体外肝细胞极性的重建

目的 使培养肝细胞在体外重建细胞极性。方法 利用三明治构型培养成鼠肝细胞并观察其形态、特异性膜区域蛋白分布并以单层胶原培养肝细胞作对照。结果 三明治构型肝细胞呈单层生长，形成肝板样结构，伴胆小管网络形成，免疫组织化学证明肝细胞膜区域蛋白呈特异性分布。而在对照组，肝细胞基本没有形成肝板样结构，肝细胞膜区域蛋白没有形成特异性分布。结论 三明治构型培养肝细胞在体外重建细胞极性，保留了体内肝细胞的特点。     

高温对罗非鱼肝细胞原代培养的影响

目的 观察温度对罗非鱼肝细胞原代培养的影响。方法 胶原酶冷消化法分离罗非鱼肝细胞，以含多种辅助因子的培养基培养。台盼蓝拒染法测细胞活力。倒置光显微镜观察罗非鱼肝细胞的形态。透射电镜观察超微结构。按不同培养温度分为A（28℃）、B（37℃）和C（39℃）三组。比较各组不同时期培养上清液中白蛋白和谷丙转氨酶含量。结果 新分离的罗非鱼肝细胞形态完整。37℃下与28℃下培养。生长良好、可存活2～3周。39℃下培养，可存活5～7d。A组和B组培养上清液中白蛋白和谷丙转氨酶含量比较差异均无统计学意义（P〉0．05）。C组和A、B组比较差异均有统计学意义（P〈0．05）。结论胶原酶冷消化法是一种较好的鱼肝细胞分离方法。罗非鱼肝细胞体外培养可耐受37～39℃高温。     

大鼠肝细胞、Kupffer和Ito细胞的分离与培养

我们采用Ｃｏｌｌａｇｅｎａｓｅ体内门静脉灌注结合体外Ｔｒｙｐｓｉｎ皿内消化制备游离肝细胞，进而采用ＰｒｏｎａｓｅＥ和Ｎｙｃｏｃｌｅｎｚ离心处理获单－Ｋｕｐｆｆｅｒ细胞和Ｉｔｏ细胞。三种细胞的产生率分别为１．２×１０８，１．１×１０７和２．３×１０７；其细胞纯度分别为９５％，８１％和８８％；存活率都在９０％以上。作者对三种细胞原代培养过程的形态特征和生物学活性进行了观察与讨论。     

胎牛血清在体外培养肝细胞极性形成的作用

目的观察胎牛血清在体外培养肝细胞极性形成的作用。方法分别利用含胎牛血清和不含胎牛血清培养液培养成鼠肝细胞,并观察血清对肝细胞形态、特异性膜区域蛋白分布以及蛋白分泌的影响,以确定胎牛血清在体外肝细胞极性形成的作用。结果不含血清组,肝细胞形成肝板样结构,肝细胞膜区域蛋白特异性分布,肝细胞保持稳定的蛋白分泌并持续增长达2周之久。而含血清组肝细胞培养3d后,分化良好的小管样结构消失,肝细胞膜区域蛋白失去特异性分布,蛋白分泌逐渐减少。结论胎牛血清可阻止肝细胞极性的形成,对于肝细胞移植及生物人工肝支持具有重要意义。     

血管内皮生长因子基因在原代培养大鼠肝细胞的转染表达

目的　研究血管内皮生长因子基因 (VEGF12 1)在原代培养大鼠肝细胞中转染表达。方法　构建 pIRES EYFP /VEGF12 1质粒 ,逆转录 聚合酶链反应 (RT PCR)、WesternBlot和荧光显微镜观察VEGF基因在原代培养大鼠肝细胞中的转染表达。结果　pIRES EYFP/VEGF12 1质粒成功构建并转染原代培养大鼠肝细胞 ,RT PCR观察到 2 5 8bp的VEGF特异性条带 ,WesternBlot检测到相对分子质量 42× 10 3 的特异性条带 ,荧光显微镜观察到转染阳性肝细胞发出黄绿色荧光。结论　pIRES EYFP/VEGF12 1质粒在原代培养大鼠肝细胞转染并表达 ,为VEGF基因修饰肝细胞移植和基因治疗奠定基础。     

