培养人胎肝细胞的形态与功能研究

目的探讨人胎肝细胞分离与培养的简易方法．方法采用体外两步灌流法分离人胎肝细胞，选择条件培养液加以培养，并进行形态学和蛋白分泌功能的动态观察．结果用该法分离的肝细胞经台盼蓝拒染试验证实存活率在９５％以上，培养时间可达２周，并保持正常形态和良好的蛋白合成分泌功能．结论用酶灌流分离和条件培养的胎肝细胞性能良好，可用作肝细胞移植的供体．     

原代培养人胎肝细胞体外感染HBV的研究

目的建立HBV感染人胎肝细胞体外培养系统。方法 首先分离、培养人胎肝细胞，然后应用HBV阳性血清感染体外培养的人胎肝细胞；每隔2d收集上清液和肝细胞，应用ELISA,免疫组化法、原位杂交法和斑点杂交法检测上清液和细胞中HBsAg和HBV DNA。结果 上清液中HBsAg在感染后2d～20d均可测出，以感染后4d～16d达高峰(A值在0．22左右)。免疫组化检测细胞中HBsAg呈阳性表达，原位杂交和斑点杂交检测细胞和上清液中HBV DNA也呈阳性表达。结论 HBV在原代培养人胎肝细胞中能稳定复制和表达至少达16d。     

不同培养条件对人胚胎肝细胞形态和表型的影响

目的:探讨适宜的人胚胎肝细胞培养条件.方法:采用胶原酶两步灌流法分离20-24 wk人胚胎肝细胞,非连续性Percoll密度梯度离心法纯化细胞,用Ham's F12普通培养基和条件培养基培养胚胎肝细胞.观察培养细胞的形态特征,免疫荧光法检测胚胎肝细胞角蛋白×18(Cytokeratin 18,CK18)和甲胎蛋(alpha-1-fetoprotein,AFP)表达,MTT法检测细胞活力和增殖能力.结果:在条件培养基培养不同时间的胚胎肝细胞始终呈现特异性多角形形态,100%表达CK18和AFP,细胞增殖活跃.活力无明显下降.而在普通培养基培养24 h时,部分细胞即呈现成纤维细胞梭样形态,仅56%细胞表达CK18,43%细胞表达AFP,在培养72 h细胞均呈现成纤维细胞梭样形态,细胞增殖及活力下降(0.25±0.03 vs 1.01±0.12,P<0.001),至培养第7天细胞CK18及AFP表达呈阴性,细胞增殖及活力显著下降(0.17±0.04 vs 0.94±0.12,P<0.001).结论:Ham's F12条件培养基体外能够显著促进人胚胎肝细胞增殖,维持胚胎肝细胞的特异性形态和表型的稳定.     

Hepatitis B virus infection and replication in primarily cultured human fetal hepatocytes

AIMTo investigate the infection and replication of hepatitis B virus(HBV)in primarily cultured human fetal hepatocytes(HFHs).METHODSThe human fetal hepatocytes were cultured in serum-free medium,HBV-positive serum was added into the medium to study the susceptibility of hepatocytes to HBV infection.The supernatant was collected for ELISA assay of HBsAg and HBeAg,and quantitative fluorescence PCR for HBV-DNA assay daily.Albumin and HBcAg,CK8 and CK18 expressions were detected by immunohistochemistry in cultured hepatocytes.Content of lactate dehydrogenate(LDH)was measured to find out the integrity of the cell membrane.RESULTSA stable hepatocyte culture system was established.HBV could infect the hepatocytes and replicate,and HBcAg expression could be detected by immunohistochemistry in hepatocyte-like cells.HBV-DNA in the supernatant could be detected from d 2 to d 18 and HBsAg and HBeAg were positive on d 3-d 18 after HBV infection.HBV in medium increased from d 0 to d 6 and subsequently decreased as the cells were progressively loosing their hepatocyte phenotypes.CONCLUSIONHBV could infect human fetal hepatocytes and replicate.This in vitro model allowed a detailed Study on early events associated with human HBV entry into cells and subsequent replication.     

