胎肝细胞

摘要： 为了利用透明质酸建立小鼠胎肝细胞3D培养体系,分离获得胚胎12～14d胎肝细胞,利用KM培养基进行初步2D肝干/祖细胞的筛选培养,并利用透明质酸及KM培养基配制水凝胶建立3D细胞培养体系.胎肝细胞在2D体系中呈现克隆状生长.分离培养获得的肝干/祖细胞,克隆在透明质酸建立的3D培养体系保持增殖活性,并进一步获得肝细胞功能特性,表现为3D培养上清中白蛋白合成和尿素水平显著增加.定量PCR结果显示,随着3D培养时间的延长,其肝细胞干性标志如AFP、CK19、EpCAM、Prox1等表达水平都大幅度降低且接近成年小鼠肝脏表达水平.本研究成功建立基于透明质酸的小鼠胎肝细胞的3D无血清培养体系,并可促进小鼠胎肝细胞的肝细胞功能进一步成熟.

摘要： 目的：观察细胞活性，客观评价CN2006型生物人工肝支持装置恒温培养箱控制系统，为生物人工肝的临床应用提供依据。方法选择16周正常引产的人胎肝，胎肝组织采用两步灌流法进行消化分离。在培养条件为37°C、5%CO2、湿度100%下培养24 h后，倒置相差显微镜观察细胞的生长及形态变化；用MTT法检测细胞活力，以2×105/ml细胞数在酶标仪上所测吸光度作为对照；同时，检测胎肝细胞清除氨的能力，设无胎肝细胞的反应器为空白对照。结果胎肝细胞培养24 h后，在倒置相差显微镜下观察发现细胞呈集落状生长。在装置内反应器中培养24 h后，胎肝细胞在装置的反应器中生长良好，而且得到了增殖。细胞在装置内反应器中培养24 h后，加入氯化铵溶液，通过在不同时间检测氨的浓度表明在装置内反应器中培养的肝细胞具有生物转化功能。结论细胞在CN2006型生物人工肝支持装置中不仅生长良好，而且还具有生物转化功能，说明设计的恒温培养系统适宜细胞生长环境，为生物人工肝的临床治疗提供了坚实的理论基础。%O bjective This article aims to observe the cell activity and objectively evaluate CN2006 biological artificial liver support device for cultivating box with constant temperature control system, providing the basis for clinical application of bioartificial liver. Methods To choose human fetal liver of normal induction with 16 weeks. Fetal liver tissue was digested and separated using the two-step perfusion method. After the cultivation for 24 h in the culture conditions of 37°C, 5%CO2, 100%humidity, observe the cell growth and morphological changes under the inverted phase-contrast microscope. Cell viability was detected by MTT, taking 2× 105/ml cell number on the enzyme labelling instrument measured absorbance as a control. Meanwhile, detect the ability of fetal liver cells in removing ammonia, and set a reactor of free fetal liver cells as blank control. Results After culturing the fetal liver cells for 24 h, cells showed the colony growth observed in the inverted phase contrast microscope. After culturing for 24 h in the device inside the reactor, fetal liver cells grew well, and got the proliferation;Adding ammonium chloride solution, it showed cultured liver cells in the device inside the reactor with functions of biological transformation by detecting the concentration of ammonia at different time. Conclusion Cells in CN2006 biological artificial liver support device do not only grow well, but also have the function of biological transformation, which shows that the culturing system with constant temperature is suitable for cell growth, and provides a solid theoretical basis for the clinical treatment of biological artificial liver.

摘要： 目的 建立大鼠肝癌细胞与大鼠胚胎肝细胞microRNA (miRNA)表达的差异谱,为miRNA在肝细胞癌变发生机制中的作用提供新线索.方法 化学致癌剂二乙基亚硝胺(DEN)建立近交系F334大鼠肝癌模型,体外原代培养大鼠肝癌细胞和正常F334大鼠胚胎肝细胞,Trizol法抽提细胞总RNA,进一步制备Hy3荧光标记的miRNA;利用Exiqon miRNA基因芯片技术,采用含2 056条探针的哺乳动物miRNA表达谱芯片对两组之间差异表达的miRNA进行分析.结果 原代培养可获得稳定传代的肝癌细胞及胎肝细胞,两者在形态上无明显差异;miRNA芯片共获得78个差异表达的miRNAs,其中68个表达上调,10个表达下调,部分miRNAs与肿瘤关系密切,其中miR-122为肝细胞癌变相关miRNA.结论 正常肝细胞中存在着与肝细胞癌变及侵袭、转移相关的miRNAs,miRNAs的表达具有明显的时序性,主要参与转录后基因表达的调控,其表达特征的改变可能导致了肝癌的发生.

