大鼠肝实质及非实质细胞分离和质量检测

目的　探索分离肝实质细胞及非实质细胞的合理条件。方法　应用胶原酶 /蛋白酶E消化分离和低速离心技术 ,以细胞活率、细胞产量和细胞纯度等指标检测细胞质量。结果　在 37℃ ,0 5g·L-1的胶原酶作用 30min ,80 0r·min-1离心条件下 ,肝实质细胞产量 (98× 10 9± 4× 10 9·L-1)、纯度 (97%± 2 % )和活率 (>90 % )最好。胶原酶和蛋白酶E合用 ,肝非实质细胞产量最高 (2 8 6× 10 9± 3 7× 10 9·L-1) ,Kupffer细胞纯度合理 (16 0 %± 3 5 % ) ,且Kupffer细胞含量符合体内正常分布。结论　本实验方法分离细胞对于进一步研究肝实质 ,尤其肝非实质细胞功能有实际意义。     

大鼠原代肝细胞分离培养的实验研究

目的:探讨胶原酶经肝门静脉灌流分离肝细胞及培养的可行性.方法:取10只SD大鼠,按照改良Seglen原位两步胶原酶灌流法分离培养原代肝细胞.以SD大鼠作为肝细胞供体,采用Ⅳ型胶原酶经门静脉灌注,下腔静脉封闭保留胶原酶消化分离肝细胞,经200目筛过滤,滤液以200 r/min离心3～5 min,重复3次,纯化肝细胞.台盼蓝拒染实验检测分离细胞活性.将10只SD大鼠分离得到的肝细胞混合,稀释50倍后接种于包被有鼠尾胶原的培养皿或培养板中,倒置显微镜下观察分离肝细胞的形态特征,糖原染色法(PAS法)鉴定分离肝细胞,MTT法测定肝细胞在培养不同时期的增殖情况.结果:平均每只大鼠肝脏分离获得(1.8±0.14) ×108/mL肝细胞,细胞活率＞85％.肝细胞内因有大量的糖原颗粒,经PAS染色后被染成红色.MTT实验结果表明在培养的第八天肝细胞增殖达到峰值,培养的第九天细胞活力开始下降.结论:采用胶原酶经肝门静脉灌流分离肝细胞是一种简单、高效的获得肝细胞的方法.     

一种简便经济的肝库普弗细胞分离方法

目的介绍一种简易、经济、高效的肝库普弗细胞分离方法,为研究库普弗细胞在肝病及全身性疾病中的作用提供细胞模型。方法参照国内外文献方法加以改良,采用前灌流液在体灌流肝脏后,再用Ⅳ胶原酶离体灌流消化,Percoll分离液行密度梯度离心分离库普弗细胞,贴壁培养;锥虫蓝测定细胞活度,并用荧光显微镜、免疫组织化学以及吞噬功能实验行细胞纯度鉴定。结果肝库普弗细胞的获得量为(5~10)×107/肝,细胞活度为90%以上,细胞纯度95%以上。贴壁后细胞呈典型的星型或三角型,免疫组织化学染色显示CD163(ED2)阳性,显微镜下可见细胞质内被吞噬的碳素颗粒。结论本方法简便、高效,不需要特殊的设备,便于推广使用;获得的细胞活度和纯度较高,可用于库普弗细胞进一步的实验研究。     

大鼠心肌细胞分离方法的改进

目的介绍一种易于判断酶解终点的分离大鼠心肌细胞的方法。方法成年SD大鼠麻醉后开胸取出心脏,分别采用传统的Langendorff灌流装置和本实验室改进的Langen-dorff灌流装置,于恒温37℃灌流消化液(含0.6%胶原酶B,0.6%BSA,30μmol.L-1Ca2+的台氏液)分离细胞,并采用IonOptix单细胞动缘检测系统检测细胞功能。结果传统的Langendorff灌流装置分离细胞,酶解时间约13～16min,但此法终点难以判断,每次分离的细胞质量差别较大,且不耐钙,收缩的稳定性较差。而以改进的Langendorff灌流装置分离细胞易于确定消化终点,细胞成活率大于80%,在含1.8mmol.L-1Ca2+的台氏液中存活率也高达50%。给予电压15V、频率1Hz、波宽4ms的持续电刺激时,其在1000s内收缩舒张功能稳定。结论经改造的Langendorff灌流装置分离的心肌细胞成活率高,可控性强,结果稳定,能够更好地用于检测细胞收缩舒张功能的实验。     

