人肝细胞的微载体培养及其意义

目的：探讨大量培养高活性人肝细胞的理想方法，为肝脏病研究提供良好的细胞材料。方法：在限制贴壁、定时振荡条件下，采用载体ｃｙｔｏｄｅｘ３为材料进行人肝细胞的微载体培养。结果：预先将体外两步灌流分离的人肝细胞振荡３０分钟后，肝细胞易与微载体相粘附．在被覆聚羟乙基异丁烯酸的培养瓶中继续间歇性振荡２４小时，约７０％的肝细胞附着于微载体之上。在使用激素限定条件培养液培养的１５天内，肝细胞保持蛋白分泌功能和多细胞粘附聚集球形体形态特征。结论：本法培养人肝细胞具有细胞数量多、密度大和活性好的特点，适宜于体外生物人工肝支持系统、肝细胞移植以及培养肝细胞因子的提取。     

人工肝生物材料人肝细胞系CL-1的微载体培养.

目的使用微载体培养人肝细胞系ＣＬ１,并观察细胞的转化及合成功能．方法使用Ｃｙｏｔｄｅｘ３微载体进行人肝细胞系ＣＬ１的高密度培养,应用光镜及扫描电镜于培养１,３,５,７,９ｄ动态观察细胞的生长情况,并同时检测培养系统的安定转化及清蛋白合成量．结果培养７ｄＣＬ１细胞密度达到最高,为２１６×１０９／Ｌ;安定转化量最高为６１９μｇ;清蛋白合成量最高为７８μｇ．结论使用微载体培养高分化人肝细胞系可达到较高的密度,且具有较好的生物转化及合成功能,可望作为组合性人工肝的生物材料     

正常原代猪肝细胞方瓶微载体静止培养

目的初步探讨微载体培养方法作为人工肝中生物材料培养方法的可能性．方法将５×１０７的肝细胞及２ｇ／Ｌｃｙｔｏｄｅｘ３微载体连同５０ｍＬ含１００ｍＬ／Ｌ猪门静脉血清及其他附助因子的ＤＭＥＭ培养基加入１００ｍＬ的培养方瓶中培养．光镜及电镜下观察细胞生长情况,检测培养上清中Ａｌｂｕｍｉｎ,Ｕｒｅａ浓度．结果接种的肝细胞在接种时即呈明显的朝Ｃｙｔｏｄｅｘ３聚集,二者极易相粘贴．在培养后２４ｈ～４８ｈ,可见典型的多细胞聚集球形成,该形态特点及Ａｌｂｕｍｉｎ,Ｕｒｅａ合成能力可保持８ｗｋ左右．结论使用微载体培养猪肝细胞具有多种优点．微载体培养的肝细胞可望作为生物人工肝的生物材料     

微载体培养人肝细胞用于人工肝的初步研究

探讨微载体培养人肝细胞用于人工肝的可能性。方法 用微载体Ｃｙｔｏｄｅｘ３培养人体肝细胞，将其置于中空纤维型生物反应器构成人工肝装置，在体外循环实验中进行生物功能及细胞活力的观察与评价。结果 体外两步法分离的人肝细胞，在辅助促聚条件下有效地粘附于微载体上，呈悬浮生长状。     

大鼠肝细胞的微载体黏附培养及其功能测定

目的探讨一种简单实用地获取大量高活性,高密度肝细胞的培养方法。方法用我们自行设计的半原位酶消化技术对成年 SD 大鼠肝细胞进行分离与微载体黏附培养,连续观察肝细胞的形态,检测肝细胞的白蛋白分泌功能和糖合成功能。并同时与贴壁培养的肝细胞比较。结果微载体黏附培养的肝细胞在存活率,保持正常形态和白蛋白分泌功能,葡萄糖合成功能方面都保持较高水平,明显优于贴壁培养的肝细胞。结论微载体培养能提供长时间保持高活性,高密度生长的肝细胞,为肝细胞移植,肝病防治以及生物人工肝等领域的研究和广泛应用提供了基础。     

中空纤维型生物人工肝用于支持治疗重型肝炎的体外初探

本实验采用混合多细胞聚集培养的人肝细胞、肝非实质细胞为生物材料，中空纤维管为生物反应器，组成人工肝装置。取重型肝炎患者血清进行体外循环，通过观察血清胆红素、白蛋白等部分生化指标和实验装置内肝细胞活力的变化，探讨生物人工肝用于支持治疗重型肝炎的可能性。...     