Establishment, characterization, and long-term maintenance of cultures of human fetal hepatocytes

Cultured human hepatocytes have broad research and clinical applications; however, the difficulties in culturing rodent and human hepatocytes are well known. These problems include the rapid loss of the hepatocytic phenotype in primary culture and the limited replicating capacity of the cultured cells. We describe the establishment of serum-free primary cultures of human fetal hepatocytes (HFHs) that retain hepatocytic morphology and gene expression patterns for several months and maintain sufficient proliferative activity to permit subculturing for at least 2 passages. Initially, HFH cultures contained 2 main cell types that morphologically resembled large and small hepatocytes. The fetal hepatocytes expressed alpha-fetoprotein (AFP), cytokeratin (CK) 19, albumin, and other hepatic proteins. Treatment of the cultures with oncostatin M (OSM) increased cell size and enhanced cell differentiation and formation of bile canaliculi, probably through an effect on hepatocyte nuclear factor (HNF) 4alpha. Approximately 1 month after plating, multiple clusters of very small cells became apparent in the cultures. These cells had very few organelles and are referred to as blast-like cells. Flow cytometric analysis of these cells showed that they express oval cell/stem cell markers such as CD90 (Thy-1), CD34, and OV-6 but do not stain with antibodies to beta(2)-microglobulin. HFH cultures maintained for 9 to 12 months produced grossly visible organoids containing ductular structures that stained for CK18, CK19, and AFP. In conclusion, HFH cultures, which might contain a population of hepatic stem cells, constitute an excellent tool for a variety of studies with human hepatocytes, including the mechanisms of viral infection.     

粒细胞集落刺激因子保护急性损伤人胎肝细胞的实验研究

目的建立D-半乳糖胺(D-galactosamine,D-GalN)对人胎肝细胞急性损伤的模型,观察粒细胞集落刺激因子(Granulocyte-colony stimulating factor,G-CSF)对人胎肝细胞损伤的保护作用。方法分别用梯度浓度的D-GalN和不同的作用时间孵育人胎肝细胞,用四唑盐比色法(MTT法)检测细胞活性,以确定最佳的人胎肝细胞急性损伤造模条件。将胎肝细胞分为4组进行不同处理:第1组为空白对照组,第2组(G组)用G-CSF处理正常细胞,第3组(ND组)和第4组(GD组)都用D-GalN进行损伤造模,但GD组加入G-CSF作为治疗,第3组加入等量的0.9%氯化钠溶液作为实验对照。最后用MTT法和乳酸脱氢酶(LDH)释放量检测各组细胞活性。结果当D-GalN浓度为10 mg/ml,作用时间为12 h时,可以杀伤90%以上的人胎肝细胞,并且可以保证有足够的药物反应时间。空白对照组和G组的细胞活性差异无统计学差异,但GD组细胞活性明显高于ND组(P0.05)。结论 D-GalN对人胎肝细胞急性损伤的造模条件为D-GalN 10 mg/ml作用12 h。G-CSF对D-GalN造成的人胎肝细胞急性损伤具有保护作用。     

体外培养妊娠早期人胎肝细胞对乙型肝炎病毒易感性的变化

目的观察妊娠早期(孕10周)人胎肝细胞在不同的体外培养条件下对乙型肝炎病毒(HBV)易感性的变化，为HBV受体的发现和鉴定打下基础。方法设计无血清培养基，分别添加不同的细胞分化诱导物，在不同时相以HBV感染原代培养10周孕龄人胎肝细胞，检测细胞形态、功能及HBV感染标志物。结果培养基中添加2．5mmol／L苯巴比妥钠，培养6d后细胞逐渐出现成熟肝细胞标志，并可被HBV感染；其他培养条件下则不能观察到HBV的成功感染。结论苯巴比妥钠可在体外培养时诱导早期人胎肝细胞分化，并出现HBV易感性。     