大鼠肝星状细胞的分离和培养

目的:本文参考Friedman等的不连续密度离心法并作改良,建立了一种经济、简便、可靠的分离大鼠肝星状细胞(HSC)的方法。方法:用链霉蛋白酶和胶原酶灌流大鼠肝脏,Nycodenz密度梯度离心分离HSC,并进行体外培养,结果:分离培养的HSC经台盼蓝拒染实验活细胞大于98％,细胞得率为3.95×10个/肝脏。结论:该方法简便、实用、稳定、可靠,而且本方法的建立为进一步研究HSC与肝纤维化的关系,尤其是细胞水平及分子生物学水平的研究奠定了基础。     

大鼠离体肝脏灌流系统的建立

目的：建立大鼠离体肝动脉／门静脉灌流系统的实验方法。方法：雌性Wistar大鼠麻醉状态下,肝动脉、门静脉和肝静脉插管;经肝动脉冲净残留血液,灌流状态下游离肝脏;定量蠕动泵调节肝动脉或门静脉的Krebs—Henseleit平衡液（或加等渗葡聚糖）的灌流流速,泰盟BL-420S生物机能实验系统监测肝动脉或门静脉的压力变化,采用Prism-4非线型可变斜率回归获得“有或无胶体渗透压”KH液灌流的流速-压力曲线及方程式,计算半效流速及其95％的可信限。结果：无胶体渗透压灌流状态下的肝脏系数无差别,肝动脉的半效流速为2217（95％CI1209～4068）μl·min^－1,对数流速-压力直线方程为Y=142．4x＋706．9;门静脉半效流速为3791（95％CI3549～4049）μl·min^－1,对数流速-压力直线方程为Y：479．6X＋5034。等效胶体渗透压灌流状态下的肝脏系数无差别,肝动脉半效流速为3754（95％CI3175～4440）μl·min^－1,对数流速-压力直线方程为Y=133．5X-719．0;门静脉半效流速为6018（95％CI5565—6508）μl·min^－1,对数流速-压力直线回归方程为Y：538．3X4-4704。半效流速接近正常大鼠生理平均值,各半效流速95％可信限涵盖大鼠正常生理状态的变化范围。有或无胶体渗透压灌流状态之间总体无差别。结论：在恒流测压模式下,等渗灌流大鼠离体肝动脉和门静脉灌流系统的视窗时程长、机能变化稳定、操作流程简便,为认识肝动脉和门静脉离体舒缩机制奠定了基础。     

大鼠离体肝脏灌流方法的改良

<正> 离体肝脏灌流(isolated liver perfusion,ILP)广泛用于药理毒理学研究,尤其在药物代谢研究方面日益受到重视。本文研究了ILP方法的改良对大鼠肝脏功能影响。常规应用的Krebs—Hcnseleit灌流液均加红细胞和血清白蛋白,这给药物的分离测定带来困难。本文用右旋糖酥代替白蛋白以维持胶渗,浓度为1.5％,并适当提高灌流速度以弥补供氧不足,流速约2～3ml·min~(-1)·g~(-1)(liver)。为减少肝脏损害,简化了手术程序,只做门静脉插管,不做下腔静脉插管及肾静脉结扎,缩短了手术时间。肝功能检查发现,灌流2h后,灌流液中LDH,     

虫草多糖对离体肝星状细胞增殖及胶原基因表达的影响

目的：探讨虫草多糖(CP)抗肝纤维化的作用机理。方法：用链酶蛋白酶和胶原酶原位灌流消化正常大鼠肝脏,Nycodenz密度梯度离心分离肝星状细胞(HSC),以MTT比色法和3H-脯氨酸(3H-Pro)掺人法观察CP对HSC增殖及胶原合成的影响。结果：在(1．0～40)μg／ml剂量范围内,CP可明显抑制HSC增殖及胶原合成,呈药物浓度依赖关系。结论：CP在体外可抑制HSC的增殖和胶原合成,提示该作用可能是其抗肝纤维化的机制之一。     

