钐在小鼠肝脏细胞中的动态观察

本实验给小鼠一次静脉注射氯化钐(70mg/kg体重)后15min至48h中的不同间期,应用电镜与X射线微区分析术对钐在肝脏枯否细胞与肝细胞中的运转进行了动态追踪。于15min 至2 h,两种细胞均以胞吞方式摄入含钐微粒,在胞质中形成吞噬体。在吞噬体中,微粒群处于由稀疏至密集的浓缩过程。小吞噬体亦互相融合。这种胞吞作用于枯否细胞极为活跃。于4—24 h,很多枯否细胞胞质充满吞噬体,细胞已经或趋于变性、崩解。肝细胞内的吞噬体则汇集于胆小管周围。于胆小管腔中可见到高电子密度微粒群,表明体内钐可经胆汁途径排出。于48 h,两种肝脏细胞巾仍见钐吞噬体沉积。     

肝脏细胞在生物活性物质代谢调控中的协作作用

肝实质细胞和非实质细胞是肝脏功能的物质和结构基础.肝实质细胞是肝脏的主要细胞类型之一,承担和执行肝脏代谢、信号转导、解毒和稳态调节等多种功能.而非实质细胞也必不可少,主要包括星型细胞、肝窦内皮细胞、枯否细胞、自然杀伤细胞和隐窝细胞等,具有物质和信号转运、吞噬、抗原提呈、免疫耐受等功能.目前,有关肝脏生理功能和病理机制的研究主要集中在组织、细胞和差异分子的水平,单细胞的生理和病理功能研究也越来越深入,而肝脏细胞协作的研究比较少,系统性总结也鲜有报道.本文从肝脏细胞相互协作的角度,对肝脏的生理功能及病理机制进行探讨,为全面了解肝细胞生理功能及肝脏疾病的致病机理提供参考.     

定向诱导小鼠ES细胞为肝脏细胞及其凝血因子Ⅷ, Ⅸ的表达

凝血因子Ⅷ, Ⅸ缺陷导致血友病A和B的发生, 肝细胞是产生凝血因子的器官, 利用肝细胞进行替代治疗是纠正凝血因子缺陷的根本办法, 但是其来源及利用存在难以克服的问题. ES细胞具有体外培养时保持未分化状态和无限的增殖能力, 具有在一定条件下可以分化发育成各个胚层细胞的潜能, 利用ES细胞源性肝细胞作为供体细胞, 解决了肝细胞来源困难、增殖能力差的难题. 体外分阶段定向诱导E14小鼠ES细胞分化为肝细胞, ICG摄取及PAS反应结果证实ES源性细胞具备肝细胞功能, 在此基础上, 利用RT-PCR法对诱导细胞初步进行了凝血因子Ⅷ, Ⅸ表达分析, 为进一步开展利用ES细胞纠正凝血因子缺乏奠定了研究基础.     

p16在HBsAg和HBx转基因小鼠肝脏肿瘤中的表达

目的:探讨p16基因在由乙型肝炎病毒基因整合引起的小鼠肝细胞癌发生发展中的表达变化规律。方法:以乙肝病毒表面抗原基因(HBsAg)及X基因(HBx)定位整合转基因小鼠及对照小鼠的肝脏组织为标本,利用North-ern印迹、Western印迹及免疫组织化学检测p16在乙肝病毒基因定位整合转基因小鼠肝脏正常组织与肿瘤组织中的差异表达。结果:p16主要在小鼠胚胎期的肝脏中表达,在新生小鼠和成年小鼠的肝脏组织中几乎检测不到其表达;在HBsAg转基因小鼠和HBx转基因小鼠的肝脏肿瘤中,p16的表达明显升高。结论:p16基因在HBsAg或HBx诱导的肝细胞癌发生过程中被重新激活,也许发挥重要的作用。     