鼠特异性Fas抗体对人鼠嵌合肝中人肝细胞增殖的促进作用

目的: 探讨人胎肝细胞移植联合使用Jo2抗体的策略,促进人胎肝细胞在小鼠肝内存活和增殖.方法:裸鼠经脾移植1×106人胎肝细胞,移植后第1天ip JO2抗体,剂量为0.2mg/kg,1次/wk,持续12 wk为实验组,裸鼠经人胎肝细胞移植后未给予JO2抗体为对照组,建立人鼠嵌合肝动物模型.采用HE染色、原位末端标记方法(TUNEL染色)观察未经人胎肝细胞移植而给予JO2抗体24 h后处死的裸鼠肝脏组织.免疫组化、逆转录聚合酶链反应(RT-PCR)检测不同时相点实验组和对照组嵌合肝中肝组织人白蛋白、特异人增殖细胞核抗原(PCNA)和人白蛋白mRNA表达.结果:未经人胎肝细胞移植而给予JO2抗体的裸鼠病理组织切片发现肝组织出血、坏死和细胞凋亡.实验组和对照组裸鼠均能存活至24 wk.移植后嵌合鼠肝组织表达人白蛋白和人PCNA阳性细胞的时间:实验组2-20 wk,对照组2-12 wk; 白蛋白mRNA表达:实验组4-16wk, 对照组4-8 wk:实验组与对照组移植后8、12 wk PCNA表达差异有显著性(25.7%±8.5% vs 13.4%±7.8%, 29.4%±5.0% vs 8.5%±2.3%,均P<0.05).结论:人肝细胞异种移植于裸鼠体内能够存活,经小剂量JO2抗体ip裸鼠,使人鼠嵌合肝中人肝细胞得以增殖,存活时间延长.     

丙型肝炎病毒体外感染胎肝细胞的初步研究

目的：探索原代培养胎肝细胞对丙型肝炎病毒（HCV）的易感性,旨在建立较为稳定实用的细胞感染模型。方法：研究血清与培养肝细胞共同孵育6－8h后,收集不同时相点标本,用逆转录聚合酶链反应（RT－PCR）分别检测细胞内或上清液中正负链RNA,免疫组化检测细胞内HCV NS3,NS5特异性抗原的表达情况,以及原位杂交检测细胞内HCV负链RNA。结果：接种感染血清3d后,即可在细胞内或培养上清液中检出HCV正负链RNA,间断检出至感染后第17天,HCV NS3,NS5特异性抗原可在感染细胞内表达,阳性物质位于乐中,原位杂交法证实细胞内存在负链RNA,也位于胞浆中,结论：原代培养的胎肝细胞对HCV不但易感,而且稳定地支持HCV复制。     

Efficient generation of human hepatocytes by the intrahepatic delivery of clonal human mesenchymal stem cells in fetal sheep

Alternative methods to whole liver transplantation require a suitable cell that can be expanded to obtain sufficient numbers required for successful transplantation while maintaining the ability to differentiate into hepatocytes. Mesenchymal stem cells (MSCs) possess several advantageous characteristics for cell-based therapy and have been shown to be able to differentiate into hepatocytes. Thus, we investigated whether the intrahepatic delivery of human MSCs is a safe and effective method for generating human hepatocytes and whether the route of administration influences the levels of donor-derived hepatocytes and their pattern of distribution throughout the parenchyma of the recipient's liver. Human clonally derived MSCs were transplanted by an intraperitoneal (n = 6) or intrahepatic (n = 6) route into preimmune fetal sheep. The animals were analyzed 56-70 days after transplantation by immunohistochemistry, enzyme-linked immunosorbent assay, and flow cytometry. The intrahepatic injection of human MSCs was safe and resulted in more efficient generation of hepatocytes (12.5% +/- 3.5% versus 2.6% +/- 0.4%). The animals that received an intrahepatic injection exhibited a widespread distribution of hepatocytes throughout the liver parenchyma, whereas an intraperitoneal injection resulted in a preferential periportal distribution of human hepatocytes that produced higher amounts of albumin. Furthermore, hepatocytes were generated from MSCs without the need to first migrate/lodge to the bone marrow and give rise to hematopoietic cells. CONCLUSION: Our studies provide evidence that MSCs are a valuable source of cells for liver repair and regeneration and that, by the alteration of the site of injection, the generation of hepatocytes occurs in different hepatic zones, suggesting that a combined transplantation approach may be necessary to successfully repopulate the liver with these cells.     