移植培养人肝细胞对急性肝衰竭小鼠的保护作用

目的探讨腹腔移植微载体粘附培养人肝细胞对Ｄ氨基半乳糖（ＤＧａｌ）诱导的急性肝衰竭小鼠的保护作用．方法采用胶原酶灌流法分离人肝细胞，胶原被覆的微载体Ｃｙｔｏｄｅｘ３加以培养，经腹腔注射２×１０６个肝细胞及载体，观察急性肝衰竭小鼠（ｎ＝３２，２５ｇ／ｋｇＤＧａｌ诱导４ｈ）７２ｈ存活率、血清ＡＬＴ、总胆红素以及肝脏病理变化．结果与只接受微载体而无粘附肝细胞的对照组比较，腹腔移植培养肝细胞对肝衰竭小鼠的存活率有明显改善（６５％ｖｓ０％），其血清ＡＬＴ（ＩＵ／Ｌ，２１７４４±２６３０ｖｓ４２６３１±４９２８，Ｐ＜００５）及总胆红素（ｍｍｏｌ／Ｌ，２６９１±３７６ｖｓ４１６８±３８３，Ｐ＜００５）较低．肝组织病理改变较轻．结论腹腔移植微载体粘附培养肝细胞可提供代谢支持，使ＤＧａｌ损伤的小鼠肝功能恢复．     

生物人工肝体外支持系统合成功能的评价

目的 对人肝细胞型体外生物人工肝进行生物合成功能的评价,藉以探讨其用于肝衰竭治疗的可能性。方法 用2×10~8个混合聚集球形培养的人肝细胞、肝非实质细胞与中空纤维型生物反应器组成体外生物人工肝支持系统(EBLSS),通过循环液(RPMI 1640)中肝细胞合成分泌产物的分析,评价其生物功能。结果 经过6小时的体外循环,循环液中总蛋白、白蛋白和甲胎蛋白含量均显著增加,尿素和葡萄糖水平也有明显改变,该循环液使体外培养大鼠肝细胞DNA合成率显著增加(与对照组比较P<0.01),实验后球形体肝细胞仍保持良好的活动。结论 由培养人肝细胞相中空纤维型生物反应器组成的体外生物人工肝具备肝脏生物合成功能,并有刺激肝细胞增殖的能力,对肝衰竭可能有一定的支持治疗作用。     

改良的乳猪肝细胞微载体粘附培养方法

目的改进常用的肝细胞手工震荡微载体粘附培养技术,提高肝细胞粘附率.方法用磁力悬浮培养容器使肝细胞轻度聚集,然后加入微载体Cytodex-3培养,在控制低转速的情况下使肝细胞更易粘附在微载体上.结果磁力搅拌粘附培养的肝细胞粘附率高(90.3%±7.7%)且较稳定(变异系数8.6%),明显优于手工震荡粘附培养(粘附率69.5%±18.3%,变异系数26.4%,P＜0.005);前2周内的白蛋白合成功能亦强于手工震荡粘附培养(P＜0.05).结论磁力搅拌粘附培养肝细胞粘附率高、重复性强、稳定性好、操作方便,且减少了肝细胞受污染的机会.     

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。     

Non-A, non-B hepatitis-like nuclear particles in nonparenchymal cells of the liver

Nuclear particles, morphologically similar to those seen in hepatocytes during non-A, non-B (NANB) hepatitis, were detected in several types of nonparenchymal cells in 10 human liver-biopsy specimens, including cases of hepatitis A and B and nonviral hepatic disease. They were also found in nonparenchymal cells of the liver in two of four normal chimpanzees and in two of four chimpanzees during experimental NANB viral hepatitis. In nonparenchymal cells the particles formed loose-to-intermediate aggregates, similar to those first described in hepatocytes during NANB hepatitis. Tightly packed aggregates, the predominant pattern in hepatocytes, were generally missing. The high prevalence of morphologically identical particles in various liver diseases and their presence in healthy livers, both in hepatocytes and in nonparenchymal cells not presumed to support the growth of hepatitis viruses, speak against their specificity for NANB hepatitis viruses. It is proposed that the particles represent a newly recognized and widespread cellular feature, of as yet unknown function.     

