大鼠肝细胞与人脐静脉内皮细胞混合共微囊化的体外研究

目的 观察人脐静脉内皮细胞(HUVECs)对共微囊化大鼠肝实质细胞的保护作用.方法 利用自制微囊发生器制备含肝细胞或肝细胞与HUVECs以10:1混合的微囊进行体外培养,同时建立非微囊化单独培养和共培养组,通过测定培养液中自蛋白、尿素的分泌量和肝细胞形态来判断肝细胞功能和活性.结果 肝细胞与HUVECs共培养或共微囊化均能提高前者的白蛋白分泌和尿素合成量(均P<0.01),存活时间延长;共微囊化组虽前7 d的白蛋白和尿素合成量较非微囊化共培养组低,但在7 d后的白蛋白和尿素合成量高于后者,且相对平稳.结论 肝细胞与HUVECs混合共微囊化能明显改善囊内肝细胞的形态、功能与寿命.     

胶原凝胶包埋肝细胞体外培养和腹腔移植

目的:改进肝细胞腹腔移植方法。方法:用胶原凝胶包埋鼠肝细胞并在体外培养。测定培养液中总蛋白(TP)和尿素氮(BUN)水平。将胶原培养液与鼠肝细胞混合后注入鼠腹腔,混合物在腹腔内形成凝胶并包埋肝细胞。用含酶的培养液将受体鼠腔内移植肝细胞洗出,再置于无葡萄糖(Glu)培养液中培养。测定培养液中Glu水平。对照组处理过程与实验组相同,但不用胶原。结果:胶原凝胶包埋肝细胞组的TP、BUN和Glu值均明显高于对照组(P<0.01)。胶原培养液与肝细胞混合物在注入腹腔24h后变为凝胶。结论:胶原凝胶可促进体外培养或腹腔移植肝细胞生长;胶原培养液腹腔注射和腹腔内胶原凝胶包埋细胞法,使肝细胞腹腔移植更简便、安全、实用。     

人胎肝细胞培养上清对大鼠肝细胞增殖作用的初步观察

目的观察培养人胎肝细胞分泌上清（ＦＨＣＳ）对培养大鼠肝细胞的影响。方法用相差显微镜动态观察培养大鼠肝细胞的形态变化，并用放免分析法检测其ＤＮＡ合成量。结果ＦＨＣＳ对鼠肝细胞的原代培养有明显作用，表现为大鼠肝细胞增殖活跃、生长旺盛，维持正常形态及存活时间延长，肝细胞ＤＮＡ合成量明显增加（Ｐ＜０．０１）。结论ＦＨＣＳ对体外大鼠肝细胞有明显的增殖刺激作用，该作用可能与分离、培养胎肝细胞过程中分泌产生的多种细胞因子和营养物质有关，并为早期胎肝细胞悬液治疗重型肝炎的可能机理。     

两种含胶原凝胶的人工肝生物反应器的功能比较

目的改进中空纤维型人工肝生物反应器。方法将大鼠肝细胞与胶原溶液混合并注入中空纤维反应器管外腔,待混合物在管外腔中形成胶原凝胶将肝细胞悬浮其中,构成胶原凝胶混悬肝细胞反应器(Ⅰ组);另将大鼠肝细胞混悬液注入涂有胶原层的中空纤维反应器管外腔,构成胶原涂层加肝细胞反应器(Ⅱ组)。两组反应器均由管内腔循环灌注培养液,置孵箱培养9 d,每24 h换液一次,每48 h取灌注液检测白蛋白(Alb)、尿素、乳酸脱氢酶(LDH)浓度,以检验反应器的肝细胞功能。采用统计软件SPSS 13.0进行统计学分析,计量数据以均数±标准差(x±s)表示,两组资料比较用配对t检验。结果Ⅰ组和Ⅱ组的Alb值均在灌注第3天达峰值,分别为(1.41±0.08)和(0.65±0.05)g/L,3~9 d各时间点Alb值Ⅰ组均明显高于Ⅱ组(P值均0.01)。Ⅰ组和Ⅱ组的尿素值分别在灌注第5天和3天达峰值,分别为(1.73±0.14)和(1.56±0.18)mmol/L,5~9 d各时间点尿素值Ⅰ组均明显高于Ⅱ组(P值均0.01)。Ⅰ组和Ⅱ组的LDH值在灌注第9天达峰值,分别为(32.03±9.13)和(70.17±25.28)U/L,1~9 d各时间点LDH值Ⅰ组均明显低于Ⅱ组(P值均0.01)。提示胶原凝胶混悬肝细胞反应器(Ⅰ组)比胶原涂层加肝细胞反应器(Ⅱ组)有更好的肝细胞合成代谢功能,肝细胞酶漏出也更少。结论胶原凝胶混悬固定肝细胞更适用于构建中空纤维型人工肝生物反应器。     