人肝细胞生长因子重组腺病毒的构建及其对人胎肝细胞细胞周期的影响

目的　利用AdEasy系统构建人肝细胞生长因子 (HGF)复制缺陷型重组腺病毒 (AdHGF) ,并观察其对人胎肝细胞细胞周期的影响。方法　将质粒 pUCHGF扩增、酶切获得人HGFcDNA片段 ,插入腺病毒穿梭载体质粒 pAdTrack巨细胞病毒 (CMV )启动子下游 ,构建重组穿梭载体pAdTrack CMV HGF ,线性化后与骨架载体AdEasy 1在细菌BJ5 183内同源重组得到腺病毒质粒 pAdHGF ,经 2 93细胞包装后得到复制缺陷型重组腺病毒AdHGF ;将AdHGF体外感染人胎肝细胞 ,以RT PCR检测HGF在胎肝细胞的表达 ;同时通过流式细胞仪测定AdHGF对胎肝细胞细胞周期的作用。结果　连接、重组后通过酶切和测序法筛选出pAdHGF ;经 2 93细胞包装 ,3d后观察到绿色荧光蛋白 (GFP)明显表达 ,氯化铯梯度离心纯化最终获得约 4× 10 10 efu/ml滴度的重组病毒 ;AdHGF体外感染人胎肝细胞 3d后 ,HGF表达明显增加 ,流式细胞仪检测结果显示感染后的胎肝细胞由G0 /G1期向S期和G2 /M期转化。结论　利用新型腺病毒载体AdEasy系统可在短期内制备同时表达GFP和HGF的重组腺病毒Ad HGF ;AdHGF体外感染胎肝细胞可显著提高HGF的表达并促进肝细胞增殖 ,这将为肝细胞移植和基因治疗肝纤维化提供新的手段。     

抑制性削减杂交鉴别早期人胎肝细胞乙型肝炎病毒感染相关基因

乙型肝炎的宫内感染涉及乙型肝炎病毒(HBV)受体问题，其机制尚不明确，一般认为胎肝细胞仍然是主要靶细胞。虽有多篇报道表明晚期(孕22～24周)胎肝细胞可在体外被HBV所感染，但目前鲜见涉及人类胎肝细胞HBV受体的研究报道。设想早期胎肝细胞由于分化程度较低，未能表达某些特定分子，因此缺乏对HBV的易感性；通过特定条件进行体外培养，在细     

Factors affecting the conversion of androstenedione to estrogens by human fetal hepatocytes in monolayer culture

The purpose of the present investigation was to characterize and determine what hormones affect the activity of aromatase in human fetal hepatocytes maintained in primary monolayer culture. The major product of aromatization of androstenedione was estrone sulfate. Optimal conditions for assay of aromatase activity in fetal liver cells were determined. The apparent Km for androstenedione was 50 nM. Aromatase activity was stimulated by glucocorticoids in the presence of fetal calf serum. The concentration of dexamethasone required for half-maximal stimulation was 10(-8) M, similar to the concentration required for half-maximal binding to glucocorticoid receptors. This action of dexamethasone was inhibited by cortisol 21-mesylate, a glucocorticoid antagonist. Aromatase activity was also stimulated by (Bu)2cAMP and cholera toxin, and was inhibited by fetal calf serum. This effect of fetal calf serum was mimicked by epidermal growth factor. However, epidermal growth factor did not mimic the permissive action of serum to stimulate aromatase activity by dexamethasone. In these respects, the regulation of aromatase activity of human fetal hepatocytes is similar to that of human adipose stromal cells. A polycyclic hydrocarbon, benzo(a)pyrene, which causes induction of aryl hydrocarbon hydroxylase activity in fetal hepatocytes, inhibited the stimulation of aromatase activity by dexamethasone. Of a number of hormones tested, including glucagon, insulin, angiotensin II, ACTH, hCG, GH, PRL, and T3, only glucocorticoids were effective in stimulating aromatase activity of human fetal hepatocytes. These results emphasize the complex and multiparameter nature of the regulation of aromatase activity in this as in other tissues.     