昆虫血细胞功能、形态及细胞免疫反应研究前沿

细胞免疫反应是昆虫先天免疫系统的重要组成部分,与体液免疫反应共同作用以防御外源物。不同类群的昆虫其血细胞种类不同,空间形态及免疫应答功能也各具特征,但在细胞免疫中大都发挥着吞噬、结节与包囊作用。本文根据国内外的研究,对昆虫血细胞的类型、功能、形态、吞噬过程、细胞表面吞噬受体、以蚜虫为代表的不完全变态昆虫免疫学和影响蚜虫免疫系统的共生体等研究动态进行了综述,以期为害虫防治提供思路和防治策略。     

小鼠ALB启动子/增强子驱动HSV-tk 对肝脏细胞的杀伤效应

利用小鼠白蛋白(ALB)启动子／增强子及单纯疱疹病毒胸苷嘧啶激酶(HSV-tk)DNA构建了载体pLLTK,以研究该载体对肝脏细胞的特异性杀伤效应。首先,为了比较载体的肝脏细胞特异转录活性,以绿色荧光蛋白(GFP)基因为报告基因构建了载体pLE(仅含小鼠ALB启动子)、pLLE(含小鼠ALB启动子和上游增强子)和pLEL(含小鼠ALB启动子和下游增强子),分别转染到人肝细胞株Hep—G2与小鼠乳腺上皮细胞株HC-11,荧光显微镜与流式细胞术分析GFP的表达。然后将载体pLLTK转染到Hep-G2研究对细胞的杀伤效应。结果发现：小鼠ALB启动子／增强子能驱动GFP肝脏特异表达;HSV-tk在Hep-G2表达使细胞具有更昔洛韦(GCV)敏感性,在GCV作用7d后,MTT分析细胞的生存率,pLLTK转染细胞表现明显的细胞死亡(53％),而阴性对照组pcDNA3．1转染细胞没有明显变化(仅2％细胞死亡)。以上结果表明所有的载体具有肝脏细胞特异性,为利用该载体产生肝脏损伤的转基因小鼠提供了细胞水平的实验依据。     

小鼠动物实验方法系列专题(二) 肝脏大部分切除实验在小鼠肝再生研究中的应用

小鼠肝大部分切除(partial hepatectomy,PH)实验是研究肝再生的一个重要的实验。本文以C57小鼠为例,对肝大部分切除实验做了较为详细的介绍。实验结果显示,在术后的1～8天,小鼠的肝脏体重比值逐渐增加,在术后的7～10天里可以达到原来肝重的90%以上,10天以后肝细胞停止分裂。正常情况下,实施肝大部分切除后,小鼠的存活率可以达到90%以上。该模型的建立为研究肝脏再生的细胞和分子生物学机制奠定了基础。     

芳香烃受体在非酒精性脂肪性肝脏疾病中的作用

非酒精性脂肪性肝脏疾病(nonalcoholic fatty liver diseases,NAFLD)是目前备受关注的一种肝脏疾病。肥胖、2型糖尿病、高脂血症等是NAFLD的重要危险因素,但其发病机理仍不十分清楚。芳香烃受体(aryl hydrocarbon receptor,AHR)是由配体激活的转录因子,其在多种重要疾病活动中发挥了重要作用。近年来多项研究表明AHR激活促进了NAFLD的发病进展,对AHR参与NAFLD发病机制的探讨将有利于进一步阐明NAFLD的发病机理,为NAFLD的防治提出新的思路。本文就AHR与NAFLD关系的研究进展做一综述,以期为该领域的研究提供新的方向。     