摘要： AIM; To investigate the human leukcyte antigen E ( HLA -E) expression in hepatocellular carci-noma cell lines. METHODS: The techniques of real - time PCR and Western blotting were used to study the HLA - E ex-pression in the 5 cell lines of hepatocellular carcinoma and a fetal liver cell line at mRNA and protein levels. RESULTS: The results of real - time PCR showed that no statistical difference of HLA - E mRNA level between fetal liver cell L02 and other 4 cell lines of hepatocellular carcinoma ( HepG2, Bel7402, PLC and MHCC97) was observed, and almost absence of HLA -E mRNA expression in Hep3B2. 1-7 cells was detected. However, the results of Western blotting showed that there was a significant statistical difference of HLA - E protein levels between L02 cells and the 5 cell lines of hepatocellu-lar carcinoma (HepG2, Bel7402, PLC, MHCC97 and Hep3B2. 1 -7) , and no HLA - E protein in Hep3B2. 1 -7 cells was detectable. CONCLUSION: Asynchronization of HLA - E expression between mRNA and protein levels was found in hepatocellular carcinoma cell lines.%目的:研究人类白细胞抗原E(HLA-E)在肝癌细胞系中的表达情况.方法:通过real-time PCR和Western blotting技术研究5种肝癌细胞系和胎肝细胞系中HLA-E mRNA 和蛋白的表达情况,进行相对定量分析.结果:Real-time PCR 检测显示,HepG2 细胞、Bel7402 细胞、PLC 细胞、MHCC97细胞和Hep3B2.1-7 细胞这5种肝癌细胞系与L02胎肝细胞系的HLA-E mRNA 水平比较,除Hep3B2.1-7细胞表达几乎接近缺失(P＜0.01)外,其余无显著差异(P＞0.05).HepG2细胞、Bel7402 细胞、PLC 细胞、MHCC97 细胞和Hep3B2.1-7 细胞与L02 细胞的HLA-E 蛋白水平比较,差异显著(P＜0.01),其中Hep3B2.1-7细胞表达缺失.结论:肝癌细胞系的HLA-E mRNA和蛋白表达不同步,可能存在转录后调控.

摘要： 肝细胞移植作为治疗暴发性肝功能衰竭及各种终末期肝病一种潜在方法,具有创伤小、安全简单、可一肝多用、重复移植、抗原性低等优点.可以用作移植的肝细胞种类很多,目前也有很多关于这方面的报道,本文就可用于移植的肝细胞的研究进展做一简单总结.