Ontogenesis of insulin processing in fetal rat hepatocytes

Summary We studied insulin processing and hepatic glycogenesis in cultured hepatocytes isolated from rat fetuses of 17, 19, and 21 days of gestation. Steady-state insulin binding increased by 250% between days 17 and 19, from 145±8 to 361±52 fmol/mg protein, and by an additional 40% (405±69 fmol/mg protein) by 21 days of gestation. At 37°C, ¹²⁵I-insulin was rapidly (t_1/2<5 min) internalized by hepatocytes at all three ages, reaching maximal levels (63–76% of the total cell-associated radioactivity) by 15 min. ¹²⁵I-labelled degradation products appeared rapidly (t_1/2<15 min) within the cells. Yet, the majority (68–77%) of the intracellular radioactivity consisted of intact ¹²⁵I-insulin, even after 4 h at 37°C. Hepatocytes pre-loaded with ¹²⁵I-insulin and then acid-stripped of surface-bound radioactivity, rapidly released both intact ¹²⁵I-insulin (retroendocytosis) and its radiolabelled degradation products. While intact insulin was initially released more rapidly (t_1/2<6 min), and reached a plateau after 15–30 min, the degradation products continued to accumulate in the medium for at least 4 h. Methylamine inhibited intracellular ¹²⁵I-insulin degradation at all three gestational ages and also blocked insulin-stimulated glycogenesis in 19- and 21-day hepatocytes, without altering basal glycogen synthesis. Insulin-stimulated glycogenesis was not induced in 17-day fetal rat hepatocytes in control or methylamine-treated cultures. We conclude that both degradative and retroendocytotic pathways for processing insulin are present in fetal rat hepatocytes by 17 days of gestation. Further, insulin-receptor processing was functionally related to the glycogenic action of insulin in responsive 19- and 21-day fetal rat hepatocytes     

Somatomedin-C stimulates glycogen synthesis in fetal rat hepatocytes

The effects of somatomedin-C/insulin-like growth factor I (Sm-C) on glycogen metabolism in cultured hepatocytes from 20-day-old rat fetuses have been examined and compared with the effects of insulin. Sm-C (25-375 ng/ml; 3.25-50 nM) stimulated dose-dependent increases in [14C]glucose incorporation into glycogen (14.4-72.9%; P less than 0.001) and total cell glycogen content (10.6-34.3%; P less than 0.01). Maximal stimulation of glycogen synthesis by Sm-C occurred at 2-4 h of incubation. Insulin (10 nM to 10 microM) also stimulated [14C]glucose incorporation but its potency was only 1/20th that of Sm-C. The time course of stimulation of glucose incorporation by insulin was identical to that of Sm-C, the dose-response curves of the two hormones were parallel, and the maximal effects of insulin were not enhanced by simultaneous exposure of cells to Sm-C. These findings suggest that Sm-C and insulin stimulate glycogenesis in fetal liver through similar or identical mechanisms. Since the potency of Sm-C was 20 times greater than that of insulin, the glycogenic action of insulin in fetal liver may be mediated through binding to a hepatic receptor which also binds Sm-C. In addition to having mitogenic effects on fetal tissues, Sm-C may have direct anabolic effects on fetal carbohydrate metabolism.     

