culture dishes coated with agar, which maintain higher ability to produce urea and albumin than monolayer cultured cells.     

Odorant signal termination by olfactory UDP glucuronosyl transferase

The onset of olfactory transduction has been extensively studied, but considerably less is known about the molecular basis of olfactory signal termination. It has been suggested that the highly active cytochrome P450 monooxygenases of olfactory neuroepithelium are termination enzymes, a notion supported by the identification and molecular cloning of olfactory-specific cytochrome P450s (refs. 13-16). But as reactions catalysed by cytochrome P450 (refs 17, 18) often do not significantly alter volatility, lipophilicity or odour properties, cytochrome P450 may not be solely responsible for olfactory signal termination. In liver and other tissues, drug hydroxylation by cytochrome P450 is frequently followed by phase II biotransformation, for example by UDP glucuronosyl transferase (UGT), resulting in a major change of solubility and chemical properties. We report here the molecular cloning and expression of an olfactory-specific UGT. The olfactory enzyme, but not the one in liver microsomes, shows preference for odorants over standard UGT substrates. Furthermore, glucuronic acid conjugation abolishes the ability of odorants to stimulate olfactory adenylyl cyclase. This, together with the known broad spectrum of drug-detoxification enzymes, supports a role for olfactory UGT in terminating diverse odorant signals.     

细胞色素P450酶系的异源表达研究

传统的药物代谢主要以实验动物为对象进行药物早期及临床研究,近几年来,结合生物化学与分子生物学的发展,药物早期代谢研究进入到药物体外代谢的研究阶段,主要围绕人肝内参与代谢的细胞色素P450家族展开.本文就近年来对此家族酶的异源表达及其在药物代谢中的应用进行了综述.     

野生型人DNA polβ高表达中国仓鼠卵巢细胞系的建立及其生物学特征

脂质体转染法将野生型人DNA聚合酶beta(polβ)重组绿色荧光蛋白表达载体转染入中国仓鼠卵巢细胞系(CHO),经G418筛选得到稳定高表达人野生型polβ的CHO细胞.通过RT-PCR方法检测转染细胞polβmRNA的表达水平,流式细胞仪测细胞周期,软琼脂实验测细胞恶性增殖能力,6-TG实验测细胞自发突变率,以探讨转染细胞的生物学特性.结果显示,转染细胞polβ的mRNA的表达较空载体转染细胞、对照细胞增加,细胞周期多被阻滞在S期,细胞软琼脂集落形成率增高,自发突变率增加,表明polβ的过表达与细胞周期的分布、细胞恶性增殖程度、遗传稳定性相关.     

凹土玉米芯垫料对大鼠肝微粒体细胞色素P450 酶系的影响

摘要: 目的观察凹土玉米芯垫料对大鼠肝脏细胞色素P450( CytP450) 、细胞色素b5( Cytb5) 含量及大鼠肝微粒 体CYP1A2 和CYP2E1 活性的影响。方法将SD 大鼠随机分为普通刨花组、玉米秸组、凹土玉米芯组和对照组,饲 养30 d、60 d、90 d 时测定大鼠肝脏微粒体CytP450、Cytb5 的含量和CYP1A2,CYP2E1 活性。结果60-90 d 时普 通刨花组、玉米秸组与空白对照组之间CytP450 含量有显著性差异( P ＜ 0. 05) ,凹土玉米芯组与对照组之间没有显 著性差异( P ＞ 0. 05) ; 肝微粒体Cytb5 含量、CYP1A2、CYP2E1 活性各组均没有明显差异( P ＞ 0. 05) 。结论凹土玉 米芯对大鼠肝脏细胞微粒体CytP450、Cytb5、CYP1A2 和CYP2E1 没有诱导或抑制作用,普通刨花组对大鼠肝 CytP450 有较强的诱导作用,玉米秸也有不同程度的诱导作用,但低于普通刨花的诱导作用。     

Cytochrome P450 Monooxygenase Immobilization as a Model of Herbicide Metabolism in vitro

To investigate herbicide metabolism in vitro by cytochrome P450 with stable enzymatic activity, cy-tochrome P450 is immobilized in silk fibroin. The enzymatic activity of immobilized cytochrome P450 is maintained above 80% after repeated batch experiments for 10 times. Moreover, the enzymatic activity of immobilized cytochrome P450 is kept as relatively high as 73.8% after storage for two months at 4℃. In addition, immobilization can improve the temperature and pH stability of cytochrome P450. Immobilized cytochrome P450 has the similar affinity Km values for herbicide chlorsulfuron and triasulfuron as the free cytochrome P450. In the case of chlorosulfuron, affinity Km value is 53μmol/L for free cytochrome P450,and 63μmol/L for immobilized cytochrome P450, respectively. In the case of triasulfuron affinity, Km value is 36μmol/L for free cytochrome P450, and 44μmol/L for immobilized cytochrome P450, respec-tivily. Immobilized cytochrome P450 will be convenient, rapid, stable and continuous for herbicide metabolism in micro-bioreactor in vitro.     

Docking and molecular dynamics studies on CYP2D6

Drug-metabolizing enzymes, also known as cytochrome P450s, are a superfamily of hemoglobin responsible for metabolizing more than 90% clinical drugs. Cytochrome P450 2D6 (CYP2D6) is a significant member of cytochrome P450s for the reason of metabolizing about 20% clinical drugs. In this paper, molecular docking and molecular dynamic simulations are used to investi- gate the active site of CYP2D6, roles of essential amino acids within the active site and time-dependent protein energy changes. The results suggest that amino acids Glu216, Asp301, Ser304 and Ala305 in the active site are likely to form hydrogen bonding interac-tions with substrates; the benzene ring of Phe120 and aromatic ring in the substrates form ∏-∏ interactions. In addition, molecular dynamics simulations prove that the catalytic conformation of CYP2D6 without ligands can be obtained by their own atomic fluctuations. The impact of ligands on protein system energy and large conformational shift is not very large. Cytochrome P450s is known for their genetic polymorphisms, which will result in severe adverse drug reactions. Ideally, we hope to use mo- lecular modeling to investigate the differences between the substrates of wild-type and mutants while they are bonded with drugs, and predict the drug metabolizing ability of mutants. Reduce the possibility for people taking drugs that they can not metabolize, therefore reduce the rate of adverse drug reactions, and eventually establish a platform of personalized drugs to largely benefit human health.     

Suppression of tumorigenicity in human colon carcinoma cells by introduction of normal chromosome 5 or 18.

Development of colon carcinomas can be associated with allelic deletions on several chromosomes, including 5q and 18q. The APC gene on 5q and the DCC gene on 18q have been identified as potential tumour suppressor genes, whose suppression contributes to colon carcinogenesis. To investigate the role of genes in these deleted regions, we have now introduced a single normal human chromosome into a human colon carcinoma cell line, COKFu, through microcell hybridization. Several clones of hybrid cells containing normal chromosome 5, and others containing normal chromosome 18, were obtained. The morphology of the hybrid cells was markedly altered: the hybrids with chromosome 5 exhibited a closely packed polygonal morphology, and the hybrid cells with chromosome 18 were flattened. The cloning efficiency of the hybrid cells in soft agar was reduced from 0.46 to 0% of that of the parental carcinoma cells, and the tumorigenicity of these hybrid cells in athymic nude mice was completely suppressed. The growth properties of the hybrid cells with chromosome 11 were not substantially changed. These results strongly suggest that the genes on normal chromosome 5 and 18 function as tumour suppressors in colon carcinogenesis.