L02细胞在2-乙酰氨基芴处理大鼠肝中的增殖

目的:应用2-乙酰氨基芴(2-acetaminofluorene, 2-AAF),研究L02细胞移植到具有正常免疫性的大鼠肝内的存活与增殖情况．方法:SD大鼠出生前宫内ip正常人L02肝细胞,诱导胎鼠对L02肝细胞产生免疫耐受,出生2 wk时分别ip小(1．2 mg／kg)、中(13 mg／kg)、大剂量(30 mg／kg)2-乙酰氨基芴或生理盐水,隔日行2／3肝切除术,同时经脾移植DiI染色后的人L02肝细胞,建立2-AAF／PH肝细胞增殖模型．采用免疫荧光、RT-PCR、免疫组化、DiI荧光示踪等方法,在不同时相分别检测人白蛋白、人白蛋白mRNA、增殖细胞核抗原(PCNA)以及在荧光显微镜下观察人L02肝细胞在鼠肝内的分布,并采用计算机图像分析系统分析不同时相PCNA阳性细胞的数密度和面积密度．结果:2-乙酰氨基芴实验组(小、中、大剂量)的各项检测,均与对照组无明显区别;于移植后1,2,4,6,8,10 wk在荧光显微镜下观察到人L02肝细胞在鼠肝内的动态分布;移植后2, 4,6,8 wk大鼠均检测出人白蛋白及人白蛋白mRNA;移植后2,4,6 wk检测出特异性人增殖细胞核抗原PCNA,移植后1,2,4,6,8 wk检测出非特异性PCNA．结论:2-AAF／PH模型中,2-乙酰氨基芴对移植L02细胞无明显增殖作用;L02细胞在2-乙酰氨基芴处理人鼠嵌合肝动物模型中存活10 wk,产生白蛋白功能持续8 wk．     

L02细胞建立人鼠嵌合肝动物模型

目的:研究L02细胞移植到具有正常免疫活性的大鼠肝内的存活情况.方法:SD大鼠出生前宫内腹腔注射正常人L02肝细胞,诱导胎鼠对人L02肝细胞产生免疫耐受,出生2wk时经脾移植DiI染色后的人L02肝细胞,建立人鼠嵌合肝动物模型.采用免疫荧光、SP免疫组化、DiI荧光示踪等方法,分别检测人白蛋白、特异性人增殖细胞核抗原PCNA(proliferatingcellnuclearantigen)以及在荧光显微镜下观察人L02肝细胞在鼠肝内的分布.结果:于移植后1,2,4,6,8,10wk在荧光显微镜下观察到人L02肝细胞在鼠肝内的动态分布;移植后2,4,6,8wk大鼠均检测出人白蛋白,4wk时分泌最多;移植后2,4,6wk检测出特异性人增殖细胞核抗原PCNA,以4wk发现PCNA阳性细胞最多.结论:移植的人L02肝细胞在具有正常免疫活性的免疫耐受鼠体内能够存活、增殖,并产生人白蛋白.     

L02细胞人鼠嵌合肝模型的建立

目的 研究正常人L02肝细胞移植到具有正常免疫活性的大鼠肝内的存活情况，建立稳定的人鼠嵌合肝动物模型。方法 SD大鼠出生前宫内腹腔注射L02肝细胞，诱导胎鼠对人L02肝细胞产生免疫耐受，出生2周后经脾移植DiI染色后的人L02肝细胞，建立人鼠嵌合肝动物模型。采用免疫荧光、免疫组织化学、DiI荧光示踪等方法，在不同时相分别检测人白蛋白和特异性人增殖细胞核抗原，在荧光显微镜下观察人L02肝细胞在鼠肝内的分布。结果 于移植后1、2、4、6、8、10周在荧光显微镜下观察到人L02肝细胞在鼠肝内的动态分布，移植后2、4、6、8周大鼠均检测出人白蛋白，移植后2、4、6周检测出特异性人增殖细胞核抗原。结论 移植的人L02肝细胞在具有正常免疫活性的免疫耐受鼠体内能够存活、增殖，并产生人白蛋白。     