糖尿病大鼠主动脉平滑肌细胞原代培养方法的改良

目的探寻一种简便、高效的糖尿病大鼠主动脉平滑肌细胞体外原代培养的方法,建立长久稳定的体外糖尿病血管平滑肌细胞模型,为研究糖尿病血管慢性病变奠定基础。方法采用链脲佐菌素腹腔注射加高脂高糖饲养自制糖尿病Wister大鼠模型20只,采用改良酶消化法体外培养主动脉平滑肌细胞。结果成功建立糖尿病大鼠模型,用改良酶消化法体外培养的血管平滑肌细胞传代生长快,细胞存活率达96％,能满足进行体外细胞实验要求。结论与传统方法相比,改良酶消化法简单、经济、成活率高。     

大鼠肝星状细胞的原代分离、培养与鉴定

目的:介绍一种合理、实用、便捷的肝星状细胞(HSCs)原代培养方法.方法:对SD大鼠进行肝脏原位灌注、酶消化,并进行间质细胞密度梯度离心.使用20%、35%和50%三层Percoll密度梯度分离纯化HSCs,分析其纯度、活力,并根据原代和传代HSCs的特征进行鉴定.结果:每只鼠肝HSCs原代培养得率为(1.5~2)×107个细胞,纯度为(97±2)%,细胞活力为(98±0.6)%.原代和传代HSCs都表达Desmin,只有传代7 d的HSCs表达α-SMA.电镜见细胞浆内大量脂滴.结论:本方法简单、实用、方便,所得结果优于其他方法.     

原代培养大鼠颌下腺细胞分离方法的比较研究

目的 探讨体外培养大鼠颌下腺细胞的分离方法并进行比较。方法 取Wistar大鼠颌下腺分别采用直接分离法与胰酶消化法分离获取颌下腺细胞，并进行原代及传代培养；相差倒置显微镜观察细胞形态，苔盼蓝染色法测细胞成活率，MTT法检测培养细胞活性，并检测24、48、72和96小时不同时间培养上清液中淀粉酶的含量，免疫组织化学鉴定细胞来源及比例。结果 直接分离法获取的细胞活性为70％，活力值为0．16土0．014，功能欠佳，经胰酶消化法所获取的细胞活性为85％，活力值为0．45土0．013，细胞形态完整、贴壁良好，且功能强；免疫组织化学校测颌下腺细胞α—SMA抗体、CK8．13抗体、keratin抗体呈阳性反应，胰酶消化法顿下腺细胞反应强度大于直接分离法。结论 胰酶消化分离获取颌下腺细胞的方法优于直接分离法。     

大鼠海马神经元的体外原代培养

细胞镇痛是生物镇痛的主要方法，以前曾将研究主要集中于能分泌镇痛物质的嗜铬细胞，但微囊化的嗜铬细胞仍无法完全避免免疫反应，神经元细胞是大脑中枢的主要细胞之一，其作为镇痛细胞移植人大脑可以减少以往的体细胞移植所带来的免疫反应。本试验旨在探索大鼠海马神经元的培养过程，观察细胞的生长情况，为转基因的时机提供依据。     

大鼠肝细胞,krpffer和Ito细胞的分离与培养

我们采用Ｃｏｌｌａｇｅｎａｓｅ体内门静灌注结合体外Ｔｙｐｓｉｎ皿内消化制备游离肝细胞。进而采用ＰｒｏｎａｓｅＥ和Ｎｙｅｏｃｌｅｎａ离心处理获－Ｋｕｐｆｆｅｒ细胞和Ｉｔｏ细胞。三种细胞的产生率分别１．２×１０＾８，１．１×１０＾７和２．３×１０＾７；其细胞纯度分别为９５％，８１％和８８％；存活率都在９０％以上。作者对三种细胞原代培养过程的形态特征和生物学活性进行了观察与讨论。     