转基因肝星状细胞株对肝细胞生长的支持作用

目的 探讨转基因肝星状细胞株CFSC／HGF对大鼠肝细胞生长的支持作用。方法将大鼠原代肝细胞培养于稳定表达肝细胞生长因子(HGF)的肝星状细胞株CFSC／HGF所构建的饲养层上,连续动态观察肝细胞的形态、超微结构、白蛋白分泌、尿素合成以及吲哚氰绿摄取排泌功能的变化,同时与传统胶原贴壁培养的肝细胞进行比较,并通过半定量逆转录聚合酶链反应(RT-PCR)检测HGF受体c-Met表达的变化。结果共培养的肝细胞体外培养至7～10d时细胞增殖、白蛋白分泌及尿素合成达到峰值,以后逐渐下降,至35d时仍保持一定的存活和功能。与传统胶原上培养的肝细胞相比,其寿命、形态和功能的维持时间明显延长;RT-PCR结果显示,与CFSC／HGF饲养层细胞共培养1周后,肝细胞表面c-Met表达上调2．23倍。结论转基因肝星状细胞株CFSC／HGF对肝细胞较长时间内保持高活性、高密度的生长有显著的支持作用,CFSC／HGF诱导的肝细胞表面c-Met表达上调可能参与了该支持作用。     

大鼠肝细胞与睾丸支持细胞混合共微囊化的体外功能研究

目的 观察大鼠肝细胞与睾丸支持细胞混合共微囊化对肝细胞生物学活性的支持作用。方法利用微囊发生器制备含肝细胞、睾丸支持细胞及两者混合细胞的微囊，根据微囊内包裹细胞种类的不同，分为微囊化肝细胞组，微囊化睾丸支持细胞组，肝细胞、睾丸支持细胞分别微囊后共同培养组和肝细胞、睾丸支持细胞混合共微囊组。观察囊内细胞形态，体外培养测定培养液中Alb与尿素分泌，判断各组囊内肝细胞活性和功能。样本比较采用重复测量数据的多元方差分析。结果体外培养96h时，光学显微镜下见微囊呈球形，表面光滑，细胞均匀、散在分布于微囊内，囊内肝细胞、睾丸支持细胞呈圆形，未贴壁。微囊化睾丸支持细胞组培养上清液中无法测及Alb与尿素。与微囊化肝细胞组相比，肝细胞、睾丸支持细胞分别微囊后共同培养组与肝细胞、睾丸支持细胞混合共微囊组肝细胞的存活时间延长，培养上清液中Alb（F=217．56，P〈0．01）和尿素（F=232．72，P〈0．01）明显增加。混合细胞共微囊组于培养第7天Alb与尿素含量最高，分别为（2．80±0．11）g／L、（1．92±0．10）μmol／L，而其他两组均于培养第3天Alb与尿素含量达到最高，分别为（2．48±0．08）g／L、（1．47±0．08）μmol／L和（2．27±0．10）g／L、（1．37±0．05）μmol／L。相对于混合细胞共微囊组，肝细胞、睾丸支持细胞分别微囊后共同培养组在培养中后期Alb、尿素含量下降趋势明显。结论肝细胞与睾丸支持细胞混合共微囊化能明显延长囊内肝细胞的寿命，维持肝细胞形态和功能。     

三明治构型大鼠原代肝细胞长期培养及功能测定

目的观察三明治构型大鼠原代肝细胞长期培养的形态学变化,并对其功能进行测定。方法采用改良原位两步法门静脉胶原酶灌注分离单肝细胞,台盼蓝拒染实验观察细胞活力,利用三明治培养构型培养成年大鼠原代肝细胞,倒置显微镜下连续观察肝细胞的形态学变化,定期收集培养细胞上清液,检测所培养肝细胞的分泌及生物转化功能,并与单层胶原培养肝细胞比较。结果平均每个鼠肝可获取(2～3)×108个肝细胞,存活率为(93±3)%;体外肝细胞培养第3天,细胞活力、清蛋白分泌功能恢复到最佳状态;三明治构型培养第7天,地西泮24h代谢量达到最高峰。三明治构型培养的肝细胞形成肝索样结构,并逐渐形成胆小管网络;在培养的21d内,清蛋白分泌、地西泮代谢始终维持较高的水平;肝细胞形态维持可达28d以上。结论三明治构型肝细胞培养体系更接近于肝细胞体内生长环境,肝细胞可在较长时间内保持良好的形态结构和功能。三明治构型不仅可以应用于肝细胞的基础研究,而且为肝细胞移植和生物人工肝治疗肝衰竭奠定了一定的基础。     