Binding of plasminogen to hepatocytes isolated from injured mice liver and nonparenchymal cell-dependent proliferation of hepatocytes

Plasmin is an essential enzyme located in the pericellular microenvironment of liver cells during liver regeneration. Previously, we reported that liver regeneration ability was significantly increased in alpha2-antiplasmin gene knockout mice as compared to wild-type mice, but it was significantly decreased in plasminogen knockout mice, or Plg/alpha2-antiplasmin gene knockout mice. The present study aimed to demonstrate direct interaction between plasminogen and mouse hepatocytes in the process of liver regeneration. Using the isolated hepatocytes from mice we analyzed following subjects: binding capacity of plasminogen to hepatocytes, plasminogen activation in the presence of hepatocytes, and proliferation ability of hepatocytes cocultured with liver nonparenchymal cells. The isolated hepatocytes from plasminogen wild-type mice bound to immobilized plasminogen. The mouse hepatocytes enhanced plasminogen activation, and impaired the inhibitory effect of alpha2-antiplasmin. The proliferation ability of hepatocytes after liver injury was studied. In plasminogen wild-type and plasminogen knockout mice, the hepatocytes cocultured with nonparenchymal cells, which were obtained from mice without CCl4 injection, showed similar proliferation abilities. On the contrary, the proliferation ability of hepatocytes cocultured with nonparenchymal cells, which were obtained from CCl4-treated plasminogen knockout mice, was significantly impaired as compared to wild-type mice. These results indicate that the plasminogen-plasmin system on the surface of mouse hepatocytes plays an important role in liver regeneration.     

Heterogeneous distribution of transferrin receptors on parenchymal and nonparenchymal liver cells: biochemical and morphological evidence

To investigate which cells of the liver express the receptor for transferrin, isolated rat liver cells produced by collagenase perfusion were fractionated by repeated differential centrifugation to produce hepatocytes (95% + 1%, mean +/- SD, n = 4) and nonparenchymal cells (97% + 1%, n = 3). Saturable, high-affinity binding of 125I-transferrin was demonstrated on intact cells at 4 degrees C, with average receptor numbers 20,900 +/- 3,160 (mean + SD, n = 4) for hepatocytes and 5,500 + 1,520 (n = 3) for nonparenchymal cells. Total cellular receptors measured in detergent permeabilized hepatocytes were 42,000 +/- 18,330 (mean +/- SD, n = 3) per cell and 14,760 +/- 7,120 (n = 3) per cell in the nonparenchymal fraction. Immunocytochemical demonstration of transferrin using antitransferrin, peroxidase antiperoxidase complex confirmed that both cell types bound transferrin. There was heterogeneity of the staining reaction since there was no detectable staining on 40% of hepatocytes and 60% of nonparenchymal cells. Microdensitometric analysis of the staining product corroborated the biochemical evidence that hepatocytes have, on average, more than three times more transferrin receptors than do nonparenchymal cells. These findings support the concept that the hepatocyte has a central role in the uptake and storage of transferrin iron.     

Transplantation of microcarrier-attached hepatocytes into 90% partially hepatectomized rats

We previously reported a method of intraperitoneal transplantation of liver cells attached to collagen-coated microcarriers, which resulted in prolonged survival and function of the transplanted cells. In the present study, we evaluated the efficacy of liver cell transplantation in providing metabolic support during acute liver insufficiency induced by 90% partial hepatectomy in rats. Ninety per cent of the liver mass (all lobes except the caudate lobe) was resected, and the rats were provided with 5% dextrose orally ad libitum upon regaining consciousness. This regimen results in severe hypoglycemia and death within 48 hr. When microcarrier-attached liver cells were transplanted into syngeneic and allogeneic recipients 3 days prior to 90% partial hepatectomy, significantly higher blood glucose levels were observed (p less than 0.01), compared to the levels in control rats which received injections of microcarriers, liver cells or medium alone. There was a marked improvement in long-term survival (40% survived longer than 28 days; p less than 0.001) in rats transplanted with microcarriers-attached cells. None of the rats given injections of microcarriers, liver cells or medium alone survived beyond 5 days. When liver cells alone or attached to microcarriers were injected intraperitoneally immediately after 90% partial hepatectomy, all rats became hypoglycemic and died within 48 hr, suggesting that vascularization of the transplant is required for function of the transplanted hepatocytes. The results indicate that intraperitoneal transplantation of microcarrier-attached hepatocytes prior to 90% partial hepatectomy in rats provides acute metabolic support resulting in improved survival.     