Transplantation of human hepatocytes into tolerized genetically immunocompetent rats

AIM: To determine whether normal genetically immunocompetent rodent hosts could be manipulated to accept human hepatocyte transplants with long term survival without immunosuppression. METHODS: Tolerance towards human hepatocytes was established by injection of primary human hepatocytes or Huh7 human hepatoma cells into the peritoneal cavities of fetal rats. Corresponding cells were subsequently transplanted into newborn rats via intrasplenic injection within 24h after birth. RESULTS: Mixed lymphocyte assays showed that spleen cells from non-tolerized rats were stimulated to proliferate when exposed to human hepatocytes, while cells from tolerized rats were not. Injections made between 15 d and 17 d of gestation produced optimal tolerization. Transplanted human hepatocytes in rat livers were visualized by immunohistochemical staining of human albumin. By dot blotting of genomic DNA in livers of tolerized rats 16 weeks after hepatocyte transplantation, it was found that approximately 2.5 X 10(5) human hepatocytes survived per rat liver. Human albumin mRNA was detected in rat livers by RT-PCR for 15 wk, and human albumin protein was also detectable in rat serum. CONCLUSION: Tolerization of an immuno-competent rat can permit transplantation, and survival of functional human hepatocytes.     

Effects of retrorsine on mouse hepatocyte proliferation after liver injury

AIM: To study the effect of retrorsine on mouse he-patocyte proliferation. METHODS: Mice and rats were treated respectively with two injections of retrorsine (as retrosine-treated group) or saline (as non-treated group) at 2 wk intervals. They received a single injection of carbon tetrachloride (CCI4) 4 wk later. On d 0, 1, 2, 3, 4, 6, 15 after CCI4 administration, the animals were killed and their livers were excised. Hematoxylin and eosin (HE) staining and Ki-67 antibody immunohistochemical analysis of liver samples were used to evaluate the pathological changes and hepatocyte proliferation. RESULTS: In rats treated with retrorsine and CCI4, the liver displayed obvious megalocytosis, proliferation of mild bile duct, small hepatocyte-forming nodule, which were not found in liver samples from non-treated group. However, in mice treated with retrorsine combined with CCI4, the liver displayed hepatocyte degeneration and necrosis in perivenous areas. There was no obvious difference between retrorsine-treated group and non-treated group. Ki-67 immunohistochemical analysis showed that in rats treated with retrorsine, the positive hepatocytes mainly found in small hepatocyte nodules, were obviously less than those in non-treated group. The mice treated with retrorsine showed that the number of Ki-67 positive hepatocytes was very high and more than that in non-treated group. CONCLUSION: Retrorsine has no effect on mouse hepatocyte proliferation.     

Human liver stem/progenitor cells decrease serum bilirubin in hyperbilirubinemic Gunn rat

AIM:To test the ability of adult-derived human liver stem/progenitor cells(ADHLSC)from large scale cultures to conjugate bilirubin in vitro and in bilirubin conjugation deficient rat.METHODS:ADHLSC from large scale cultures were tested for their phenotype and for their capacity to conjugate bilirubin in vitro after hepatogenic differentiation.In vivo,Gunn rats[uridine diphosphate-glucuronosyltransferase 1A1(UGT1A1)deficient animal]were injected with ADHLSC and cryopreserved hepatocytes(positive control).Two,4,13 and 27 wk posttransplantation,transplanted Gunn rat bilirubin serum levels were determined by high performance liquid chromatography.Human cell engraftment of trans-planted cells was assessed 27 wk post-transplantation using immunohistochemistry and RTqPCR.RESULTS:Large scale culture conditions do not modified ADHLSC phenotype,ADHLSC were able to specifically conjugate bilirubin.ADHLSC were intraportally injected into Gunn rats and blood UCB was measured at different times post-transplantation,infused-Gunn rats exhibited a metabolic effect 3 mo post-transplantation and maintained over a 6 mo period.ADHLSC engraftment into Gunn rat’s liver was demonstrated by RTqPCR and immunohistochemistry against albumin and UGT1A1.CONCLUSION:ADHLSC from large scale cultures are efficient in conjugating bilirubin in vitro and in restoring a deficient metabolic function(reducing bilirubin level)in hyperbilirubinemic rats.     

