Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.     

Effect of partial hepatectomy on the interrenal tissues of Xenopus laevis (Daudin)

Summary Partial hepatectomy was carried out on Xenopus laevis to investigate its influence on the endocrine system. In addition to other endocrine effects, a marked hypertrophy and stimulation of the interrenal gland was observed. Activated cells contain mitochondria with extended and irregularly coiled tubules embedded in a low electron dense matrix. Hepatectomy induces two phases of proliferation [3 and 35 days postoperative (p.o.)]. After 106 days p.o. giant mitochondria possessing narrow and closely packed, parallel tubules surrounded by an electron dense matrix indicate a phase of inactivation. The smooth endoplasmic reticulum and the Golgi apparatus proliferate after hepatectomy.During activation the high lipid content seen in controls is decreased significantly.This investigation was supported by the Deutsche Forschungsgemeinschaft     

Increased activity of rat liver N2-guanine tRNA methyltransferase II in response to liver damage

Alterations in rat liver transfer RNA (tRNA) methyltransferase activities have been observed after liver damage by various chemicals or by partial hepatectomy. The qualitative and quantitative nature of these activity changes and the time course for their induction have been studied. Since homologous tRNAs are essentially fully modified in vivo, E. coli tRNAs were used as in vitro substrates for the rat liver enzymes in these studies. Each of the liver-damaging agents tested rapidly caused increases in activities of the enzyme(s) catalyzing methyl group transfer to tRNAs that have an unmodified guanine at position 26 from the 5′ end of the molecule. This group of tRNAs includes E. coli tRNA^Nfmet, tRNA^Ala₁, tRNA^Leu₁, or ^Leu₂, and tRNA^Ser₃ (Group 1). In each case N²-methylguanine and N²,N²-dimethylguanine represented 90% or more of the products of these in vitro methylations. The product and substrate specificity observed are characteristic of N²-guanine methyltransferase II ($\text{S-}\text{adenosyl}\text{-}\text{L}\text{-}\text{methionine:tRNA}$ (guanine-2)-methyltransferase, EC 2.1.1.32). In crude and partially purified preparations derived from livers of both control and treated animals this enzyme activity was not diminished significantly by exposure to 50°C for 10 min. The same liver-damaging agents induced little or no change in the activities of enzymes that catalyze methyl group transfer to various other E. coli tRNAs that do not have guanine at position 26 (Group 2). The results of mixing experiments appear to rule out the likelihood that the observed enzyme activity changes are due to stimulatory or inhibitory materials present in the enzyme preperations from control or treated animals. Thus, our experiments indicate that liver damage by each of several different methods, including surgery or administration of chemicals that are strong carcinogens, hepatotoxins, or cancer-promoting substances, all produce changes in liver tRNA methyltransferase activity that represent a selective increase in activity of N²-guanine tRNA methyltransferase II. It is proposed that the specificity of this change is not fortuitous, but is the manifestation of an as yet unidentified regulatory process.     

移植途径对大鼠骨髓间充质干细胞归巢及肝功能恢复的影响

目的 探讨移植途径对骨髓间充质干细胞（MSCs）归巢及促进肝切除大鼠肝再生的影响.方法 建立肝切除大鼠模型,随机分为3 组,即肝切除对照组、尾静脉移植组和门静脉移植组.移植组分别经尾静脉和门静脉注射DAPI 标记的MSCs 约1.5×106/ 只,分别于第3 天和第9 天后采血清检测肝功能,第9 天处死大鼠取肝脏标本,并通过荧光显微镜观察两种移植途径对MSCs 向肝脏迁移的影响.结果 门静脉移植组（18.1 ± 3.4）个细胞/100 倍视野到肝脏归巢及定植的 MSCs 多于尾静脉移植组（7.6 ± 2.0）个细胞/100 倍视野,差异有统计学意义（P 〈 0.01）.术后第9 天各组大鼠肝功能均有好转,丙氨酸氨基转移酶（ALT）及天冬氨酸氨基转移酶（AST）3 组之间对比差异无统计学意义（F = 2.822,1.046,P = 0.057,0.365,P 〉 0.05）;但两移植组与单纯肝切除组比较血浆白蛋白（ALB）均有明显升高,差异具有统计学意义（F = 6.259,P = 0.006）;尾静脉移植组与门静脉移植组两移植组之间相比,差异无统计学意义（P 〉 0.05）.结论 移植途径对 MSCs 归巢、定植到肝脏有一定影响,门静脉途径优于外周静脉,MSCs 移植对肝大部切除大鼠肝功能恢复具有促进作用.     

