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。     

A new method to immortalize primary cultured rat hepatocytes

BACKGROUND: In conventional methods of establishing hepatocyte cell lines, the immortalizing gene alone is introduced into hepatocytes. We designed a new method in which not only the immortalizing gene, the simian virus-40 large T-antigen (SV-40 Tag) gene, but also a drug-resistant gene, under the control of an albumin enhancer/promoter, were introduced into hepatocytes to efficiently obtain immortalized hepatocyte cell lines. METHODS: The plasmid pAPUR contains the puromycin-resistant gene under the control of an albumin enhancer/promoter, and the pSVTag contains the early region of SV-40 enhancer/promoter and the SV-40 Tag gene. Both pAPUR and pSVTag were transferred into isolated rat hepatocytes by electroporation. After these cells were cultured on a collagen-coated dish for 24 hours, puromycin selection was started. Expression levels of albumin, alpha-fetoprotein (AFP), SV-40 Tag, and cytokeratin 19 (CK 19) in the transformed cells were evaluated by western analysis, immunocytochemical staining, and RT PCR. RESULTS: Approximately 3 weeks after transfection, five or six colonies appeared on the dish. Twenty strains were obtained by cloning these cells. All strains that were similar to immature hepatocytes expressed albumin and SV-40 Tag, although CK 19 was not detected. AFP expression was detected in 33% of these strains. CONCLUSIONS: All clones cotransfected by pAPUR and pSVTag expressed albumin. Our new method may be useful to establish hepatocyte cell lines.     

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     

原代猪肝细胞培养体系及培养模式的初步探讨

目的探索一高密度、高效率的原代猪肝细胞培养方法,为生物人工肝的生物材料提供一种有效的培养模式.方法采用改良Seglen二步胶原酶灌注法分离肝细胞,分别将5×106的肝细胞加入胎牛血清、猪门静脉血清的培养体系中进行方瓶静止培养及聚球培养.结果肝细胞在接种后呈明显的Cytodex-3聚集趋势.门静脉血清培养体系中肝细胞的Albumin、Urea合成能力明显高于胎牛血清组.在培养后的前3周门静脉血清培养体系中聚球细胞模式优于微载体静止培养模式.结论与胎牛血清培养体系相比,猪门静脉血清培养体系更适合原代猪肝细胞高密度培养.聚球培养是一种较为理想的原代猪肝细胞培养模式.     

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.     

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     

Comparison of methods for isolating primary hepatocytes from mini pigs

Successful porcine hepatocyte isolation is crucial for the development of bioartificial liver devices and hepatocyte transplantation. Serva collagenase NB grades are formulated collagenases that are suitable for various tissue isolation applications. N‐acetylcysteine (NAC) can improve the viability of human hepatocytes. The aim of this study was to compare the effectiveness of two collagenases and effect of NAC on hepatocyte isolation from porcine liver tissue. Porcine hepatocytes were isolated using the perfusion method from Bama mini pigs assigned to the Serva NB 4 group (n=6), the Serva NB 8 group (n=6), or the NB 8+NAC group (n=6). Viability and yield were defined as fresh hepatocytes and their spheroids formation after 24‐hour rocker culture in serum‐free medium. Metabolic function was assessed by gene expression, albumin, and urea synthesis. All procedures resulted in successful hepatocyte isolation. Cells from the NB 8+NAC group had (97.8±1.9)% viability, which was higher than the NB 8 group with (94.4±2.4)% and the NB 4 group with (94.5±3.2)% (P<.001). The final cell yield reached (11.8±1.0)×10⁹ cells in the NB 8+NAC group, compared to (9.5±2.1)×10⁹ cells in the NB 8 group (P<.01) and (9.1±1.1) ×10⁹ cells in the NB 4 group (P<.001). The secretion of albumin was superior in the NB 8+NAC group at a concentration of (425.8±35.3) ng/mL compared to the NB 8 group (339.1±32.6) ng/mL (P <.001) and NB 4 group (293.6±43.3) ng/mL (P <.01). The injury of hepatocytes also decreased in the NB 8+NAC group (P<.01). The data are presented as means ± SD. Formulated collagenase Serva NB 8 and NAC could improve the porcine hepatocyte isolation, resulting in higher yields of viable cells.     