Diminution of toxic copper accumulation in toxic milk mice modeling Wilson disease by embryonic hepatocyte intrasplenic transplantation

AIM: To observe the therapeutic effect of intrasplenic transplantation with embryonic hepatocytes on amelioration of hereditary copper accumulation in toxic milk (TX) mouse modeling Wilson disease. METHODS: Donor hepatocytes were harvested from 14-d fetal liver of a pregnant homogeneous DL mouse. These cells were successively cultured, labeled with fluorescein dye Hoechst 33342 for 24 h, and sequentially infused into the spleen parenchyma of the recipient TX mice. No host immunosuppression measures were taken. Two and four weeks after transplantation, the recipients were killed for routine histologic investigation and immunohistochemistry study up to 4 wk after transplantation. The serum copper and ceruloplasmin concentrations of the recipient mice were determined by graphite furnace atomic absorption spectroscopy. RESULTS: In the following 2nd and 4th wk after transplantation, the donor hepatocytes could be visualized in the livers of 47.3% recipients. The serum ceruloplasmin and copper concentrations increased by 1.6-fold after 2 wk and 2.0-fold times after 4 wk respectively, which ultimately rose from about 30% of the normal level to nearly 60% (P<0.01). The hepatic copper concentration decreased 7.2%, 4 wk after transplantation. Pathologic examination showed that there were many actively proliferative hepatocyte precursor cells with specific embryonic hepatocyte marker AFP migrated into hepatic sinusoids of the recipients. A large number of cells carrying hepatocytes marker and albumin were observed in the recipient spleen tissues. CONCLUSION: Embryonic hepatocytes are capable of differentiating into mature hepatocytes in vivo. After transplantation, the hereditary abnormalities of copper metabolism in TX mice could be corrected partially by intrasplenic transplantation of homogeneous embryonic hepatocytes.     

Intrasplenic hepatocellular transplantation corrects hepatic encephalopathy in portacaval-shunted rats.

The aim of this work was to evaluate the effect of intrasplenic hepatocellular transplantation on hepatic encephalopathy in an experimental model of chronic liver failure induced by end-to-side portacaval shunt in the rat. Inbred male Wistar Furth rats were divided into three groups: rats subjected to portacaval shunt (n = 10), rats subjected to portacaval shunt and intrasplenic hepatocellular transplantation of 10(7) hepatocytes isolated from livers of syngeneic rats (n = 10) and sham-operated rats (n = 10). Behavior tests were performed in a blind fashion at 3 wk, at 2 mo and at 3 mo after surgery. Spontaneous activity and nose-poke exploration by individual rats were studied in automated open field boxes equipped with infrared cells. Each cell beam interruption was automatically recorded on a microcomputer and transformed into a score index (counts/hour). Plasma levels of amino acids, ammonia and total biliary acids were measured. Portacaval shunt rats showed reduced spontaneous activity and nose-poke exploration scores. Intrasplenic hepatocellular transplantation significantly increased spontaneous activity after 2 mo and improved nose-poke exploration after 3 wk. At 3 mo, spontaneous activity and nose-poke exploration in portacaval shunt/intrasplenic hepatocellular transplantation rats were not significantly different from those of sham rats. Increases in plasma ammonia levels after portacaval shunt were not corrected. Amino acid imbalance and bile acid concentration in plasma were partially corrected by intrasplenic hepatocellular transplantation. These data show that intrasplenic hepatocellular transplantation can correct the neurological symptoms of hepatic encephalopathy in an experimental model of chronic liver failure and suggest that intrasplenic hepatocellular transplantation might be of therapeutic interest in chronic liver failure.     