原发性肝癌患者肝切除术前营养风险筛查及营养支持对营养风险患者康复效果的对照研究

摘要 目的：观察原发性肝癌 (PLC) 肝切除术前营养风险,并研究营养支持对营养风险患者康复效果的影响。方法：选择我院2021年7月~2021年12月期间收治的PLC患者99例,采用营养风险筛查2002 (NRS2002) 量表评估患者的营养风险,单因素及多因素Logistic回归分析PLC患者肝切除术前发生营养风险的影响因素。根据有无营养风险分为营养风险组（n=64）和无营养风险组（n=35）,其中营养风险组患者采用随机数字表法分为术前未接受营养支持组和术前接受营养支持组,各32例。其中术前未接受营养支持组术前不接受营养支持,术前接受营养支持组术前接受营养支持,对比术前未接受营养支持组、术前接受营养支持组的康复效果。结果：经营养风险筛查结果显示,99例PLC患者中有64例术前就已存在营养风险。单因素分析结果显示,无营养风险组、营养风险组在肿瘤直径、贫血、临床分期、家庭月收入、年龄、并发乙肝方面对比存在明显差异（P<0.05）。年龄为60~岁、家庭月收入<3000元、临床分期为Ⅱ期、贫血、并发乙肝均是PLC患者肝切除术前发生营养风险的危险因素（P＜0.05）。术前接受营养支持组的住院时间、首次排气时间、排便时间均短于术前未接受营养支持组（P<0.05）。两组术后7 d白蛋白、前白蛋白、血红蛋白水平均下降,但术前接受营养支持组高于术前未接受营养支持组（P<0.05）。术前未接受营养支持组、术前接受营养支持组的并发症总发生率组间对比无统计学差异（P>0.05）。结论：PLC患者肝切除术前发生营养风险的比例较高,且受到多种因素影响,术前给予营养支持可促进患者术后恢复,有效调节患者术后营养水平。     

半肝阻断联合肝静脉阻断在复杂肝癌精准肝切除中的应用

目的:探讨半肝血流阻断联合肝静脉阻断技术在精准肝切除中的应用价值。方法:回顾性分析我科2013年1月至2014年12月共120例行半肝血流阻断联合肝静脉阻断的精准肝切除病例,作为治疗组。另选单用第一肝门阻断的复杂肝癌半肝切除患者318例为对照。结果:治疗组在控制术中出血和输血量方面优于单用半肝血流阻断的对照组,治疗组术中平均出血量为650±46 m L,平均输血量为410±76 m L,差别有统计学意义(P0.05);治疗组在平均手术时间和平均住院时间方面均少于对照组,但两组差别无统计学意义(P0.05)。结论:半肝血流阻断联合肝静脉阻断技术在复杂肝癌的精准肝切除术中合理使用能有效的减少术中出血,提高了手术安全,降低了术中输血量,最大限度保留剩余肝细胞体积,减少了缺血再灌注的损失。     

盐酸纳布啡联合舒芬太尼用于腹腔镜下肝癌切除术后静脉自控镇痛的临床观察

目的:探讨盐酸纳布啡联合舒芬太尼用于腹腔镜下肝癌切除术后静脉自控镇痛(PCIA)的临床疗效。方法:选取2016年1月至2018年1月安康市中心医院收治的104例行腹腔镜下肝癌切除术患者,按照随机数字表法将患者分为对照组(n=52)和研究组(n=52)。对照组术后给予舒芬太尼PCIA,研究组术后给予盐酸纳布啡联合舒芬太尼PCIA。比较两组术后6h、24h、48h的视觉模拟量表(VAS)评分和数字镇静量表(NSS)评分。检测并比较两组麻醉诱导前、术毕、术后6h、24h及48h血清中P物质(SP)、神经肽Y(NPY)、多巴胺(DA)、C反应蛋白(CRP)、α1-酸性蛋白(AAG)、结合珠蛋白(HP)、白细胞介素-2(IL-2)、白细胞介素-6(IL-6)及白细胞介素-8(IL-8)水平。记录两组术后48h内不良反应发生情况。结果:研究组术后6h、24h、48h的VAS评分均明显低于对照组(P0.05);两组各时间点的NSS评分比较差异均无统计学意义(P0.05)。研究组术后6h、24h、48h的血清SP、NPY、DA、CRP、AAG、HP、IL-6及IL-8水平均明显低于对照组,血清IL-2水平明显高于对照组(P0.05)。术后48h内,两组不良反应发生率比较差异无统计学意义(P0.05)。结论:腹腔镜下肝癌切除术后采用盐酸纳布啡联合舒芬太尼PCIA方案,可缓解全身炎症反应,有效镇痛,且安全性较好。     