Growth and metabolic activity of immortalized porcine hepatocytes in extracorporeal hollow-fiber liver assist devices

The development of a cell based extracorporeal liver assist device offers a promising clinical approach to bridge individuals suffering from acute liver failure to transplant. However, a major drawback of the existing technology is the lack of a continuous supply of well differentiated hepatocytes. Although some investigators have used primary porcine cells, this approach demands costly, labor-intensive isolation procedures and yields cells with inconsistent detoxification capacity. The limitations of primary cells led us to develop the HepLiu immortalized porcine hepatocyte cell line for use in liver assist devices (LADs). HepLiu cells are nontumorigenic and exhibit multiple hepatic detoxification functions including diazepam and acetaminophen metabolism. To investigate the suitability of HepLiu cells for artificial liver support, morphology, as well as xenobiotic metabolism, was studied in perfused polysulfone hollow-fiber LADs. HepLiu cells were cultured in the intercapillary space of a prototype LAD, and the metabolism of diazepam, acetaminophen, and 7-ethoxycoumarin was evaluated over 25 days in culture. Our results indicated that HepLiu cells proliferated rapidly following inoculation of the LAD until Day 10 when proliferation appeared to cease. Ultrastructural analysis demonstrated that HepLiu cells retained many of the features of primary hepatocytes including desmosomes that sealed bile canalicular-like structures and junctional complexes (intermediate, gap junctions) that appeared concentrated in the paracanalicular areas. Unlike primary porcine hepatocytes, HepLiu cells retained drug metabolic function throughout the 25 day culture period. Diazepam metabolism by HepLiu cells was consistently higher than that of primary cells. Acetaminophen metabolism persisted throughout the 25 day period albeit at a much lower level than the primary cells exhibited on Days 1 or 2. In conclusion, we have shown that HepLiu cells proliferate to occupy the intercapillary space of perfused hollow-fiber LADs following inoculation, and retain their metabolic capacity for Phase I and Phase II detoxification reactions in perfusion culture. Our findings suggest that HepLiu cells may provide an alternative to primary porcine hepatocytes as the cellular component of bioartificial liver support systems.     

Comparison of four minimally invasive methods of laparoscopic vagotomy in a porcine model

Background: An experimental study in a porcine model was undertaken to evaluate the currently available techniques of laparoscopic vagotomy. Methods: Four groups of pigs were studied. Under general anesthesia, the animals were submitted to either bilateral vagotomy, bilateral highly selective vagotomy, posterior truncal vagotomy with anterior highly selective vagotomy, or Taylor's procedure. Gastric acid secretion and intestinal motility were evaluated before and after the surgical procedure. The feasibility of the four different techniques was assessed by means of a personal difficulty score. Results: All four procedures produced significant acid secretory reduction. Multivariate analysis showed that the factor most affecting the outcome was the difficulty score. Conclusions: Taylor's procedure was the easiest and safest technique. It also produced the best functional results for secretion and motility. Received: 19 December 1996/Accepted: 30 June 1997     

四种胆道姑息性引流手术的疗效比较

目的:应用4种胆道姑息引流手术处理恶性梗阻性黄疸病人的疗效比较。方法:收集1997年1月至2006年12月我院收治的153例因各种原因而分别行胆管T管外引流术、胆囊空肠Roux-en-Y吻合术、胆管空肠Roux-en-Y吻合术及胆管空肠T管架桥内引流术的恶性梗阻性黄疸病人,对4种胆道姑息引流手术的手术时间、术中出血量、手术退黄效果、肝功能恢复情况、手术死亡率、并发症率、术后住院天数、住院费用、术后生活质量以及病人生存期进行比较分析。结果:本组获随访者123人,随访率为80.39%。胆管空肠T管架桥内引流术组在平均手术时间[(100.31±34.29)min]、术中平均出血量[(223.97±120.79)ml]、术后平均住院天数(17.84±8.30d)和平均住院费用(22836.6±14112.24元)方面均显著低于胆管空肠Roux-en-Y吻合术组,而同胆管T管外引流术组和胆囊空肠Roux-en-Y吻合术组比较无显著性差异。4种胆道姑息引流手术在退黄效果、肝功能恢复、手术死亡率和并发症率方面经统计学检验亦无显著性差异。结论:胆管空肠T管架桥内引流术兼具T管外引流手术与胆肠内引流手术的优点,具有疗效确切、操作简便、手术创伤小、恢复快等特点。尤其适用于高龄和术前一般情况较差无法耐受长时间手术的病人。     

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.     

