IL-1 receptor antagonist (IL-1Ra) does not inhibit the production of C-reactive protein or serum amyloid A protein by human primary hepatocytes. Differential regulation in normal and tumour cells

The synthesis of some class 1 acute-phase proteins (APP), including C-reactive protein (CRP) and serum amyloid A (SAA) protein is completely blocked by the IL-1 receptor antagonist (IL-1Ra), whereas the production of fibrinogen, a class 2 APP, is increased by IL-1Ra in hepatoma cells, but this has never been tested in human hepatocytes in primary culture. Since previous studies on the contributions of cylokine inhibitors in connective tissues diseases suggested that IL-1 and tumour necrosis factor-alpha (TNF-α) might play an important role in the regulation of CRP, we decided to examine in more detail the respective roles of IL-1β, IL-6, and TNF-α and their inhibitors in the production of APP by human primary hepatocytes versus the hepatoma cell line PLC/PRF/5. In the hepatoma cell line, IL-1β and/or TNF-α had synergistic effects with IL-6 on the production of CRP and SAA. In contrast, these cytokines were devoid of effect in normal hepatocytes. The production of fibrinogen was increased by IL-6 and decreased by IL-1 (and TNF-α) in both cell types. The secretion of CRP and SAA by primary hepatocytes incubated with a cytokine-rich mononuclear cell-conditioned medium was totally unaffected by IL-1Ra or anti-TNF-α antibodies. In contrast, the addition of IL-1Ra increased the production of fibrinogen by both hepatoma cells and primary hepatocytes incubated with the mononuclear cell-conditioned medium. We therefore conclude that IL-1β and TNF-α do not exert any significant effect on the synthesis of CRP and SAA by human primary hepatocytes.     

Microfluidic environment for high density hepatocyte culture

We present a microfluidic bioreactor for culturing high-density arrays of hepatocytes in a tissue-like micro-architecture. The microfluidic environment mimicked physiological liver mass transport, enabling sustained culture of high density cells (>2,000 cells/mm²) without nutrient limitation for over 1 week. The key feature of this design was a microporous microfluidic barrier that formed a sieved-pocket to concentrate cells during loading. Nutrient depletion within the cell mass was avoided by maintaining a continuous flow of medium (10 μl/day) that diffused across the porous barrier. Human hepatoma cells (HepG2/C3A) remained viable and functional as demonstrated by fluorescent viability assays and secretion of albumin for the one-week culture period.     

RNA干扰技术沉默CDC25a基因对人肝癌细胞HepG2增殖的影响

 目的:研究细胞分裂周期素25a（cell division cycle 25a,CDC25a）基因沉默后对于人肝癌细胞系HepG2增殖的影响。同时探讨该影响发生的可能作用机制。 方法: 使用RNA干扰技术沉默人肝癌HepG2细胞的CDC25a基因,采用实时荧光定量PCR技术检测肝癌细胞中的CDC25a 及其作用基因cyclin E及CDK2的 mRNA表达水平,Western blotting检测CDC25a的蛋白表达水平,并采用MTT法、Giemsa染色法及流式细胞技术检测细胞的增殖情况。 结果: CDC25a 的mRNA及蛋白表达水平在RNA沉默组细胞中的表达低于阴性对照组及正常对照组细胞（P＜0.05）。Cyclin E及CDK2 的mRNA表达水平在沉默组低于阴性对照及正常对照组（P＜0.05）。MTT法、Giemsa染色法结果显示沉默组细胞增殖能力低于阴性对照组及正常对照组细胞（P＜0.05）,流式细胞技术结果显示沉默组细胞阻滞在G1期。 结论: LV-CDC25a-RNAi重组体感染HepG2细胞可以有效抑制CDC25a基因的表达,使人肝癌HepG2细胞增殖受到抑制,提示CDC25a基因可能是肝癌治疗的关键靶点。     

Experimental Study on the Inhibitory Effect of Sodium Cantharidinate on Human Hepatoma HepG2 Cells

Backgroud

Cantharidin, and its derivatives can not only inhibit the proliferation of tumor cells, but can also induce tumor cell apoptosis. It shows cantharidin exhibits a wide range of reactivity in anticancer. The objective of this paper was to study the inhibitory effect of sodium cantharidinate on human hepatoma HepG2 cells.