昆明小鼠心室肌细胞分离方法及动作电位、L型钙通道电流记录

目的 探讨耐钙昆明小鼠心室肌细胞的急性分离方法及AP、L型钙通道电流的记录。方法 采用三步灌流法灌流,首先灌流无钙台氏液,再换成含Ⅱ型胶原酶0.1 mg.ml_-1、胰蛋白酶0.01 mg.ml_-1、牛血清白蛋白0.2mg.ml_-1的无钙台氏液灌流,消化液灌流期间,每隔5min加入20μl 的20mM CaCl₂,以观察流出液是否有单个心肌细胞来判断消化终点,最后灌流含1 mg.ml_-1牛血清白蛋白的KB液,采用全细胞膜片钳记录方式记录到动作电位及L型钙通道电流。结果 可获得80～90%杆状心肌细胞,复钙后,仍有60%细胞保持静止,细胞表面干净整洁,折光性强,边缘和横纹清晰,立体感强获得60%左右的耐钙心室肌细胞,并记录到典型的AP、L型钙通道电流。结论 该分离方法分离的细胞具有耐钙性和正常电生理特性。     

小鼠离体灌流肝生活力的估价

以不含血红蛋白的灌液对小鼠灌流肝的生活力作了估价。供氧灌流150min内LDH活力和蛋白质浓度均稳定,但在缺氧灌流情况下,酶活力和蛋白质浓度分别在40和60min后开始持续升高。葡萄糖水平在供氧与缺氧条件下均由高渐渐降低,但前者高于后者。钾、钠离子和pH在二种灌流情况下均无明显变化。电镜和病理所见,缺氧时主要出现胞浆内普遍的大空泡,肝组织有坏死等,供氧灌流无以上变化。以乙醇作为灌流药物,与LDH活力变化有极好的剂量效应关系。小鼠与大鼠有类同的灌流肝实验效果,供氧150min灌流肝脏能保持正常活力。     

Isolation and purification of immune cells from the liver

Isolating and purifying liver immune cells are crucial for observing the changes in intrahepatic immune responses during the development of liver diseases and exploring the potential immunological mechanisms. Therefore, the aim of this study was to provide an optimal protocol for isolating immune cells with a high yield and less damage. We compared mechanical dissection and collagenase digestion, and the results were represented by the proportion of lymphocytes, Kupffer cells and neutrophils. The apoptosis rates of liver immune cells resulted by different isolation protocols were compared by Annexin V-staining using flow cytometric analysis. Our data indicated that the enzymatic digestion in vitro was more efficient than the mechanical dissection in vitro with a suitable collagenase IV concentration of 0.01%, and the purification of liver immune cells by a one-step density gradient centrifugation in 33% Percoll had the definite advantage of a higher proportion of the target cells. We also provided evidence that enzymatic digestion in vitro method was superior to collagenase digestion in situ for liver T lymphocytes, NK cells and NKT cells isolation and purification. This protocol was also validated in human liver samples. In conclusion, we developed an optimal protocol for isolating and purifying immune cells from mouse and human liver samples in vitro by 0.01% collagenase IV and 33% Percoll density gradient centrifugation with the advantages of higher cell yields and viability. This method provides a basis for further studying liver immune cells and liver immunity with a wide range of applications.     

Isolation of hepatocytes from postnatal mice

A method for the isolation of hepatocytes from postnatal (1- to 3-week-old) mice has been developed. Cell isolation was carried out by retrograde perfusion of the liver with a collagenase-containing bicarbonate buffer. Viable cells were separated by selective adsorption onto collagen membranes. Cell viability was assessed by measuring ATP and glutathione content, lactate:pyruvate ratio, and the rate of protein synthesis. Comparisons of these parameters were made with those in cells isolated from adult mice and with values in whole liver. These hepatocytes were capable of metabolizing acetaminophen to its known hepatic conjugates and were susceptible to acetaminophen toxicity. This procedure for isolating mouse hepatocytes should be useful for the study of hepatic drug metabolism and its relationship to toxicity in the postnatal mouse.     