IFNγ对培养大鼠肝细胞DNA和胶原的影响

目的观察ＩＦＮγ对培养大鼠肝细胞ＤＮＡ和胶原的影响．方法应用３Ｈ胸腺嘧啶核苷和３Ｈ脯氨酸掺入量及掺入抑制率测定的方法．结果在５ｋＵ／Ｌ～１００ｋＵ／Ｌ浓度范围内的ＩＦＮγ能明显抑制培养大鼠肝细胞ＤＮＡ（１００ｋＵ／Ｌ为６４６％）和胶原（１００ｋＵ／Ｌ为６７４％）合成，抑制程度与浓度和时间呈正相关（Ｐ＜００１）．结论ＩＦＮγ抗肝纤维化作用的机制之一是能抑制肝细胞增殖及胶原合成     

新型编织型生物反应器内大量乳猪肝细胞的组织化培养

目的:使反应器内培养的肝细胞能在较长时间保持肝细胞的特殊功能和活力,提高生物反应器的功效并为反应器内储存和运输肝细胞提供可能。方法:将新生实验型小猪肝细胞与微载体共同培养,待肝细胞与微载体充分粘附形成微载体-球形聚集体后,将其置入新型编织型生物反应器的外腔,用培养液循环式人工毛细管培养系统进行培养,在培养液内加入氯化氨、醋胺酚检测生物反应器的转化功能,行无血清培养检测肝细胞的白蛋白的合成功能,观察肝细胞的酶漏出量。锥虫蓝染色观察肝细胞的活力,电镜观察其超微结构。结果:生物反应器内培养的肝细胞在1周内能保持较高的活力,并保持着较高的氯化氨、醋胺酚的生物转化及白蛋白合成功能,酶的漏出量也少。肝细胞超微结构示内质网、线粒体等细胞器丰富,核内染色体分布均匀,肝细胞间的微绒毛形成胆小管样结构。结论:肝细胞与微载体及肝细胞之间的聚集,形成直径大小不等的肝细胞-微载体球形聚集体,这种由肝细胞重新结合而成的聚集体类似于体内的肝组织结构,在新型编织型生物反应器内的中空纤维网架的支持下,肝细胞聚集体均匀地分散其中,为肝细胞的生长提供了一个类似体内内环境的三维空间,因而在无氧合器供氧的情况下,肝细胞也能在1周内保持较好的形态和功能。     

中药抗肝纤维化的细胞机制

1 降低肝细胞胶原合成,减轻肝细胞坏死,保护肝细胞功能,清除肝纤维化诱因 肝细胞既能合成胶原基质,也是非胶原基质纤维连接蛋白(FN)和层粘连蛋白(LN)的主要细胞来源。采用原位杂交方法观察,新分离的和原代培养的肝细胞内含有胶原及前胶原mRNA,CCI_4损害的大鼠肝细胞内也有Ⅰ、Ⅲ、Ⅳ型胶原mRNA合成。     

Primary hepatocyte culture in collagen gel mixture and collagen sandwich

AIM: To explore the methods of hepatocytes culture in a collagen gel mixture or between double layers of collagen sandwich configuration and to examine the functional and cytomorphological characteristics of cultured hepatocytes. METHODS: A two-step collagenase perfusion technique was used to isolate the hepatocytes from Wistar rats or newborn Chinese experimental piglets. The isolated hepatocytes were cultured in a collagen gel mixture or between double layers of collagen sandwich configuration respectively. The former was that rat hepatocytes were mixed with type I rat tail collagen solution till gelled, and the medium was added onto the gel. The latter was that swine hepatocytes were seeded on a plate precoated with collagen gel for 24 h, then another layer of collagen gel was overlaid, resulting in a sandwich configuration. The cytomorphological characteristics, albumin secretion, and LDH-release of the hepatocytes cultured in these two models were examined. RESULTS: Freshly isolated rat hepatocytes were successfully mixed and fixed in collagen gel, and cultured in the gel condition. During the culture period, the urea synthesized and secreted by rat hepatocytes was detected throughout the period. Likewise, newborn experimental piglet hepatocytes were successfully fixed between the double layers of collagen gel, forming a sandwich configuration. Within a week of culture, the albumin secreted by swine hepatocytes was detected by SDS/PAGE analysis. The typical cytomorphological characteristics of the hepatocytes cultured by the above two culture models were found under a phase-contrast microscope. There was little LDH-release during the culture period. CONCLUSION: Both collagen gel mixture and double layers of collagen sandwich configuration can provide cultural conditions much closer to in vivo environment, and are helpful for maintaining specific hepatic functions and cytomorphological characteristics. A collagen gel mixture culture may be more eligible for the study of bioartificial livers.     