Materials and Methods

MTT assay was used to detect the proliferation of HepG2 cells, and immunohisto-chemical method was used to detect the change in VEGF, protein level, and to determine the inhibitory effect of sodium cantharidinate on human hepatoma HepG2 cells.

Results

As results, sodium cantharidinate significantly inhibited the growth of HepG2 cells in a time-and dose-dependent manner.

Conclusion

We conclude that sodium cantharidinate has an inhibitory effect on human hepatoma HepG2 cells.     

核转录因子HMBOX1对肝癌细胞生物学特征的影响

目的:比较肝癌细胞系与肝细胞系中HMBOX1的表达水平,探讨其在肝癌发生、发展中的作用。方法:RT-PCR检测HMBOX1在多种肝癌细胞系和正常肝细胞系中的表达。利用分子生物学方法构建pEGFP:HMBOX1融合表达载体,采用脂质体方法转染HepG2肝癌细胞系,MTT方法及流式细胞技术评价转染后细胞增殖和细胞周期的变化。基因芯片技术分析表达载体转染HepG2后肿瘤相关信号通路的变化。结果:PCR结果显示肝癌细胞系HMBOX1 mRNA表达水平明显低于正常肝细胞系;pEGFP:HMBOX1融合表达载体转染HepG2后,细胞的增殖能力和周期未出现显著变化;基因芯片的结果提示转染表达载体的HepG2细胞,肿瘤和免疫信号通路相关基因的表达发生显著变化。结论:HMBOX1在多种肝癌细胞系表达下调,可能参与了多条与肿瘤和免疫相关信号通路的调节,为进一步阐明肝癌的发生机制提供了新的实验依据。     

RPMI-1640和DMEM培养基中肝癌细胞系BEL-7402与HepG-2的生长状态比较

背景：DMEM和RPMI-1640是两种最常用的商品化培养基,二者对肝癌细胞生长的影响尚未见直接的对比研究。 目的：比较两种常用培养基对人肝癌BEL-7402与HepG-2细胞系体外生长和增殖效果的影响,从中选择更适合的培养基。 方法：分别应用高糖DMEM与RPMI-1640完全培养液培养BEL-7402和HepG-2细胞,于培养的0,24,48,72,96,  120 h用酸性磷酸酶检测法测定细胞生长和增殖速率,并于倒置显微镜下观察细胞的形态。 结果与结论：结果发现BEL-7402和HepG-2细胞在RPMI-1640培养基中的生长增殖速率均明显高于DMEM培养基 (P < 0.01)。镜下观察证实细胞在RPMI-1640培养基中的黏附和伸展状态更好。因此,建议首选RPMI-1640培养基进行肿瘤细胞体外培养。     

Up-regulation of drug-metabolizing enzyme genes in layered co-culture of a human liver cell line and endothelial cells

Primary human hepatocytes are used extensively to study drug-metabolizing enzymes such as the cytochrome P450 (CYP) enzymes. However, the activities of these enzymes decrease rapidly during culture. In the present study, using a thermo-responsive culture dish, layered co-culture was achieved by placing a bovine pulmonary artery endothelial cell (BPAEC) sheet onto the human hepatoma cell line HepG2. In the BPAEC/HepG2 layered co-culture system, real-time polymerase chain reaction analysis showed that the expression levels of various CYP enzymes were more than 10 times greater 21 days after layering than with a HepG2 monolayer. The expression levels of CYP1B1, CYP2C9, CYP2E1, and CYP3A4 were up-regulated in a time-dependent manner, gradually increasing from day 10 after layering, and continuing to increase until at least day 21. The gene expression levels of the various CYP enzymes were almost identical to that of human liver. These results suggest that our layered co-culture system enhances the function of HepG2 cells and that our BPAEC/HepG2 layered co-culture system can serve as a useful model for the in vitro evaluation of CYP regulation.     