The Fate of Plasmid DNA After Intravenous Injection in Mice: Involvement of Scavenger Receptors in Its Hepatic Uptake

Purpose. We examined the stability and disposition characteristics of a naked plasmid DNA pCAT as a model gene after intravenous injection in mice to construct the strategy of in vivo gene delivery systems. Methods. After the injection of pCAT to the mice, stability, tissue distribution, hepatic cellular localization, and effect of some polyanions on the hepatic uptake were studied. Results. The in vitro study demonstrated that the pCAT was rapidly degraded in mouse whole blood with a half-life of approximately 10 min at a concentration of 100 µg/ml. After intravenous injection, pCAT was degraded at a significantly faster rate than that observed in the whole blood, suggesting that pCAT in vivo was also degraded in other compartments. Following intravenous injection of [³²P] pCAT, radioactivity was rapidly eliminated from the plasma due to extensive uptake by the liver. Hepatic accumulation occurred preferentially in the non-parenchymal cells. The hepatic uptake of radioactivity derived from [³²P] pCAT was inhibited by preceding administration of polyanions such as polyinosinic acid, dextran sulfate, maleylated and succinylated bovine serum albumin but not by polycytidylic acid. These findings indicate that pCAT is taken up by the liver via scavenger receptors on the non-parenchymal cells. Pharmacokinetic analysis revealed that the apparent hepatic uptake clearance was fairly close to the liver plasma flow. Conclusions. These findings provide useful information for the development of delivery systems for in vivo gene therapy.     

非髓性淋巴细胞12删除对B16黑色素瘤动物模型生存期的影响

目的利用C57BL/6小鼠制备黑色素瘤动物模型,选择合适的造模条件并观察非髓性淋巴细胞删除（NMLD）对该动物模型生存期的影响。方法黑色素细胞株B16制备成不同浓度的细胞悬液,分为1×105/ml、1×106/ml、5×106/ml和1×107/ml 4组。注射于小鼠背部,每组小鼠15只。于注射7 d后采用不同方法进行全身照射,达到NMLD。观察小鼠的出瘤时间、生存期及小鼠生活习性的改变。结果 4组小鼠出瘤率均为100%。浓度越高出瘤时间及生存期越短,生活习性改变越明显。结论选择背部为注射部位,较方便。细胞浓度为1×106/ml制备的动物模型,出瘤时间和生存时间更适于实际工作;NMLD对黑色素细胞瘤动物模型的生存期没有显著影响。     

In vitro metabolic products of RWJ-34130, an antiarrythmic agent, in rat liver preparations

The in vitro metabolism of RWJ-34130, an antiarrhythmic agent, was conducted using rat hepatic 9000 x g supernatant (S9) and microsomes in an NADPH-generating system, and the rat liver perfusion. The 100 and 20 microg ml(-1) concentrations of RWJ-34130 aqueous solution were used for microsomal incubation and liver perfusion, respectively. Unchanged RWJ-34130 (approximately 77-78% of the sample in both S9 and microsomes) plus a major metabolite, RWJ-34130 sulfoxide (20% of the sample in both S9 and microsomes) were profiled, isolated and identified from both hepatic S9 and microsomal incubates (60 min) using HPLC and mass spectrometry (MS), and by comparison to a synthetic RWJ-34130 sulfoxide, which was synthesized by reacting RWJ-34130 with MCPBA (meta-chloroperoxy benzoic acid). No unchanged RWJ-34130 was detected in the 3 h liver perfusate, however, 1-phenyl-2-oxo-pyrrolidine was profiled, isolated and identified as a major hydrolyzed metabolite of liver perfusate. RWJ-34130 is not extensively metabolized in vitro in rat hepatic S9 and microsomes. All HPLC metabolic profiles of hepatic S9 and microsomal samples (30 min, 60 min) were qualitatively and nearly quantitatively identical.     