Cryopreservation and gel collagen culture of porcine hepatocytes

AIM: To study the method of cryopreserving porcine hepatocytes and gel collagen culture measure after its cryopreservation. METHODS: Hepatocytes, isolated from Chinese expedmental suckling mini-pigs by two-step perfusion with collagenase using an extra corporeal perfusion apparatus, were cryopreserved with 50 mL/L to 200 mL/L DMSO in liquid nitrogen for 4 mo, then thawed and seeded in 1 or between 2 layers of gel collagen. The expression of porcine albumin message RNA, cellular morphology and content of aspartate aminotransferase (AST) and urea nitrogen (UN) were examined during culture in gel. RESULTS: Viability of 150 mL/L DMSO group thawed hepatooltes was (83&#177;4)%, but after purification, its viability was (90&#177;5)%, attachment efficiency was (86&#177;7)%, the viability of thawed hepatocytes was near to fresh cells. When the thawed hepatooltes were cultivated in gel collagen with culture medium adding epidermal growth factor, the hepatocytes grew in various administrative levels in mixed collagen gel, and bunchy in the sandwich configuration cultures. For up to 10 days‘ culture, the typical cellular morphological characteristics of cultivated hepatocytes could be observed. The leakage of AST was lower during culture in gel than that in common culture. At the same time, the UN synthesized by cells cultivated in mixed gel collagen was higher than that in other groups. CONCLUSION: Storage in liquid nitrogen can long keep hepatocytes‘ activities, the concentration of 150 mL/L DMSO is fit for porcine hepatooltes‘ cryopreservation. Thawed hepatocytes can be cultivated with collagenous matrix, which provides an environment that more closely resembles that in vivo and maintain the expression of certain liver-specific function of hepatocytes.     

Evaluation of diffusion in gel entrapment cell culture within hollow fibers

AIM: To investigate diffusion in mammalian cell culture by gel entrapment within hollow fibers. METHODS: Freshly isolated rat hepatocytes or human oral epidermoid carcinoma (KB) cells were entrapped in type I collagen solutions and statically cultured inside microporous and ultrafiltration hollow fibers. During the culture time collagen gel contraction, cell viability and specific function were assessed. Effective diffusion coefficients of glucose in cell-matrix gels were determined by lag time analysis in a diffusion cell. RESULTS: Significant gel contractions occurred in the collagen gels by entrapment of either viable hepatocytes or KB cells. And the gel contraction caused a significant reduction on effective diffusion coefficient of glucose. The cell viability assay of both hepatocytes and KB cells statically cultured in hollow fibers by collagen entrapment further confirmed the existence of the inhibited mass transfer by diffusion. Urea was secreted about 50% more by hepatocytes entrapped in hollow fibers with pore size of 0.1 μm than that in hollow fibers with MWCO of 100 ku. CONCLUSION: Cell-matrix gel and membrane pore size are the two factors relevant to the limited mass transfer by diffusion in such gel entrapment of mammalian cell culture.     

Cellular interactions promote tissue-specific function, biomatrix deposition and junctional communication of primary cultured hepatocytes

Hepatocytes, prepared from normal adult rat liver, were seeded onto a collagen substratum and cultured alone or in the presence of rat liver endothelial cells. When hepatocytes were cultured alone in a hormonally defined serum-free medium, decreased albumin production and rapid morphological deterioration of bile canaliculi structures and gap junctions occurred within 4 to 5 days. In contrast, hepatocytes cocultured with liver mesenchymal cells remained morphologically intact and biochemically functional for at least 4 weeks. They reorganized into small islands, continued to secrete high levels of albumin, did not express alpha-fetoprotein (a fetal marker), and remained strongly dye coupled. All of the hepatocytes synthesized albumin and retained their gap junctional channels. No junctional communication was observed between hepatocytes and endothelial cells. Long fibers containing fibronectin, Type I collagen and laminin distributed over the hepatocytes were induced in coculture but never appeared in hepatocytes cultured alone. Moreover, supplementation of the hormonally defined medium with phenobarbital and dimethyl sulfoxide, both of which improve the life span and functional activities of cultured hepatocytes, failed to induce reticulin fiber formation in pure culture of hepatocytes. The modulation of albumin secretion, biomatrix deposition and junctional communication observed in hepatocytes cultured with sinusoidal liver cells was also obtained when hepatocytes were in association with various epithelial or mesenchymal cells [rat liver epithelial cells (T51B), mouse embryonic fibroblasts (NIH 3T3), human or rat dermal fibroblasts and bovine aorta endothelial cells (AG 4762)].     