Relationship between Insulin binding and glycogenesis in cultured fetal hepatocytes

Summary Binding of ¹²⁵I-insulin and the stimulatory effect of insulin on ¹⁴C-glucose incorporation into glycogen have been studied in cultured fetal rat hepatocytes. Measurement of both variables was possible at 37 °C because of the slow rate of insulin degradation in the medium. ¹²⁵I-insulin binding approached maximum after 10 min, thus largely preceding the insulin glycogenic effect which became significant after 45 min. Maximal effect was observed after 3 h with 10 nmol/l insulin when 16,000 specific sites per hepatocyte were occupied and half-maximal response was achieved with 0.3 nmol/l insulin (2,900 sites/hepatocyte). Dissociation of bound insulin was rapid (t1/2=3 min) and accelerated by the availability of native insulin. In hepatocytes preincubated with insulin, binding was measured after 30 min incubation in the absence of hormone which allowed the liberation of most (95%) of the bound insulin. No modification of insulin binding was observed over extended periods (2–24 h) of exposure to 10 nmol/l insulin, when the glycogenic effect of insulin showed striking variations, notably a cessation of the effect between 4 and 12 h. Thus the time-dependence of the glycogenic effect of insulin cannot be related to a defect in insulin binding in cultured fetal hepatocytes.     

大鼠肝癌细胞与胎肝细胞间抑癌微小RNAs的表达差异

微小RNAs(miRNAs)是一类短序列、非编码、具有调控功能的单链小分子RNA[1],通过与其靶mRNA分子互补结合,在翻译水平上特异性抑制基因表达,参与调控生物生长和发育等许多复杂的生命过程[2,3],异常的miRNAs表达可能与人类许多疾病乃至肿瘤的发生和发展都密切相关[3,4].已知的miRNAs参与癌症发生的机制包括细胞黏附、血管生成、蛋白质水解、细胞信号系统、细胞增殖、侵袭转移和细胞死亡[5].miRNAs在肝癌组织中异常表达,它可能参与了肝细胞癌变及肝癌转移的病理过程[6].本研究应用Exiqon miRNA基因芯片技术,建立大鼠肝癌细胞和胎肝细胞之间miRNAs的差异表达谱,探索肝癌诊断和预后的新指标.DOI:10.3760/cma.j.issn.1007-3418.2009.02.016     

纯化大鼠胚肝细胞脾内移植增殖的实验研究

目的探讨脾内移植后纯化胚肝细胞的增殖能力。方法免疫吸附法纯化胚肝细胞后脾内移植,图像分析和流式细胞仪检测脾内移植后胚肝细胞的增殖。结果移植后3、7、14d胚肝细胞的面积密度分别为(1603±337)μm2/mm2、(3788±605)μm2/mm2、(8129±1025)μm2/mm2,增殖期细胞比率为14.25%±4.11%、16.07%±5.35%、17.32%±5.17%,均明显高于成年肝细胞,差异有显著性(P＜0.01)。结论纯化后的胚肝细胞比成年肝细胞更易于在脾内增殖。     

An in vitro system for infection with hepatitis B virus that uses primary human fetal hepatocytes.

An in vitro culture of human fetal hepatocytes has been employed for infection by hepatitis B virus (HBV) virions that are produced by an established human hepatoma cell line, HB 611. HBV surface antigen and e antigen were released into the medium 3-4 days after infection, and production continued thereafter. RNA synthesis with similar kinetics was observed. Viral DNA replication started 2 days after infection, and replicative HBV DNA that included relaxed circles, single-stranded minus strands, and closed circles accumulated during 16 days of incubation. Immunofluorescent study using fluorescein isothiocyanate-labeled rabbit antisera directed against HBV core antigen revealed that this antigen is present in the nuclei in 12% of the infected cells. Particles containing HBV DNA were detected in the culture medium and were infectious. Thus, this in vitro infection system closely mimics infection in vivo and it allows detailed studies on early events associated with human HBV entry into cells and subsequent replication and integration.