Long-term albumin infusion improves survival in patients with cirrhosis and ascites： An unblinded randomized trial

AIM: To investigate the effects of long-term albumin administration on survival, recurrence of ascites and onset of other complications. METHODS: One hundred consecutive patients admitted for first-onset ascites were randomized to receive diuretics plus human albumin 25 g/wk in the first year and 25 g every two wk thereafter (group 1) or diuretics alone (group 2). The primary endpoint was survival without liver transplantation. Secondary endpoints were recurrence of ascites and occurrence of other complications. RESULTS: Median follow-up was 84 (2-120) mo. Albumin-treated patients had significantly greater cumulative survival rate (Breslow test =7.05, P=0.0078) and lower probability of ascites recurrence (51% versus 94%, P< 0.0001). Chronic albumin infusion resulted in a mean increase in survival of 16 mo. CONCLUSION: Long-term albumin administration after first-onset ascites significantly improves patients' survival and decreases the risk of ascites recurrence.     

Granulomatous enterocolitis induced in rats by purified bacterial cell wall fragments

This study was designed to determine if poorly biodegradable bacterial cell wall components can produce chronic intestinal inflammation. A sterile aqueous suspension of sonically disrupted group A or group D streptococcal cell wall fragments was injected intramurally into the small intestine and cecum of 100 rats. Gross findings in rats killed at intervals of 1 day to 6 mo included intestinal thickening, adhesions, and mesenteric contraction. Acute histologic inflammation subsided by 2 wk, but chronic granulomatous inflammation persisted for 6 mo in the rats injected with group A streptococcal cell wall fragments and 3 mo in the rats injected with group D streptococcal cell wall fragments. Ninety-six control rats identically injected with human serum albumin or phosphate-buffered saline demonstrated mild acute inflammation that resolved, with only 1 rat having chronic intestinal inflammation. Granulomas in the intestine, mesentery, and mesenteric lymph nodes developed in 46% of the rats injected with group A fragments and 45% of the rats injected with group D streptococcal cell wall fragments, compared with 20% of the controls injected with albumin and 4% of the controls injected with phosphate-buffered saline. Group A streptococcal antigen was detected by immunofluorescence at the site of inflammation for 4 mo, and possible reactivation of acute inflammation was seen up to 6 mo after injection. We conclude that bacterial cell wall fragments are capable of producing chronic granulomatous inflammation in the intestinal wall if present in appropriate particle size and concentration. We speculate that cell walls from the enteric microflora may leak across a permeable mucosa in chronic inflammatory bowel disease to initiate and sustain local and systemic inflammation.     

Repopulation of adult and neonatal mice with human hepatocytes: a chimeric animal model

We report the successful transplantation of human hepatocytes in immunodeficient, fumarylacetoacetate hydrolase-deficient (fah(-/-)) mice. Engraftment occurs over the entire liver acinus upon transplantation. A few weeks after transplantation, increasing concentrations of human proteins (e.g., human albumin and human C3a) can be measured in the blood of the recipient mouse. No fusion between mouse and human hepatocytes can be detected. Three months after transplantation, up to 20% of the mouse liver is repopulated by human hepatocytes, and sustained expression of lentiviral vector transduced gene can be observed. We further report the development of a hepatocyte transplantation method involving a transcutaneous, intrahepatic injection in neonatal mice. Human hepatocytes engraft over the entire injected lobe with an expansion pattern similar to those observed with intrasplenic transplantation.     