摘要： Objective To observe the effect of moderate intensity aerobic exercise and fetal liver cell infusion on the repairing and regenerating liver injury in rat with obstructive jaundice. Methods Forty SD rats were divided into sham control (C) , obstructive jaundice induced liver damage (OB) , fetalliver cells (FL) and fetal liver cells plus exercise intervention (FL+E) groups. From the gestational 12 to 16 day-old SD rat embryos, fetal liver cells were prepared. FL group and FL+E group were given fetal liver cell through hepatic portal vein. FL+E group ran on a treadmill at the speed of 16.8m/min with 5% slope, five days a week. 45 minutes a day for 12 weeks. After 12 weeks, rats' livers were excised. The liver cell morphology (HE stain) , serum total bilirubin (TBIL) , glutamic-pyruvic transaminase (ALT) , glutamic-oxaloacetic transaminase (AST) contents, epidermal growth factor of dry tissue (EGF) . Hepatocyte growth factor (HGF) , multidrug resistance (MPR2) protein, FXR and MRP2 mRNA expression were observed. Results Morphology of liver cells in the C group appeared to be normal. OB group showed extensive vacuolar degeneration and necrosis of liver cells. There was no obvious necrosis of liver cells in FL+E group. FL group showed a small amount of spotty necrosis of liver cells. Serum TBIL, AST and ALT contents in FL and FL+E groups were significantly lower than that in OB group (P < 0.05) . HGF and EGF expressions in FL and FL+E groups were significantly lower (P < 0.01) and MRP2 expression was significantly higher (P < 0.05) than that in OB groups. Conclusion Fetal liver cell infusion and fetal liver cell infusion in combination with moderate intensity exercise have significant repair and regeneration effect on the liver injury induced by obstructive jaundice, probably through the promotion of expression of liver cell regeneration factors.%目的:观察胎肝细胞输注移植联合中等强度有氧运动干预对大鼠阻塞性黄疸致重症肝损伤的修复与再生的影响.方法:40只SD大鼠随机分为假手术、阻塞性黄疸致肝损伤、胎肝细胞及胎肝细胞联合运动干预4组,后3组均采用改良的胆总管结扎法构建大鼠阻塞性黄疸致重症肝损伤模型.自孕龄12～16天的SD孕鼠胚胎中提取肝脏组织,加工制备胎肝细胞.两胎肝细胞组于模型构建过程中的胆总管结扎后进行胎肝细胞肝门静脉输注移植.运动组进行坡度5％、速度16.8 m/min、每天45 min、每周5天、共12周的中等强度跑台训练.12周后大鼠禁食过夜,随后分离出肝组织并取材,应用HE染色法观察大鼠肝细胞形态；生物化学法测定血清总胆红素( TB IL)、谷丙转氨酶(ALT)、谷草转氨酶(AST)含量；免疫组化技术检测肝组织表皮生长因子(EGF)、肝细胞生长因子(HGF)、多药耐药蛋白(MPR2)的蛋白表达；RT-PCR技术检测EGF、FXR、MRP2 mRNA表达.结果:假手术组肝细胞形态结构正常,阻塞性黄疸致肝损伤组可见肝细胞广泛空泡样变性、片状坏死,胎肝细胞组肝细胞少量点状坏死,胎肝细胞联合运动组肝细胞未见明显坏死.胎肝细胞组和胎肝细胞联合运动组大鼠血清TBIL、AST、ALT含量显著低于阻塞性黄疸致肝损伤组(P＜0.05).胎肝细胞组和胎肝细胞联合运动组大鼠HGF、EGF阳性表达显著低于阻塞性黄疸致肝损伤组(P＜0.01),而MRP2阳性表达显著高于阻塞性黄疸致肝损伤组(P＜0.05).胎肝细胞组和胎肝细胞联合运动组大鼠EGF mRNA表达显著低于阻塞性黄疸致肝损伤组(P＜0.01),而MRP2、FXR mRNA含量表达显著高于阻塞性黄疸致肝损伤组(P＜0.05).结论:胎肝细胞肝门静脉输注移植以及胎肝细胞肝门静脉输注移植联合中等强度有氧运动对阻塞性黄疸导致的重症肝损伤有明显的修复与再生作用,其机制可能是通过促进肝细胞再生相关因子的表达和肝细胞的增殖,从而发挥修复与再生作用.

