一种改良大鼠肝细胞分离和原代培养技术

目的 探讨并改进大鼠肝细胞分离和培养技术.方法 Ⅲ型胶原预铺培养板,改良原位胶原酶两步灌流法分离肝细胞,适宜密度接种.含10%胎生血清的PuDMI 1640培养基培养;观察细胞产量、活率、贴壁和生长情况评价方法的可行性.结果 肝细胞分离时间明显缩短;胶原酶用量减少1/3;每鼠可获得(1.81±0.65)X 108个肝细胞;活率为(87.46±6.90)%;细胞接种4 h即可贴壁,可存活2～3周.结论 该法操作简便、经济,细胞产量和存活率高,贴壁和生长良好,可满足各种实验需求.     

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     

A hybrid artificial liver system composed of primary cultured canine hepatocytes

The usefulness of newly device hybrid artificial liver system was evaluated in anhepatic dogs. The artificial liver module was composed of 60 to 80gm. primary cultured canine (Beagle) hepatocytes which were attached to 200 borocillicate glass plates. Total hepatectomy were done through cavo-caval and porto-caval shunt method using Anthron catheter to 14 dogs. Dogs were divided into following three groups. Group I; no treatment (n = 6) Group II; plasma perfusion (n = 4) Group III; treated with the artificial liver systems (n = 4) The survival times were 21.3 +/- 5.6, 27.8 +/- 4.0, and 55.0 +/- 11.3 hours in group I, II, and III, respectively. The longest survival time was 65 hours in one of group III dogs. APTT levels in group I and II increased more than 100 sec. within 24 hours. On the other hand it was maintained within 50 sec. during 54 hours treatment in group III. Ammonia levels in group I and II extremely increased over 2000ng/dl. In group III, it was less than 400ng/dl for 54 hours. Plasma amino acid levels in group I and II (Glutamine, Arginine, AAA) revealed significantly higher than in group III at 18 hours after operation. It is concluded that the newly device hybrid artificial liver system was useful for in vivo liver support.     

一种肝细胞联合冷冻保存的新方法

目的寻找一种效果更好的冻存液,进一步提高冻存肝细胞的活率和活性.方法用体重5～8 kg健康杂种乳猪作为肝细胞供体,用胶原酶原位灌注法分离肝细胞,将肝细胞分别以三种不同的冻存液(二甲基亚砜、二甲基亚砜+羟乙基淀粉、二甲基亚砜+羟乙基淀粉+1.3 mmol/L 氯化钙)在液氮中冷冻保存1个月.1个月后快速复温肝细胞,做活率、活性和形态学检测,部分肝细胞在含Ca2+和不含Ca2+两种不同的培养液里培养5 d,并观察其功能表达情况.结果不同冻存液保存的猪肝细胞其活率、活性和形态学表现均有所不同,其中单用DMSO(二甲基亚砜)的冷冻液效果最差;其次是HES(羟乙基淀粉)+DMSO组;HES+DMSO+1.3 mmol/L Cacl2联合冷冻液效果最好.复温后在含Ca2+培养液里培养的肝细胞其活率和白蛋白浓度比不含Ca2+的明显要好.结论 HES+DMSO+1.3 mmol/L Cacl2联合冷冻液显著提高了肝细胞的冻存质量.     

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。     

三明治构型原代大鼠肝细胞培养细胞极性重建及功能的研究

目的 研究三明治构型培养大鼠原代肝细胞的形态学变化、极性重建过程并对其功能进行测定.方法 改良原位两步法门静脉胶原酶灌注分离单肝细胞,三明治构型培养肝细胞,观察肝细胞的形态学变化,测定特异性膜区域蛋白的重新分布,检测白蛋白mRNA及肝细胞功能.结果 平均每个鼠肝可获取(2～3)×108个肝细胞,活率在93%±3%,纯度在96%±3%.培养6～8 h后,肝细胞排列成肝索样结构.3 d后,白蛋白mRNA表达明显增强.4 d后,DPPIV几乎完全集中在胆小管膜区.5 d后,胆小管完全连接成网络.形态维持达49 d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.     