治疗用卡介苗抑瘤小鼠模型的建立

目的  建立治疗用卡介苗抑瘤小鼠模型,以研究治疗用卡介苗的抗肿瘤疗效。方法  分别将适宜浓度的治疗用卡介苗溶液(20、10和5 mg/ml）、环磷酰胺溶液（7.2 mg/ml）和灭菌生理氯化钠溶液与适宜浓度的小鼠肝癌H22细胞（1×107个/ml）混合制成治疗用卡介苗处理液、阳性对照液和阴性对照液,皮下接种BALB/c小鼠,接种后4周,测定小鼠的肿瘤大小和重量,计算每组小鼠的平均肿瘤重量和瘤重抑制率,采用t检验对测定的结果进行统计学分析。结果  与阴性对照组相比,10 mg/ml治疗用卡介苗处理组的肿瘤重量明显减轻（t=3.41,P＜0.05）,肿瘤体积明显缩小（t=4.88,P＜0.05）,表明采用治疗用卡介苗处理可使肿瘤生长受到抑制。结论  建立的治疗用卡介苗抑瘤小鼠模型可用于卡介苗的抗肿瘤疗效研究。     

A rapid method of preparing hepatic parenchymal cells for studying drug metabolism

Parenchymal cells were prepared from the livers of male Sprague-Dawley rats by collagenase perfusion and purified by a self-generating Percoll gradient. The method consisted of mixing 31% Percoll and 5 x 10(6) cells/ml, followed by centrifugation at 10,000 x g for 10 min. A self-generated gradient provided a rapid and efficient recovery of highly viable parenchymal cells. The parenchymal cells were determined to be very stable during incubation at 37 degrees C for at least 2 h. Cell integrity was evaluated by trypan blue dye exclusion, lactate dehydrogenase leakage, and membrane peroxidation. In addition, drug metabolism and conjugation were evaluated as markers of intracellular integrity. With increasing p-nitroanisole (pNA) concentration, the formation of p-nitrophenol (pNP) increased. The rate of sulfation was maximal at a pNA concentration of 0.25 mM and decreased greatly above 1.0 mM. Glucuronidation increased from 0.25 mM to a maximum rate of 2.0 mM pNA. Above 1.0 mM pNA, nonconjugated pNP increased proportionately to the decrease in sulfation. These results indicate that the cell integrity was maintained, and that these cells can be used as a model for studying drug metabolism.     

Novel galactosylated liposomes for hepatocyte-selective targeting of lipophilic drugs

Novel galactosylated neutral liposomes containing cholesten-5-yloxy-N-(4-((1-imino-2-beta-D-thiogalactosylethyl)amino)butyl)formamide (Gal-C4-Chol) as a "homing" device were developed for hepatocyte-selective drug targeting. Distearoylphosphatidylcholine (DSPC)/cholesterol (Chol) (60:40) and DSPC/Chol/Gal-C4-Chol (60:35:5) liposomes were prepared and labeled with [3H]cholesteryl hexadecyl ether (CHE). [3H]Prostaglandin E1 (PGE1) and [14C]probucol were incorporated in liposomes as model lipophilic drugs. After intravenous injection of the liposomes, mice were sacrificed at suitable time periods, and the lung, liver, kidney, spleen, and heart were excised. DSPC/Chol/Gal-C4-Chol liposomes rapidly disappeared from the blood, and 85% of the dose had accumulated in the liver within 10 min compared with hepatic accumulation of DSPC/Chol liposomes of 12%. The liver was perfused with collagenase, and liver parenchymal cells (PC) and liver nonparenchymal cells (NPC) were separated by centrifugal differentiation to determine the cellular distribution. The PC/NPC ratios for DSPC/Chol/Gal-C4-Chol and DSPC/Chol liposomes were 15.1 and 1.1, respectively. The hepatic uptake of DSPC/Chol/Gal-C4-Chol liposomes, but not that of DSPC/Chol liposomes, was significantly inhibited by the predosing of galactosylated bovine serum albumin. [14C]Probucol and [3H]PGE1 incorporated in DSPC/Chol/Gal-C4-Chol liposomes was also efficiently delivered to the liver. In conclusion, newly developed galactosylated liposomes have been proven to be a useful carrier for hepatocyte-selective targeting that will have many practical applications.