肝细胞提取物生物学活性研究

为深入研究肝细胞提取物的生物学活性,以正常人肝细胞株、肝癌细胞株、肝星状细胞株及原代培养的肝星状细胞为研究靶细胞,观察在不同浓度肝细胞提取物作用下,上述细胞的增殖活性(MTT法)、细胞凋亡活性、细胞外基质成分透明质酸(HA)产生水平及I型胶原基因启动子活性的影响,同时利用流式细胞术比较正常成人外周血单个核细胞(PBMC)在肝细胞提取物作用下,活性T淋巴细胞(CD4+/CD69+)的变化。结果发现肝细胞提取物对正常人肝细胞系(张氏肝细胞)具有明确刺激增殖作用,而对原代培养的肝星状细胞具有抑制增殖作用;肝星状细胞株HSC-T6的研究中,肝细胞提取物具有抑制其细胞外基质成分HA产生、抑制α1(I)型胶原基因启动转录的活性。60μg/ml的肝细胞提取物对肝癌细胞株Hep G2具有明确的诱导凋亡作用。正常成人PBMC经肝细胞提取物作用后活性T淋巴细胞(CD4+/CD69+)数量显著上升。研究表明,肝细胞提取物具有促进肝细胞再生、抑制肝纤维化发生和抗肿瘤作用,并首次发现具有免疫调节作用。     

人胚胎干细胞对肝癌HepG2 细胞体外抑制作用的研究

目的:研究体外共培养情况下人胚胎干细胞H9 对肝癌HepG2 细胞的抑制作用。方法:建立人胚胎干细胞(H9) 与肝癌HepG2 细胞共培养体系,显微镜下观察胚胎干细胞对肿瘤细胞生物学行为的影响,流式细胞仪检测H9 细胞对肿瘤细胞的凋亡与周期的影响,Transwell 小室法检测H9 细胞对HepG2 细胞侵袭和迁移的影响,基因芯片分析共培养后HepG2 细胞全基因组的表达谱变化。结果:结果发现共培养过程中,肝癌HepG2 细胞生长受到抑制,随共培养时间延长,细胞数量逐渐减少,出现老化或凋亡迹象;流式细胞术检测发现HepG2 细胞凋亡率显著增加,细胞周期被阻滞于G0/ G1 期;Transwell 实验发现HepG2 细胞侵袭、迁移力均降低;基因芯片结果发现HepG2 细胞全基因组表达谱发生了显著变化,差异基因涉及多条信号通路。结论:人胚胎干细胞H9 体外对人肝癌细胞HepG2 有一定程度的抑制作用。     

An Autocrine Activity Capable of Substituting for Serum in Cell Cultures

Abstract

Provided that high cell densities (above 10⁶/ml) are maintained, a factor-dependent murine hemopoietic progenitor cell line (FDC-PI) will proliferate in serum-free medium. The conditioned medium (CM) from high-density FDC-PI cells permits the serum-free survival of FDC-PI cells even at low density, indicating the existence of a diffusible autocrine factor. The requirement of FDC-PI for a colony-stimulating factor (either IL-3 or GM-CSF) is not abrogated by culturing the cells at high cell density or in the conditioned medium. Furthermore, the CM from FDC-PI enhances the mitogenic stimulation of normal human skin fibroblasts (HSF) by epidermal growth factor (EGF): i.e., the lag period before entry into the cell cycle is shortened by up to 6 hr. The fibroblasts themselves secrete an activity into serum-free medium that appears to be required during mitogenic stimulation by EGF. The HSF-CM also allows FDC-PI cells to survive and proliferate serum-free at low cell densities. Low concentrations of fetal calf serum or human plasma (0.2-2%) have the same effect as FDC-PI-CM and HSF-CM. We have tested many of the known growth factors, and none of them mimicked the autocrine serum replacing activity (ASRA). The activity in human plasma elutes from a gel-filtration column with an apparent molecular weight of 60,000. It appears as if cultured normal cells and cell lines produce molecules capable of complementing the growth factors required for the survival and proliferation of a range of cells in serum-free cultures.     