High density lipoprotein catabolism in primary cultured hepatocytes from daunomycin-induced nephrotic rats

We investigated into HDL (high density lipoprotein) catabolism with primary cultured hepatocytes to elucidate the causes of increased HDL apolipoproteins in the plasma of daunomycin-induced nephrotic rats (D rats). The phospholipid, triglyceride, cholesterol, cholesteryl ester and apolipoprotein contents in HDL increased in D rats compared with control rats (C rats). The uptake (binding plus internalization) of (125)I-HDL from D rats to two groups of hepatocytes was significantly greater than that of (125)I-HDL from C rats. Uptake of (125)I-HDL from D rats to D rats' hepatocytes was significantly greater than that of (125)I-HDL from C rats to C rats' hepatocytes. The degradation of (125)I-HDL from D rats was greater than that of (125)I-HDL from C rats using two groups of hepatocytes. These results demonstrated that the uptake and degradation of HDL to D rats' hepatocytes were greater than those of HDL to C rats' hepatocytes. The increased HDL apolipoprotein content in the plasma of D rats may not be due to decreased uptake and degradation of HDL in hepatocytes compared with C rats. Copyright Copyright 1999 S. Karger AG, Basel     

Functional characterization of serum-free cultured rat hepatocytes for downstream transplantation applications

Although ex vivo culture of hepatocytes is known to impair functionality, it may still be considered as desirable to propagate or manipulate them in culture prior to transplantation into the host liver. The aim of this study was to clarify whether rat hepatocytes cultured over different periods of time proliferate and retain their hepatocyte-specific functions following transplantation into the recipient liver. Rat hepatocytes were cultured under serum-free conditions in the presence of hepatocyte and epidermal growth factors. Cells derived from wild-type donor livers were transplanted into the livers of CD26-deficient rats. Cell proliferation and the expression of hepatocyte-specific markers were determined before and after transplantation. Cell number increased threefold over a culture period of 10 days. The expression of connexin 32 and phosphoenolpyruvate carboxykinase declined over time, indicating the loss of hepatocyte-specific functions. Hepatocytes cultured over 4 or 7 days and then transplanted proliferated in the host parenchyma. The transplanted cells expressed connexin 32, cytokeratin 18, and phosphoenolpyruvate carboxykinase, indicating the differentiated phenotype. The loss of hepatocyte-specific functions during culture may be restored after transplantation, suggesting that the proper physiological environment is required to maintain the differentiated phenotype.     

Comparison of bioenergetic activity of primary porcine hepatocytes cultured in four different media

Primary hepatocytes have extensively been used in biochemical, pharmacological, and physiological research. Recently, primary porcine hepatocytes have been regarded as the cells of choice for bioartificial liver support systems. The optimum culture medium for hepatocytes to be used in such devices has yet to be defined. In this study we investigated the effectiveness of four culture media in driving energy metabolism of primary porcine hepatocytes. The media selected were William's E medium, medium 1640, medium 199, and hepatocyte medium. Cells (3 x 10(10); viability 87 +/- 6%) were isolated from weanling piglets and seeded on 90-mm plates in the above media supplemented with antibiotics and hormones at a density of 8 x 10(6) viable cells per plate. Using 1H NMR spectroscopy we looked at indices of glycolysis, gluconeogenesis. ketogenesis, and ureagenesis on days 2, 4, and 6 of the experiments (n = 9). We also studied urea and albumin synthesis and total P450 content. The examined metabolic pathways of the hepatocytes were maintained by all media, although there were statistically significant differences between them. All media performed well in glycolysis, ureagenesis, and albumin synthesis. William's E medium and medium 199 outperformed the rest in gluconeogenesis. Medium 199 was best in ketogenesis. Overall, medium 199 was the best at driving energy metabolism from its constituent substrates and we think that it preferentially should be used in the culture of primary porcine hepatocytes.     