体外培养条件下肝细胞功能研究

目的 探讨一种新的细胞培养模型以使体外培养的肝细胞更好地恢复肝功能。方法 利用三明治构型培养肝细胞,观察体外肝细胞蛋白合成,生物转化及其mRNA表达并与传统单层胶原培养肝细胞相比较。结果 三明治构型培养肝细胞保持稳定的蛋白分泌功能并持续增长,且有良好的生物转化功能,而传统单层胶原培养肝细胞蛋白分泌功能微弱,7d后基本消失,肝细胞生物转化功能差。原位杂交显示三明治构型培养较单层胶原培养肝细胞具有更强的白蛋白、P450-bmRNA表达,结论 三明治构型培养肝细胞较传统单层胶原培养肝细胞具更强的白蛋白、P450-bmRNA表达。结论 三明治构型培养肝细胞较传统单层胶原培养肝细胞更类似体内肝细胞,可体外重建细胞极性。     

Cryopreservation of isolated primary rat hepatocytes: enhanced survival and long-term hepatospecific function

OBJECTIVE: To investigate the long-term effect of cryopreservation on hepatocyte function, as well as attempt to improve cell viability and function through the utilization of the hypothermic preservation solution, HypoThermosol (HTS), as the carrier solution. SUMMARY BACKGROUND DATA: Advances in the field of bioartificial liver support have led to an increasing demand for successful, efficient means of cryopreservation of hepatocytes. METHODS: Fresh rat hepatocytes were cryopreserved in suspension in culture media (Media-cryo group) or HTS (HTS-cryo group), both supplemented with 10% DMSO. Following storage up to 2 months in liquid nitrogen, cells were thawed and maintained in a double collagen gel culture for 14 days. Hepatocyte yield and viability were assessed up to 14 days postthaw. Serial measurements of albumin secretion, urea synthesis, deethylation of ethoxyresorufin (CYT P450 activity), and responsiveness to stimulation with interleukin-6 (IL-6) were performed. RESULTS: Immediate postthaw viability was 60% in Media-cryo and 79% in HTS-cryo, in comparison with control (90%). Albumin secretion, urea synthesis and CYT P450 activity yielded 33%, 55%, and 59% in Media-cryo and 71%, 80%, and 88% in HTS-cryo, respectively, compared with control (100%). Assessment of cellular response to IL-6 following cryopreservation revealed a similar pattern of up-regulation in fibrinogen production and suppression of albumin secretion compared with nonfrozen controls. CONCLUSIONS: This study demonstrates that isolated rat hepatocytes cryopreserved using HTS showed high viability, long-term hepatospecific function, and response to cytokine challenge. These results may represent an important step forward to the utilization of cryopreserved isolated hepatocytes in bioartificial liver devices.     

Tissue reconstruction in primary cultured rat hepatocytes on asialoglycoprotein model polymer

Adult rat hepatocytes attached on an asialoglycoprotein model polymer, poly-N-p-vinylbenzyl-D-lactonamide (PVLA), formed anchored multilayer aggregates that had stable three-dimensional structure when epidermal growth factor (EGF) and insulin were added to the culture medium. The formation of multilayer aggregates depended on the concentrations of EGF and insulin added. Furthermore, the formation was synergistically accelerated by the presence of both hormones. Cells in the aggregates expressed a higher level of albumin secretion and lower proliferative ability than those in monolayer cultures on collagen. It seemed likely that the cells in multilayer aggregates experienced stable differentiated states resembling those in vivo through the formation of multilayer aggregates. The culture system described here has potential use for the study of the process of liver regeneration and the development of hepatic module systems such as a bioreactor and a hybrid artificial liver.     