磷脂酰肌醇3-激酶信号通路在姜黄素诱导肝细胞转录因子Nrf2核转位中的作用

目的研究磷脂酰肌醇3-激酶（PI3K）信号通路在姜黄素诱导人肝细胞Nrf2核转位中的作用。方法用30μmol/L姜黄素和Wortmannin（PI3K抑制剂）干预L02肝细胞12 h,Western blot观察Nrf2核转位水平;将L02肝细胞分为对照组、模型组、干预组和抑制剂组,对照组用RPMI1640正常培养,模型组用100 U/L葡萄糖氧化酶（GO）干预2 h,干预组用30μmol/L姜黄素干预12 h后给予100 U/L GO干预2 h,抑制剂组先用0.1μmol/L Wortmannin预处理1h,余处理同干预组。流式细胞术检测细胞内活性氧簇（ROS）。分光光度法检测细胞MDA、GSH及培养液LDH水平,速率法检测细胞培养液AST的水平。结果姜黄素明显诱导Nrf2核转位,其诱导作用可被Wortmannin部分阻断。模型组ROS、MDA、AST、LDH水平较对照组显著升高（P〈0.01）,干预组ROS、MDA、AST、LDH水平较模型组显著降低（P〈0.01）,抑制剂组ROS、MDA、AST、LDH水平显著高于干预组,低于模型组（P〈0.01）。模型组GSH水平较对照组显著降低（P〈0.01）,干预组GSH水平较模型组显著升高（P〈0.01）,抑制剂组GSH水平显著低于干预组（P〈0.01）,高于模型组（P〈0.01）。结论 PI3K信号通路是姜黄素诱导Nrf2核转位的重要信号通路,通过抑制该通路可减弱姜黄素对肝细胞氧化应激损伤的保护作用。     

Failure of regeneration of the steatotic rat liver: disruption at two different levels in the regeneration pathway

Hepatic resection or transplantation in patients with fatty liver is associated with increased morbidity and mortality. The regenerative capacity of fatty livers after major tissue loss is unknown. Interleukin 6 (IL-6) is a potent inducer of hepatic regeneration in normal and ischemic livers. Therefore, we studied hepatic regeneration at day 1, day 2, and day 4 in a model of 70% hepatectomy in obese and lean Zucker rats, and obese Zucker rats pretreated with recombinant interleukin 6 (rIL-6). The mitotic cycle in hepatocytes was investigated by 4 different markers of regeneration representing distinct phases of mitosis (proliferating cell nuclear antigen [PCNA] = G(1) phase, bromodeoxy uridine [BrdU] = S phase, mitotic index, and regenerated liver weight = M phase). Obese Zucker rats had significantly decreased regenerative capacity compared with lean Zucker rats (PCNA, BrdU, mitotic index, regenerated liver weight) at days 1 and 2 after surgery. Four days after resection fatty animals showed an increase in the mitotic index indicating a delay of regeneration in steatotic livers. Animal survival after 70% hepatectomy was significantly decreased in obese rats compared with lean animals. Pretreatment of obese animals with rIL-6 normalized PCNA expression (G(1) phase) in steatotic hepatocytes but failed to increase DNA synthesis (BrdU, S phase), mitosis (mitotic index and regenerated liver weight, M phase), and animal survival. These results indicate major impairment of hepatic regeneration in steatotic livers. Two different blockages of regeneration must be present, one rIL-6 sensitive, at the level of IL-6 or upstream, and a second, rIL-6 resistant, at the level of G(1)/S-phase transition.     

Hepatocyte transplantation: Studies in preclinical models

Summary Transplantation of allogeneic or genetically modified autologous hepatocytes may be an alternative to whole-liver transplantation for the treatment of hereditary metabolic liver diseases. Human hepatocytes have already been transplanted in patients, demonstrating the safety and feasibility of both approaches. Although a few cases of allogeneic transplantation have resulted in long-term engraftment and function, only a partial and transient correction of the disease was achieved. This may partly result from a lack of proliferation of transplanted cells. In rodents, transplanted hepatocytes do not proliferate in adult quiescent livers and repopulate recipient livers only when they display a proliferative advantage over resident hepatocytes. Most of these models are not transposable to humans, however. Our aim is to develop preclinical approaches to hepatocyte transplantation in nonhuman primates. We have defined a strategy that increases the engraftment efficiency of transplanted hepatocytes by inducing their proliferation together with that of resident hepatocytes. We have also immortalized simian fetal hepatic progenitor cells and shown that these cells do not proliferate in situ after transplantation into the livers of immunodeficient mice. By contrast early human hepatoblasts repopulate mouse livers more efficiently. However, if we consider the number of cells to be transplanted (one to several billion), the means of expanding and differentiating stem or progenitor cells other than hepatocytes will have to be determined prior to envisaging treating patients. Presented at the 42nd Annual Meeting of the SSIEM, Paris, 6–9 September, 2005. Competing interests: None declared     