细胞凋亡在肝脏衰老中的作用

细胞凋亡是细胞的一种程序性死亡过程,存在死亡受体主导的外源性凋亡途径和细胞器触发的内源性凋亡途径。细胞凋亡主要清除异常细胞以保证机体的正常生理功能,其作用与凋亡程度有关。肝脏衰老是一种随年龄增长的自然进程,通常伴随着肝脏合成与分解代谢等功能的降低。肝脏的高强度代谢活动使其容易受到外源性和内源性的应激刺激,进而激活肝脏细胞p53等基因,通过相关的信号通路发生自噬、细胞周期阻滞和DNA修复等,最终细胞可能清除应激,也可能因DNA损伤导致衰老、凋亡甚至癌变。细胞凋亡和衰老在肝脏中有保护与损害双重作用。一方面,凋亡和衰老可使细胞避免癌变;另一方面,凋亡可加剧因衰老造成的肝脏功能减退,而且凋亡失衡时可能导致非酒精性脂肪肝疾病、肝纤维化、肝硬化、肝癌等肝脏衰老相关的疾病。本文就近年来有关细胞凋亡在肝脏衰老期间可能存在的保护、损害作用以及在老年性肝病发生机制中的作用进行了分析和总结。     

小鼠骨髓树突状细胞对白念珠菌吞噬、杀伤作用的研究

目的体外分离培养小鼠骨髓树突状细胞(Dendritic Cell,DC)并与白念珠菌共同孵育,研究不同浓度的DC对白念珠菌的吞噬、杀伤作用,并研究随着共孵育时间的延长DC表型的变化及其吞噬、杀伤白念珠菌能力的变化。方法无菌条件下分离培养C57小鼠骨髓DC;倒置显微镜下观察DC的形态;Cellometer Mini细胞计数仪测定DC生长曲线及细胞直径变化;流式细胞仪检测DC及白念珠菌刺激后DC表面标志物CD11c及其表面分子MHCⅡ、CD40、CD80及CD86的表达;光学显微镜、激光共聚焦显微镜及流式细胞仪测定DC对白念珠菌的吞噬及杀伤作用。结果体外培养10d小鼠骨髓DC,倒置显微镜观察可见典型DC形态;随着培养时间的延长,细胞逐渐增大,培养第10天时,细胞直径分布最多的为10.2μm;生长曲线显示,DC呈倍数增长,第2~9天为细胞对数生长期,第9天左右增殖曲线达到顶峰。培养第10天,未经白念珠菌刺激的DC表面CD11c分子及CD80分子高水平表达,CD40、CD86及MHCⅡ低表达;经白念珠菌刺激后,DC表面CD11c分子、CD80、CD40、CD86及MHCⅡ均高表达,呈成熟DC表型。激光共聚焦(Laser Scanning Confocal Microscope,LSCM)及流式细胞仪(Flow Cytometer,FCM)检测DC对白念珠菌吞噬作用,当DC与白念珠菌的比例为1∶1时,随着共培养时间的延长(30min~1.5h),吞噬率无明显变化(P=0.063);当DC与白念珠菌比例为2∶1、1∶2及1∶4,随着DC细胞所占比例的减小,DC细胞吞噬作用增强,当比例为1∶4时,DC吞噬作用最强,且随着共培养时间的延长(30min~1.5h),吞噬率也明显上升,差异有统计学意义(P=0.003;P=0.002;P=0.002)。显微镜检测DC对白念珠菌杀伤率,可见随着时间的延长,各实验组的杀伤率下降(P=0.000);当DC∶白念珠菌=1∶1时,较2∶1时,杀伤率明显下降,但随着白念珠菌比例在一定范围内的增高(从1∶1到1∶4),杀伤率明显增高,差异有统计学意义(P=0.000)。结论小鼠骨髓细胞体外经GM-CSF诱导可培养DC具有较高的纯度,呈未成熟细胞表型;且每只C57小鼠可获得3×107总数的细胞,可满足一些实验的DC用量。经白念珠菌刺激后DC表型成熟;在一定的比率范围内,DC对白念珠菌有较强的吞噬及杀伤作用,其吞噬作用随着DC与白念珠菌比例的减小而增强。且随着两者作用时间的延长,DC对白念珠菌的吞噬作用逐渐增强而杀伤作用逐渐减弱。     

Cultivation of embryonic mouse liver epithelium]

Mouse embryo liver epithelial cells maintained in vitro were connected with each other by highly permeable cell-to-cell contacts, synthesized alpha-fetoprotein and possessed the property of contact inhibition of phagocytosis. Methods of cultivation of these cells are presented in this paper.     