摘要： 背景:目前体外诱导骨髓间充质干细胞分化为肝细胞样细胞的研究主要集中在不同诱导因子的诱导作用,而微环境的诱导作用研究较少.目的:观察肝细胞生长因子、胎肝细胞对骨髓间充质干细胞体外诱导分化为肝细胞样细胞的影响.方法:采用密度梯度离心法分离大鼠骨髓间充质干细胞,反复贴壁法纯化扩增骨髓间充质干细胞;采用Ⅰ型胶原酶消化法分离孕3周大鼠胚胎肝脏细胞,差速贴壁法纯化肝脏细胞;阴性对照组骨髓间充质干细胞只加入含体积分数为10%胎牛血清的L-DMEM培养基.诱导组在阴性对照组基础上加入一定浓度肝细胞生长因子或者与胎肝细胞共培养进行诱导分化.结果与结论:诱导组白蛋白、甲胎蛋白水平均高于非诱导组(P < 0.01),诱导组糖原染色阳性,免疫细胞化学染色CK-18阳性,而非诱导组糖原染色、CK-18均阴性.结果显示肝细胞生长因子和胎肝细胞均可在体外诱导大鼠骨髓间充质干细胞分化为具有肝细胞样细胞表型和功能的类肝细胞.%BACKGROUND: The study of the induction of differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into hepatocyte-like cells majorly concentrates on the induction of different inducing factors, and pays little attention on the induction of micro environment. OBJECTIVE: To investigate the differentiation of rat BMSCs into hepatocyte-like cells. METHODS: The rats BMSCs were isolated by density gradient centrifugation and purified by adherence cultivation, morphology feature of BMSCs were observed by microscope. The fetal liver cells were isolated by adherence cultivation after digesting the rats embryo liver of 3 weeks with collagenase. The negative control group was cultured in L-DMEM containing 10% fetal bovine serum. The purified BMSCs in the induced group were cultured in L-DMEM containing 10% fetal bovine serum with hepatocyte growth factor (HGF) and fetal liver cells. RESULTS AND CONCLUSION: Compared with those in non-induced BMSCs, the levels of alpha-fetoprotein and albumin in the induced-BMSCs were higher (P < 0.01). Both glycogen and CK-18 were positive in the induced BMSCs. BMSCs can differentiate into hepatocyte-like cells with hepatic phenotype and function in the presence of HGF and fetal liver cells, which may be used as a kind of cell resources to treat severe hepatic disease.

摘要： 流式细胞分选（fluorescence—activated cells sorling，FACS）和免疫磁珠分选（magnetic affinity cells sorting，MACS）是干细胞分选的两种最常用的方法，这两种方法都需借助特异的表面标志。

摘要： 背景:肝脏来源的胞外膜粒可介导细胞间通讯、调控肝细胞的增殖与分化,在肝病诊断、肝癌防治、肝脏再生和细胞移植等技术的研究应用中均有重要价值.但由于对膜粒的发生和类型归属缺乏认识,制约了该领域的进展.隧道纳米管是介导细胞间通讯的另一新发现的结构,是否存在于肘细胞间尚无报道.目的:通过对比人正常胎肝细胞和肝癌细胞表面超微结构的特点,寻找两种细胞的差异,揭示胞外膜粒的生物发生过程,判断其类型归属,明确隧道纳米管是否存在.方法:用扫描电镜观察处于增殖周期中的贴壁培养胎肝L02和肝癌BEL-7402细胞样品,分析其细胞表面超微结构的共性和差异.结果与结论:首次显示人胎肝L02和肝癌BEL-7402细胞表面均具有大量微绒毛样突起,其特征与静止期细胞的典型微绒毛有显著差异.L02细胞上微绒毛样突起的平均长度和密度均超过BEL-7402细胞,可作为区分两种细胞的指标.两种细胞上的微绒毛样突起均既可产生纳米膜粒,又可转化为隧道纳米管.纳米膜粒的产生方式有出芽和串珠样缢缩两种类型.总之,高分辨率扫描电镜图像可作为鉴定肝脏来源细胞表面超微结构特征和胞外膜粒来源的金标准.