原代猪肝细胞无血清培养

目的研究原代猪肝细胞无血清培养及肝细胞的功能。方法采用EGTA和胶原酶P两步法经肝静脉逆行灌注分离乳猪肝细胞,在无血清培养基中培养,并对不同培养时间的肝细胞生化合成及生物转化功能进行检测。结果肝细胞产量为（1．5±0．1）×10^10／肝,活率为（90．3±1．5）％,接种培养后肝细胞增生旺盛,LDH漏出第2天达到高峰,然后逐渐下降并稳定于一定水平。随着时间的延长及肝细胞数量的增加,自蛋白合成及利多卡因转化率逐渐增加,呈时间及肝细胞数量的依赖关系。结论采用本法分离获取的猪肝细胞产率高、活性强、具有良好的生物合成及生物转化功能,可作为生物人工肝较理想的肝细胞来源。     

Repolarization of hepatocytes in culture

We have evaluated the biochemical, morphological, and functional redevelopment of polarity in freshly isolated hepatocytes cultured using a double layer collagen gel sandwich technique. Western blot analysis showed increased cellular levels of the cell adhesion protein uvomorulin as cultured hepatocytes repolarized. Immunofluorescence studies using antibodies against domain-specific membrane proteins showed polarity as early as 48 hours, although the pattern of the polymeric Immunoglobulin-A receptor (pIgA-R) differed from in vivo liver. Electron microscopy showed developing bile canaliculi at 1 day. However, the functional presence of tight junctions was absent at 1 day, but present at 5 days. We further showed functional polarity to be present at 4 days by documenting the ability of cultured hepatocytes to metabolize and excrete fluorescein diacetate into visible bile canaliculi. We conclude that hepatocytes cultured appropriately develop morphological and functional polarity. Hepatocyte culture is therefore a useful tool for the study of mechanisms responsible for the development of polarized function.(Hepatology 1997 Jan;25(1):167-72)     

Culture of mouse gallbladder epithelial cells by microexplant culture on collagen gel]

We established a new culture method of mouse gallbladder epithelial cells. Explantation of a tiny fragment of the mouse gallbladder (microexplant) on the collagen gel allowed the epithelial cells to proliferate on the collagen gel for more than 4 weeks. The epithelial cells spread in a monolayer sheet from the microexplant and extended onto the surface of the collagen gel 0.31 +/- 0.02 mm per day. Whereas, contaminated stromal cells proliferated under the epithelial cell layer and into the collagen gel. At the peripheral zone of epithelial sheet, the shape of the epithelial cells was squamous, and the cells were frequently positive for BrdU immunostaining, indicating the high proliferative activity. In contrast, the cells in the central part of epithelial sheet were cuboidal or low columnar in shape and showed well cell-polarity and mucus-secretory activity similar to the gallbladder epithelia in vivo. It was indicated that the cultured epithelial cells in this system preserved the morphological and functional differentiation to the physiological gallbladder epithelia. This newly established culture method has the characteristics of both the organ and the cell culture, and might be useful for the morphological and functional studies of the gallbladder epithelia in vitro.     

Effect of insulin on glycogen and protein synthesis in monolayer cultures of hepatocytes from normal and alloxan diabetic rats

Summary The effects of insulin on net glycogen synthesis and amino acid incorporation into protein were studied in cultured hepatocytes from adult normal and alloxan diabetic rats. Insulin stimulated glycogen synthesis in monolayer cells throughout a four day culture period and enhanced leucine incorporation into protein more effectively in normal cells with high glycogen levels than in cultured diabetic cells. These differences correlate well with the observed cellular ultrastructures which were maintained much better in the presence of insulin. Restoration of the morphological changes of alloxan diabetic hepatocytes to normal liver cell structures can be observed at any time during the culture period by giving insulin continuously.