白细胞介素-1β对原代培养大鼠肝细胞的细胞毒性作用

目的利用原代培养大鼠肝细胞 ,研究白细胞介素 1β(IL 1β)对肝细胞的作用。 方法无菌条件下原位胶原酶灌注Wistar大鼠肝脏分离肝细胞。观察IL 1β对肝细胞释放LDH ,肝细胞增殖(3 H标记的胸腺嘧啶掺入法 )以及肝细胞能量代谢的影响 (细胞内ATP含量和培养液中酮体比KBR)。结果在 6种培养条件下 ,各IL 1β组LDH活性均显著高于对照组 (培养条件Ⅰ～Ⅵ ,各对照组LDH活性分别为 :2 2± 2 ;2 5± 4;18± 5 ;12± 4;15± 5 ;11± 4,各IL 1β组分别为 :36± 3;43± 5 ;34± 6 ;31± 4;31± 5 ;2 2± 3,P值均小于 0 0 5 ) ;在培养条件Ⅱ情况下 ,IL 1β显著减少了原代培养大鼠肝细胞胸腺嘧啶的掺入量〔对照组 :(2 34 5 6± 312 3)Dpm/ 0 2 5× 10 6个细胞 ,IL 1β组 :(15 34 0±2 5 34 )Dpm/0 2 5× 10 6个细胞 ,P <0 0 1)〕 ;在培养条件Ⅳ、Ⅴ、Ⅵ情况下 ,IL 1β显著降低了培养细胞内ATP的含量(培养条件Ⅳ、Ⅴ、Ⅵ ,各对照组细胞内ATP的含量分别为 :10 0± 1 1;11 0± 2 3;11 5± 1 5 ,各IL 1β组分别为 :6 5± 0 5 ;5 9± 1 3;5 6± 1 2 ,P值均小于 0 0 5 ) ;在培养条件Ⅳ、Ⅴ、Ⅵ情况下 ,IL 1β显著降低了KBR(培养条件Ⅳ、Ⅴ、Ⅵ ,各对照组细胞内ATP的含量分别为 :1 0 1± 0 2 1;0 85± 0 13;0     

三明治构型培养肝细胞——一种潜在的生物人工肝单位

目的：探索一种新的生物人工肝单位。方法：双层胶原三明治构型培养肝细胞,并观察其形态分化的特点及蛋白合成功能,并与传统单层胶原培养肝细胞相比较。结果：三明治构型培养的肝细胞48h后形成肝板样结构,伴胆小管网络,肝细胞蛋白分泌功能稳定、持续增高,生存时间达2个月之久;而对照组基本没有形成肝板样结构,蛋白分泌功能差,7d后基本消失,细胞趋向死亡。结论：三明治构型培养的肝细胞在体外重建细胞极性,形态及功能更接近体内肝细胞,是一种潜在的生物人工肝单位。     

体外肝细胞极性重建中cofilin的去磷酸化及其调节

目的观察SSH1L对肝细胞cofilin去磷酸化的调节作用。方法通过体外三明治构型培养肝细胞诱导cofilin的去磷酸化，应用免疫共沉淀法进行SSH1L的体外酶活性分析。进一步对体外培养大鼠肝细胞进行野生型和无活性型SSH1L质粒的转染以实现SSH1L在肝细胞的过表达，观察肝细胞的cofilin去磷酸化水平。结果三明治构型培养肝细胞96h和1周后，与对照组相比，cofilin的去磷酸化增加，SSH1L酶活性增强。野生型SSH1L在肝细胞的过表达与无活性SSH1L过表达相比，P-cofilin去磷酸化显著增加。结论三明治构型培养肝细胞存在cofilin的去磷酸化，且为SSH1L活性所调节。     

白细胞介素1β对大鼠肝细胞毒性作用的细胞内信号传导途径

目的研究白细胞介素-1β(IL-1β)对原代培养大鼠肝细胞细胞毒性作用的细胞内信号传导机制. 方法使用雄性Wistar大鼠,原位胶原酶灌注分离肝细胞.应用比色法测定乳酸脱氢酶(LDH)活性,Wes tern Blot方法分析c-Jun N末端激酶(JNK)、p38激酶的表达,凝胶电泳移动抑制实验检测激活物蛋白-1(AP-1)的结合活性. 结果 IL-1 β促进原代培养大鼠肝细胞LDH释放(IL-1β刺激组与对照组LDH活性分别为21.9%±3.6% 和11.0%±1.8%,P＜0.01);IL-1β通过激活JNK途径,激活转录因子AP-1,对原代培养大鼠肝细胞产生细胞毒性作用,而同时激活的p38激酶途径对这一过程起负性调节作用. 结论 IL-1β通过激活JNK途径,激活转录因子AP-1,对原代培养大鼠肝细胞产生细胞毒性作用.     