摘要： Objective To explore the miRNA (microRNA) differential expression profile between hepatocellular carcinoma and embryonic hepatocyte in the rats to provide new evidence that miRNAs are involved in the molecular pathogensis of HCC. Methods The liver cancer model was established by low dose DEN in the rats. Then the hepatocellular carcinoma and embryonic hepatocytes were cultured by primary culture in vitro. The total RNAs were extracted by Trizol and the miRNAs were stained by Hy3. Hybridization was performed by applying the cell miRNAs to mammalian miRNA chip. Data analysis was conducted by using software of SAM of version 3. 0. Results A total of 78 differential expressed miRNAs were found and they included 68 over-expression and 10 low-expression miRNAs. Data analysis revealed that some miRNAs were associated with some different tumors, especially the has-miR-21. Conclusion There are some miRNAs being related to hepatocellular carci-nogenesis and invasion and metastasis in the normal hepatocytes. The regularity of the orderly miRNA expression is obvious and the change of expression characteristics of miRNAs may be involved in the pathogensis of HCC.%目的 建立大鼠肝癌细胞与大鼠胚胎肝细胞microRNA(miRNA)表达的差异谱,为研究miRNA在肝细胞癌变发生机制中的作用提供新线索.方法 化学致癌剂二乙基亚硝胺(DEN)建立近交系F334大鼠肝癌模型.体外原代培养大鼠肝癌细胞和正常F334大鼠胚胎肝细胞,Trlzol法抽提细胞总RNA,进一步制备Hy3荧光标记的miRNA;利用ExiqonmiRNA基因芯片技术.采用含2056条探针的哺乳动物miRNA表达谱芯片对两组之间差异表达的miRNA进行分析,其实验数据用SAM3.0软件进行差异显著性分析.结果 原代培养可获得稳定传代的肝癌细胞及胎肝细胞,两者在形态上无明显差异;miRNA芯片共获得78个差异表达的miRNAs,其中68个表达上调(foldchange>2),10个表达下调(foldchange<0.5),部分miRNAs与肿瘤关系密切,其中has-miR-21为肝癌癌变相关miRNA.结论 正常肝细胞中存在着与肝细胞癌变及侵袭、转移相关的miRNAs,miRNAs的表达具有明显的时序性,其表达特征的改变可能导致了肝癌的发生.

摘要： 中毒性肝衰竭(toxic hepatic failure, THF)凶险、进展快、死亡率高.较为有效的肝脏移植、肝细胞移植或生物人工肝治疗因供体日益紧缺，难于挽救众多濒危的患者。因此。寻找充足数量同种肝细胞成为肝功能替代疗法解决THF的关键问题。最近研究显示，从胎肝脏分离获得细胞存在诸多优点，特别是采用端粒酶逆转录酶(hTERT)调控端粒酶活性可达到获得永生化胎肝细胞的目的，在体外将表达hTERT的载体对人胎肝细胞进行转染，检测了hTERT在胎肝细胞的表达和活性，观察了培养转染细胞增殖能力、细胞形态学和功能变化，现进行了报告。

摘要： 目的：探讨人胎肝细胞分离、培养的简单方法并证实其具有近似正常肝细胞功能。方法：取水囊引产中期(4～6月龄)妊娠死胎，用两步灌注法分离胎肝细胞，观察细胞形态及存活情况，并以正常成人肝组织作正常对照，检测胎肝细胞五种基因的表达，进行光密度分析。结果：经台盼蓝染色、FDA荧光染色观察，细胞培养后11天内均存活良好，最长可存活18天：新鲜分离、培养3、5、7、lO天组胎肝细胞均可表达GAPDH（Glyceraldehyde Phosphate Dehydrogenase磷酸甘油醛脱氢酶)、ALB(Albumin白蛋白)、GS(Glutamine Synthetase谷氨酰胺合成酶)、CK(Cytokeratin细胞角蛋白)，经光密度分析证实，上述四种基因在培养肝细胞和正常成人肝细胞中的表达无明显统计学差异；AFP基因表达明显强于正常肝细胞，并且于培养5天后呈显著的减弱。结论：经分离、培养的人胎肝细胞存活良好，具有和正常成人肝细胞类似的基因表达。

摘要： 本发明公开了一种原代肝细胞冻存液、肝细胞冻存的方法及肝细胞复苏的方法，属于原代肝细胞冻存与复苏领域。所述原代肝细胞冻存液，由如下体积百分数的组分组成：45％溶液A、10％‑45％胎牛血清、10％二甲基亚砜和0％‑35％水，所述溶液A由如下组分组成：D‑葡萄糖、羟乙基淀粉、乳糖酸、聚乙烯吡咯烷酮、五水D‑棉子糖、氢氧化钾、磷酸二氢钾、七水硫酸镁、腺苷、还原性谷胱甘肽、别嘌醇、牛磺熊去氧胆酸和甘草酸二氨。本发明还公开了一种肝细胞冻存的方法及肝细胞复苏的方法。本发明的原代肝细胞冻存液可以降低原代肝细胞在冻存过程中的损伤、提高解冻后原代肝细胞活力与贴壁效率，可广泛用于肝脏基础研究与新药研发过程中。