体外培养肝细胞胆汁分泌功能的研究

目的：使体外培养肝细胞获得胆汁分泌功能.方法：利用三明治构型培养成SD大鼠肝细胞并观察其胆小管结构及其胆汁分泌功能的形成,并以单层胶原培养肝细胞作对照。结果：免疫细胞化学显示三明治构型肝细胞培养24h后,胆小管结构初步形成,随着培养时间延长,胆小管结构更加清晰,120h后形成胆小管网络,而在对照组肝细胞,胆小管结构形成后,随着培养时间延长逐渐模糊,120h后胆小管结构消失,没有形成胆小管网络。其次,荧光二乙酯的肝细胞代谢显示,三明治构型肝细胞培养96h后即有胆汁分泌功能,而在对照组,肝细胞没有胆汁分泌功能。结论：三明治构型培养肝细胞在体外重建体内胆小管结构,保持了体内肝细胞胆汁分泌功能的特点。     

猪肝细胞与骨髓间充质干细胞最适共培养体系的建立

目的：建立猪肝细胞与骨髓间充质干细胞（MSCS）最适宜共培养体系,为生物人工肝的构建提供理想的细胞来源。方法：抽取中华实验猪（n=3）髂前上棘骨髓,采用密度梯度离心法分离单个核细胞,贴壁传代培养至第3代;采用原位两步胶原酶法分离猪肝细胞后与MSCS按1∶1,2∶1,5∶1和10∶1的比例混合培养,观察各组共培养肝细胞形态和功能的变化水平。结果：所获肝细胞纯度>99%,存活率>95%。共培养组肝细胞迅速黏附于MSCS表面,在三维空间呈球形聚集生长;肝细胞与MSCS间出现细胞连接,超微结构与正常肝细胞接近。肝细胞/MSCS 以2∶1共培养组的肝细胞清蛋白分泌水平、尿素合成能力和细胞色素P450为各组中之最佳,自第1天培养起均显著高于对照组（P<0.05）,并在第2天达到高峰,而且下降趋势较缓慢。结论：猪肝细胞/MSCS 按2∶1组成的最适共培养体系可最大程度地维持肝细胞功能,为构建功能性生物人工肝提供高效细胞材料。     

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.     

Comparison of four energy-based vascular sealing and cutting instruments: A porcine model

Aim To compare the safety and efficacy of four energy-based vascular sealing and cutting instruments. Methods Blood vessels of various types and diameters were harvested from four pigs using four instruments: Harmonic ACE™ (Ethicon Endo-Surgery, Cincinnati, OH), LigaSure™ V and LigaSure Atlas™ (Valleylab, Inc., Boulder, CO; a division of Tyco Healthcare), and EnSeal™ vessel fusion system (SurgRx, Inc. Redwood City, CA). The diameters of the vessels, speed and adequacy of the cutting and sealing process, and bursting pressures were compared. An additional set of specimens was sealed and left in situ for up to 4 h after which the vessels were harvested and histopathologically analyzed for the degree of thermal injury. Results The bursting pressures were significantly higher with EnSeal™ compared to all other instruments (p < 0.0001). The sealing process was significantly shorter with Harmonic ACE™ and significantly longer with LigaSure Atlas™ (p <0.0001). The mean seal width was larger with the LigaSure Atlas™ compared to the other instruments, and it was smaller with EnSeal™ and Harmonic ACE™. Less radial adventitial collagen denaturation was present with EnSeal™ and LigaSure™ V than with the other two instruments; there were no significant differences in collagen denaturation although proximal thermal injury to the smooth muscle in the media of the vessel wall was less common with LigaSure Atlas™ than with the other instruments; however, the numbers were too small for statistical analysis. Conclusions The bursting pressures with EnSeal™ were significantly higher than with all the other instruments. Harmonic ACE™ was the fastest sealing instrument and LigaSure Atlas™ was slowest. EnSeal™ created less radial thermal damage to the adventitial collagen of the vessels and LigaSure Atlas™ created less thermal damage to the media of the vessels. The clinical significance of these findings is unknown. Accepted for podium presentation at the SAGES meeting, April 2007, Las-Vegas