Insulin-mimetic effects of vanadate in primary cultures of rat hepatocytes

To evaluate possible mechanisms by which insulin inhibits hepatic apolipoprotein B (apoB) secretion, we incubated primary cultures of rat hepatocytes with sodium orthovanadate, a phosphotyrosine phosphatase inhibitor and insulin-mimetic agent. Vanadate (10 microM) and insulin (10 nM) inhibited the medium accumulation of apoB (secretion) by 21 and 37%, respectively, without increasing intracellular apoB. The effects of insulin and vanadate together were not additive. Both insulin and vanadate enhanced intracellular glycogen accumulation by 82 and 37%, respectively. Unlike insulin, vanadate, at a concentration that inhibited apoB secretion (10 microM), had no effect on intracellular lipogenesis, inhibited the secretion of newly synthesized hepatic proteins, and had a delayed onset and termination of action on inhibition of apoB secretion. At higher concentrations (40 and 80 microM), vanadate stimulated intracellular lipogenesis. In conclusion, our data indicate that vanadate mimics insulin action in hepatocytes with regard to the inhibition of medium accumulation of apoB. These data are consistent with the hypothesis that inhibition of apoB secretion may be secondary to an increase in phosphotyrosine content at its site of synthesis. The kinases responsible for this effect have not been identified. Several effects of vanadate, however, are different from those of insulin, suggesting a differential sensitivity to vanadate, a divergence of the signal transfer by insulin and vanadate at the insulin-receptor or postreceptor level, or both.     

Isolated rat adrenal perfusion: A new method to study adrenal function

To study the function of the rat adrenal glands directly, a method was developed whereby the adrenal glands are selectively isolated and perfused in situ. Under basal conditions the perfused adrenals synthesized 570 ± 68 (mean ± SEM) μg corticosterone per gram adrenal tissue in 90 min compared to 933 ± 132 μg/g adrenal tissue (P < 0.05) when the stimulus, ACTH, was added to the perfusate. Because nonperfused control adrenals contain only 25 ± 3 μg corticosterone per gram tissue, the perfused adrenals synthesized between 20 (basal) and 40 (stimulated) times (both P < 0.01) as much corticosterone as was present initially. Moreover, the amount synthesized was equal to that synthesized by the intact animal and considerably greater than that reported using other experimental methods. In addition, almost all of the corticosterone synthesized during perfusion was also secreted during perfusion.     

A new source of hepatocytes for transplantation

INTRODUCTION: The most effective treatment for acute or chronic liver failure is orthotopic liver transplantation. Worldwide there is a shortage of organs for transplantation. This shortage has called for research into new treatments for management of patients with liver failure. One such treatment is hepatocyte transplantation. During liver resections considerable amounts of normal liver are unavoidably resected. We aim to harvest these hepatocytes and to filter the tumor cells from them to provide a source for transplantation. MATERIALS AND METHODS: After liver resection, the largest vessel at the resected liver edge was identified and cannulated. Seglen's two-stage technique of perfusing the liver with EDTA and collagenase was performed to harvest the hepatocytes. Ep-CAM Ags are consistently present on the surface of epithelial cells and in particular in colorectal cancer cells. Therefore, MOC31 antibodies (selective Abs for Ep-CAM) attached to magnetic beads were used to target the tumor cells. These tumor cells are selectively removed using a magnet. CEA staining was then used to ensure the hepatocyte collection was tumor cell free. Five million hepatocytes were rosetted with one million HT29 CRC cells to assess the immunomagnetic filtration technique. RESULTS: The hepatocyte harvesting resulted in 864,000 viable hepatocytes to be harvested per gram of liver. Histochemical staining using CEA demonstrated 75% of the HT29 cells in the hepatocyte collection were removed after one use of magnetic beads. CONCLUSION: We have demonstrated the successful initial stages of harvesting tumor-free hepatocytes from liver resected for malignancy.     

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.