摘要： 本发明公开了一种三步法序贯式诱导胎肝干细胞向成熟肝细胞分化的方法，属于生物技术领域，包括以下步骤：(1)胎肝干细胞的获得与鉴定：a)胎肝干细胞的分离和扩增；b)胎肝干细胞的鉴定；(2)胎肝干细胞的诱导分化：a)在干冰上低温收集正常肝脏组织研磨匀浆后制成的组织悬液，离心后收集上清液加入Williams’E细胞培养基，并对步骤(1)中的胎肝干细胞进行培养以获得肝祖细胞；b)将步骤a)所获得的细胞用加入Notch信号通路抑制剂的Williams’E培养基，继续培养以获得肝样细胞；c)将步骤b)所获得的细胞用加入含HGF的细胞快速扩增培养基培养，以获得有功能的肝细胞，能快速、准确、高效地将我们前期获得的胎肝干细胞诱导分化为具有功能的肝细胞。

摘要： 本发明公开了一种原代肝细胞冻存液、肝细胞冻存的方法及肝细胞复苏的方法，属于原代肝细胞冻存与复苏领域。所述原代肝细胞冻存液，由如下体积百分数的组分组成：45％溶液A、10％‑45％胎牛血清、10％二甲基亚砜和0％‑35％水，所述溶液A由如下组分组成：D‑葡萄糖、羟乙基淀粉、乳糖酸、聚乙烯吡咯烷酮、五水D‑棉子糖、氢氧化钾、磷酸二氢钾、七水硫酸镁、腺苷、还原性谷胱甘肽、别嘌醇、牛磺熊去氧胆酸和甘草酸二氨。本发明还公开了一种肝细胞冻存的方法及肝细胞复苏的方法。本发明的原代肝细胞冻存液可以降低原代肝细胞在冻存过程中的损伤、提高解冻后原代肝细胞活力与贴壁效率，可广泛用于肝脏基础研究与新药研发过程中。

摘要： 本发明提供特异性识别形成集落的同时增殖的人肝细胞的单克隆抗体、用该抗体分离人肝细胞的方法、将分离的增殖性人肝细胞分化诱导为功能性人肝细胞的方法。另外本申请的方法提供通过上述方法获得的增殖性人肝细胞和功能性肝细胞，以及含有该细胞的细胞试剂盒以及杂合型人工肝脏。

摘要： 本发明公开了一种新的小鼠胎肝细胞系PL08（CCTCC No.C201297）在红细胞前体细胞体外培养与增殖中的应用。该细胞系作为基质细胞，能很好地支持脐带血红细胞前体细胞的体外培养与增殖，为临床脐带血红细胞前体细胞提供更丰富的细胞资源。

摘要： 本发明公开了一种新的小鼠胎肝细胞系PL08（CCTCC？No.C201297）在红细胞体外保存中的应用。该细胞系作为基质细胞，能很好地支持脐带血造血干细胞的体外培养，为临床使用脐带血造血干细胞提供更丰富的细胞资源。

摘要： 本发明公开了一种新的小鼠胎肝细胞系PL08（CCTCC No.C 201297）在红细胞体外保存中的应用。该细胞系作为基质细胞，能很好地支持红细胞的体外保存，可以应用于体外常温保存成熟人体红细胞。

摘要： 本发明公开了一种新的小鼠胎肝细胞系PL08（CCTCC NO:C201297）在红细胞前体细胞体外培养与增殖中的应用。该细胞系作为基质细胞，能很好地支持脐带血红细胞前体细胞的体外培养与增殖，为临床脐带血红细胞前体细胞提供更丰富的细胞资源。

摘要： 本发明公开了一种新的小鼠胎肝细胞系PL08（CCTCC NO:C201297）在红细胞体外保存中的应用。该细胞系作为基质细胞，能很好地支持红细胞的体外保存，可以应用于体外常温保存成熟人体红细胞。

摘要： 本发明公开了一种新的小鼠胎肝细胞系PL08（CCTCC No.C201297）在红细胞体外保存中的应用。该细胞系作为基质细胞，能很好地支持脐带血造血干细胞的体外培养，为临床使用脐带血造血干细胞提供更丰富的细胞资源。