Short-term metabolic fate of [13N]ammonia in rat liver in vivo

The short-term metabolic fate of [13N]ammonia in the livers of adult male, anesthetized rats was determined. Following a bolus injection of tracer quantities of [13N]ammonia into the portal vein, the single pass extraction was approximately 93%, in good agreement with the portal-hepatic vein difference of approximately 90%. High performance liquid chromatographic analysis of deproteinized liver samples indicated that labeled nitrogen is exchanged rapidly among components of: mitochondrial aspartate aminotransferase and glutamate dehydrogenase reactions and cytoplasmic aspartate aminotransferase and alanine aminotransferase reactions (t1/2 for the exchange of label toward equilibrium is on the order of seconds). Comparison of specific activities of glutamate and ammonia suggests that at 5 s most labeled glutamate was mitochondrial, whereas at 60 s approximately 93% was cytosolic; this change is presumably brought about by the combined action of the mitochondrial and cytosolic aspartate aminotransferases and the aspartate carrier of the malate-aspartate shuttle. Specific activity measurements of glutamate, alanine, and aspartate are in accord with the proposal by Williamson et al. (Williamson, D.H., Lopes-Vieira, O., and Walker, B. (1967) Biochem. J. 104, 497-502) that the components of the aspartate aminotransferase reaction are in thermodynamic equilibrium, whereas the components of the alanine aminotransferase reaction are in equilibrium but compartmented in the rat liver. Despite considerable label in citrulline at early time points, no radioactivity (less than or equal to 0.25% of the total) was detected in carbamyl phosphate, suggesting very efficient conversion to citrulline with little free carbamyl phosphate accumulating in the mitochondria. Our data also show that some portal vein-derived ammonia is metabolized to glutamine in the rat liver, but the amount is small (approximately 7% of that metabolized to urea) in part because liver glutamine synthetase is located in a small population of perivenous cells "downstream" from the urea cycle-containing periportal cells. Finally, no tracer evidence could be found for the participation of the purine nucleotide cycle in ammonia production from aspartate. The present work continues to emphasize the usefulness of [13N]ammonia for short-term metabolic studies under truly tracer conditions, particularly when turnover times are on the order of seconds.     

Progressive polyploidy in mouse liver following repeated hepatectomy]

Mouse liver regeneration after partial hepatectomy results in sharp changes of ploidy classes towards the increase of high ploidy cells and the decrease low ploidy ones. These changes retain during three months. Each following partial hepatectomy (till 3 times) intensifies the hepatocyte polyploidy with appearance of cells with 32--64 ploidy nuclei. The cell polyploidization stimulated by repeated regenerations is similar to that observed in normal postnatal liver growth.     

Chromocenters of interphase nuclei in the mouse liver contain satellite DNA]

In isolated interphase mouse liver nuclei after hypotonic treatment only the chromocenters belonging to the pericentromeric heterochromatin remain in dense form while the main mass of a chromatin is completely decondensed. The centromeric nature of these chromocenters is demonstrated by their capability for C-banding and for hybridization with a satellite mouse DNA.     

Immunohistochemical study of cardiolipin and phosphatidylinositol in mouse liver]

Localization of two phospholipid haptens--cardiolipin and phosphatidylinositol--in mouse liver sections was studied by the indirect method of fluorescent antibodies. Two types of liver sections--paraffin and cryostat, and two type of fixation--in acetone, and in the acetone, buffer, and formalin mixture--were used. Antiphospholipid sera stain specifically the plasma membrane of hepatocytes and predominantly the membrane region overlooking the blood capillary. A possibility of detecting the specific phospholipid haptens depends on the method of obtaining the sections and their fixation. Two types of immunization give two types of antiphospholipid sera which differ by the stability, by the possibility of monospecific antibodies isolation from them on lipid immunosorbents, and by the types of liver section staining.