Formation of human hepatocytes by human hematopoietic stem cells in sheep

We took advantage of the proliferative and permissive environment of the developing preimmune fetus to develop a noninjury large animal model in sheep, in which the transplantation of defined populations of human hematopoietic stem cells resulted in the establishment of human hematopoiesis and led to the formation of significant numbers of long-lasting, functional human liver cells, with some animals exhibiting levels as high as 20% of donor (human) hepatocytes 11 months after transplantation. A direct correlation was found between hepatocyte activity and phenotype of transplanted cells, cell dose administered, source of cells used on a cell-per-cell basis (bone marrow, cord blood, mobilized peripheral blood), and time after transplantation. Human hepatocytes generated in this model retained functional properties of normal hepatocytes, constituted hepatic functional units with the presence of human endothelial and biliary duct cells, and secreted human albumin that was detected in circulation. Transplanting populations of hematopoietic stem cells can efficiently generate significant numbers of functional hepatic cells in this noninjury large animal model and thus could be a means of ameliorating or curing genetic diseases in which a deficiency of liver cells or their products threatens the life of the fetus or newborn.     

Colonization of albumin-producing hepatocytes derived from transplanted F344 rat bone marrow cells in the liver of congenic Nagase's analbuminemic rats

BACKGROUND/AIMS: We investigated whether bone marrow cells (BMCs) of normal rats can be transformed in albumin-producing hepatocytes in analbuminemic rat livers. METHODS: BMCs (2 x 10(7)) from F344 rats (F344) were infused via the portal vein into the livers of congenic Nagase's analbuminemic rats (F344alb) immediately after 70% hepatectomy (PH). Alternatively, F344alb were hematopoietically reconstituted with F344 BMCs by whole body irradiation and BMC transplantation before PH. The recipients were examined for albumin positive (alb +) hepatocytes and albumin mRNA in the livers as well as serum albumin levels 4 weeks later. Sry3 in situ hybridization was done for the livers of female F344alb that received male F344 BMCs. RESULTS: Livers of untreated F344alb contained a few single and double alb+hepatocytes, but these did not form clusters after PH. Clusters (>3 alb + hepatocytes) were detected in livers of the recipients which were transplanted with BMCs immediately after PH as well as the reconstituted F344alb with or without PH. Normal albumin mRNA was detected in the recipient livers, and serum albumin levels were increased. Sry3 was identified in the alb+clusters in the female recipients. CONCLUSIONS: Transplanted BMCs from normal rats can increase clusters of albumin-producing hepatocytes within the liver of analbuminemic rats.     

Prevention of diabetic glomerulopathy in streptozotocin diabetic rats by insulin treatment

Summary A single antibody radioimmunoassay has been used to measure albumin excretion in 3 groups of female Wistar rats. Two groups had Streptozotocin diabetes and were treated daily with insulin for 6 months. In one of the diabetic groups good glycaemic control was attempted and throughout the 6 months plasma glucose levels were fairly close to normal (92 ± 33 mg/100 ml at 2300 h and 186 ± 9 mg/100 ml at 0800 h). In the other diabetic group poor control was intended and the group had consistent high plasma glucose levels (576 ± 89 mg/100 ml and 460 ± 43 mg/100 ml). The third group was a non-diabetic control group. — Albumin excretion was measured on two occasions: before the induction of diabetes and after 6 months of diabetes. The geometric mean albumin excretion increased from 0.38 to 2.56 mg/24 h in the 18 non-diabetic controls. In the 20 diabetic rats in good control the geometric mean albumin excretion increased from 0.37 to 1.58 mg/ 24 h (NS compared with controls) and in the group of 22 rats in poor control albumin excretion increased from 0.35 to 6.54 mg/24 h. — The increase in albumin excretion in rats in poor control differed significantly both from that of the non-diabetic controls (2p = 0.023) and from that of the well-controlled diabetic rats (2p = 0.00011).