Concentrations of 3,4-Dihydroxyphenylalanine and Catecholamines and Metabolites in Brain in an Anhepatic Model of Hepatic Encephalopathy

Abstract: Alterations in the catecholaminergic neurotransmitter systems have been shown to occur in hepatic failure and may contribute to development of hepatic encephalopathy. In the present study we used the rat after complete hepatectomy as a model for study of changes that occur in brain in acute liver failure. We attempted to identify processes in the synthesis, storage, and metabolism of catecholamine neurotransmitters that might be changed during liver failure by measuring levels of, together with those of norepinephrine and dopamine, the precursor (3,4-dihydroxyphenylalanine) and the neuronal metabolites of dopamine and norepinephrine (3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylglycol, respectively) in different regions of brains of control rats and of rats after hepatectomy. We found that in most brain regions of hepatectomized rats there were increases in the concentration of 3,4-dihydroxyphenylalanine or of dopamine but decreases in the concentrations of norepinephrine or of 3,4-dihydroxyphenylglycol. The particulate/supernatant ratios of catecholamines are indices of retention of neurotransmitters in storage sites. These ratios were not different in brain regions between control rats and hepatectomized rats, suggesting that vesicular retention of catecholamines in brain was not impaired after hepatectomy. The data suggest that inhibition of dopamine-β-hydroxylase might be a characteristic of hepatic failure.     

Plasma lysophosphatidic acid level and serum autotaxin activity are increased in liver injury in rats in relation to its severity

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological actions. We have reported that LPA stimulates hepatic stellate cell proliferation and inhibits DNA synthesis in hepatocytes, suggesting that LPA might play some role in the liver. We have found that plasma LPA level and serum autotaxin (ATX) activity were increased in patients with chronic hepatitis C. However, the clinical significance of LPA and its synthetic enzyme, autotaxin (ATX), is still unclear. To determine whether the increase of plasma LPA level and serum ATX activity might be found generally in liver injury, we examined the possible modulation of them in the blood in rats with various liver injuries. Plasma LPA level and serum ATX activity were increased in carbon tetrachloride-induced liver fibrosis correlatively with fibrosis grade, in dimethylnitrosamine-induced acute liver injury correlatively with serum alanine aminotransferase level or in 70% hepatectomy as early as 3 h after the operation. Plasma LPA level was correlated with serum ATX activity in rats with chronic and acute liver injury. ATX mRNA in the liver was not altered in carbon tetrachloride-induced liver fibrosis. Plasma LPA level and serum ATX activity are increased in various liver injuries in relation to their severity. Whether increased ATX and LPA in the blood in liver injury is simply a result or also a cause of the injury should be further clarified.     

Roles of alpha1- and beta-adrenergic receptors in adrenergic responsiveness of liver cells formed after partial hepatectomy

The adrenergic receptor involved in the action of epinephrine changed dramatically during the process of active proliferation which follows partial hepatectomy. In control or sham-operated animals, the stimulation of glycogenolysis, gluconeogenesis and ureogenesis by epinephrine was mediated through alpha₁-adrenergic receptors. In contrast, in hepatocytes obtained from animals partially hepatectomized 3 days before experimentation, the receptor involved in the stimulation of these metabolic pathways by epinephrine was of the beta-adrenergic type. Interestingly, the adrenergic receptor involved in the metabolic actions of epinephrine, in hepatocytes from rats partially hepatectomized 7 days before experimentation was again of the α₁-subtype. Thus, it appears that during the process of liver regeneration which follows partial hepatectomy there is a transition in the type of adrenergic receptor involved in the hepatic actions of catecholamines from β in the initial stages to later α₁. A similar transition seems to occur as the animal ages. Cyclic AMP accumulation in response to β-adrenergic stimulation was significantly enhanced in hepatocytes obtained from rats partially hepatectomized 3 days before the experiment, as compared to control hepatocytes or cells obtained from animals operated 7 days before experimentation. This enhanced β-adrenergic sensitivity is probably related to the increased number of β-adrenergic receptors observed at this stage. However, a clear dissociation between cyclic AMP levels and metabolic effects was evidenced when the different conditions were compared. The number and affinity (for epinephrine or prazosin) of α₁-adrenergic receptors did not change at any stage of the process, which indicates that the markedly diminished α₁-adrenergic sensitivity observed in hepatocytes obtained from rats partially hepatectomized 3 days before experimentation is probably due to defective generation or intracellular processing of the α₁-adrenergic signal, rather than to changes at the receptor level.