重组人硫氧化还原蛋白对原代培养肝细胞的影响

目的探讨重组人硫氧化还原蛋白(rhTrx)对原代培养肝细胞生存生长的影响。方法逆转录聚合酶链反应法扩增出hTrx cDNA,并在原核表达系统中表达。IgM还原实验和胰岛素还原实验测定rhTrx的生物学活性。3H胸腺嘧啶核苷(TdR)掺入试验和细胞乳酸脱氢酶(LDH)释放试验检测rhTrx对SD大鼠原代培养肝细胞生存生长的影响。结果克隆出hTrx开放阅读框cDNA并获得高效表达,可溶性目的蛋白回收率为23．20％,蛋白纯度为96．30％。IgM还原和胰岛素还原实验均显示制备的rhTrx具有较好的生物学活性(t=7．5860,P<0．01)。加入rhTrx培养的肝细胞生长旺盛,并维持了良好的细胞形态。rhTrx可促进原代培养肝细胞的DNA合成,并以剂量依赖方式明显抑制乙醇造成的肝细胞损害。结论制备出具有生物学活性的rhTrx,hTrx对原代培养肝细胞的生存具有促进作用。     

乙醇对体外生长肝细胞功能的影响

目的探讨一种新的细胞培养模型以观察乙醇对培养肝细胞功能的影响。方法利用三明治构型培养成鼠肝细胞并观察酒精对其形态、胆汁分泌功能以及蛋白合成功能的影响。结果首先荧光二乙酯的肝细胞代谢显示,三明治构型肝细胞培养96h后有明显的胆汁分泌功能,而在酒精干预后,胆汁分泌功能消失。其次三明治构型肝细胞96h后具有较好的蛋白合成功能,而给予酒精干预后,肝细胞蛋白合成功能受到损害而明显下降。结论酒精可造成肝细胞损伤,导致肝细胞功能严重下降。本实验为在细胞水平研究酒精性肝病的发病机理提供了一个体外模型,具有重要的临床意义。     

Long-term function of cryopreserved rat hepatocytes in a coculture system

The goal of this study was to investigate postpreservation long-term function of cryopreserved primary rat hepatocytes using the hepatocyte/3T3-J2 fibroblast coculture system. The long-term function of thawed hepatocytes cocultured with fibroblasts was evaluated and compared with hepatocytes cultured without fibroblasts. Fresh isolated primary rat hepatocytes were frozen at a controlled rate (-1 degrees C/min) up to -80 degrees C, and then stored in liquid nitrogen for up to 90 days. Thawed hepatocytes were thereafter cocultured with 3T3-J2 murine fibroblasts and cocultivation was monitored for 14 days. The viability of fresh isolated hepatocytes was 91.4%, and that of cryopreserved hepatocytes was 82.1%. Cellular morphology and polarity, which were determined by the localization of actin filaments and connexin-32, were successfully maintained in cryopreserved hepatocytes following cryopreservation. Albumin and urea synthesis reached the maximum level and became stable after day 7 in coculture in both fresh and cryopreserved hepatocytes. Urea synthesis of cryopreserved hepatocytes was maintained 89.0% of nonfrozen fresh control, and albumin production of cryopreserved hepatocytes was 63.7% of control in coculture. Cytochrome P450 activity, which was measured by deethylation of ethoxyresorufin, was also maintained in cryopreserved hepatocytes at 88.6% of nonfrozen fresh control in coculture. The retention of synthetic and detoxification activities was verified to be well preserved during extended low-temperature storage (90 days). Both fresh control and cryopreserved hepatocytes cultured without fibroblast did not retain their synthetic and detoxification functions in long-term culture. These data illustrate that, through the utilization of our cryopreservation procedure, primary hepatocyte function was successfully maintained when placed into coculture configuration following thawing.