Hepatic and extrahepatic clearance of circulating human lactoferrin: an experimental study in rat

Abstract: Lactoferrin, unlabelled or ¹²⁵I-labelled by 2 different methods, was given intravenously to rats. Blood, tissue and liver cell radioactivity was measured. Both of the radiolabelled preparations were eliminated by the liver, and some deposited extrahepatically. One preparation formed large aggregates – here 90% of the hepatic uptake occurred in the Kupffer cells. The other preparation, consisting mostly of protein monomers but also dimers/oligomers/microaggregates, was taken up by hepatocytes (63% of total liver uptake), liver endothelial cells (22%) and Kupffer cells (15%). On a per cell volume basis, lactoferrin uptake was much more efficient by nonparenchymal cells compared to hepatocytes, which explains why immunomorphological staining only revealed lactoferrin in the nonparenchymal liver cells. The study demonstrates that radio-iodination of lactoferrin can affect its properties and handling, which may be important regarding contradictory reports on hepatic lactoferrin uptake. We conclude that both hepatocytes and nonparenchymal liver cells are involved in the blood clearance of lactoferrin, probably to a great extent owing to nonspecific mechanisms. Extrahepatic deposition and exposure (for instance on vessel walls/glomeruli) suggests that lactoferrin can be available to circulating anti-lactoferrin autoantibodies in autoimmune disease.     

Experimental study of bioartificial liver with cultured human liver cells

ＭＥＴＨＯＤＳＴｈｅｌｉｖｅｒｓｕｐｐｏｒｔｅｘｐｅｒｉｍｅｎｔｏｆＥＢＬＳＳｃｏｎｓｉｓｔｉｎｇｏｆａｇｇｒｅｇａｔｅｓｃｕｌｔｕｒｅｄｈｕｍａｎｌｉｖｅｒｃｅｌｓ,ｈｏｌｏｗｆｉｂｅｒｂｉｏｒｅａｃｔｏｒ,ａｎｄｃｉｒｃｕｌａｔｉｏｎｕｎｉｔ...     

体外生物人工肝支持作用的实验研究

目的 构建理想的体外人工肝支持系统，探讨其对暴发性肝衰竭的支持作用。方法 用经体外两步灌流法分离，限制贴壁技术球形聚集混合培养的人肝细胞－肝非实质细胞与中空纤维型生物反应器及由血液透析仪改装的辅助循环装置共同构建的ＥＢＬＳＳ，对门腔静脉分流加入肝血液阻断法建立的ＦＨＦ模型犬进行人工肝支持实验。结果 在平均约１０＾８肝细胞－肝非实质细胞ＥＢＬＳＳ的支持下，治疗组犬存活时间较无细胞空白循环对照组延长近     

In vivo uptake of chylomicron [3H]retinyl ester by rat liver: evidence for retinol transfer from parenchymal to nonparenchymal cells.

We have studied hepatic uptake of chylomicron retinyl ester. Chylomicrons were obtained from intestinal lymph of rats that were given retinol in groundnut oil by intraduodenal injection. When lymph was injected intravenously into normal rats, the radioactivity was cleared from blood with t1/2 approximately equal to 10 min. Retinyl ester was taken up initially by the liver, which, after 30 min, contained 80-90% of the radioactivity injected. Initially, most of the radioactivity was in hepatocytes, but after 30 min it disappeared from these cells and reappeared in nonparenchymal liver cells. After 2 hr these cells contained more radioactivity than the hepatocytes. When lymph was injected into vitamin A-deficient rats or rats given vitamin A in the form of retinoic acid, the plasma clearance and initial hepatic uptake of radioactivity were similar to that found in control animals. However, the nonparenchymal cells in these animals did not accumulate radioactivity. The current data suggest that vitamin A (in chylomicron remnants) is taken up initially by hepatocytes and then is released from these cells and delivered mainly to nonparenchymal liver cells in normal animals. In vitamin A-deficient rats, the vitamin is transferred from the hepatocytes to extrahepatic tissues.