An autocrine activity capable of substituting for serum in cell cultures

Provided that high cell densities (above 10(6)/ml) are maintained, a factor-dependent murine hemopoietic progenitor cell line (FDC-P1) will proliferate in serum-free medium. The conditioned medium (CM) from high-density FDC-P1 cells permits the serum-free survival of FDC-P1 cells even at low density, indicating the existence of a diffusible autocrine factor. The requirement of FDC-P1 for a colony-stimulating factor (either IL-3 or GM-CSF) is not abrogated by culturing the cells at high cell density or in the conditioned medium. Furthermore, the CM from FDC-P1 enhances the mitogenic stimulation of normal human skin fibroblasts (HSF) by epidermal growth factor (EGF): i.e., the lag period before entry into the cell cycle is shortened by up to 6 hr. The fibroblasts themselves secrete an activity into serum-free medium that appears to be required during mitogenic stimulation by EGF. The HSF-CM also allows FDC-P1 cells to survive and proliferate serum-free at low cell densities. Low concentrations of fetal calf serum or human plasma (0.2-2%) have the same effect as FDC-P1-CM and HSF-CM. We have tested many of the known growth factors, and none of them mimicked the autocrine serum replacing activity (ASRA). The activity in human plasma elutes from a gel-filtration column with an apparent molecular weight of 60,000. It appears as if cultured normal cells and cell lines produce molecules capable of complementing the growth factors required for the survival and proliferation of a range of cells in serum-free cultures.     

人剪切修复基因XPD肝癌对人HepG2细胞中Rb和MAD2表达的影响*

 目的：探讨人剪切修复基因着色性干皮病D组基因（xeroderma pigmentosum group D, XPD)转染人肝癌HepG2细胞后视网膜母细胞瘤蛋白(retinoblastoma,Rb)和有丝分裂阻滞缺陷蛋白 2（mitotic arrest deficient 2, MAD2）表达的变化及对细胞增殖的影响。方法：将人工合成的pEGFP-N2-XPD重组质粒通过LipofectamineTM 2000转染HepG2细胞。实验分为4组,分别为无转染HepG2细胞组（空白对照组）、脂质体转染HepG2细胞组（脂质体组）、空载质粒pEGFP-N2转染细胞组（N2 组）和重组质粒pEGFP-N2-XPD转染细胞组（XPD组）。分别用RT-PCR和Western blotting法检测各组细胞中XPD、Rb及MAD2 mRNA和蛋白的表达量,用MTT法检测各组细胞的增殖活力,流式细胞术检测各组的细胞凋亡情况及细胞周期。结果：与其它3组相比,XPD组XPD mRNA及蛋白表达量明显升高（P<0.05）,Rb mRNA及蛋白表达量明显升高（P<0.05）,而MAD2 mRNA及蛋白表达量显著降低（P<0.05）。XPD组细胞增殖活力显著降低,细胞凋亡率明显升高（P<0.05）。XPD组细胞停滞在G1期,难以进入S期。结论：XPD可以抑制MAD2的表达和肝癌细胞的增殖,促进Rb的表达。     

Human Liver Stem Cell-Derived Microvesicles Inhibit Hepatoma Growth in SCID Mice by Delivering Antitumor MicroRNAs

Microvesicles (MVs) play a pivotal role in cell-to-cell communication. Recent studies demonstrated that MVs may transfer genetic information between cells. Here, we show that MVs derived from human adult liver stem cells (HLSC) may reprogram in vitro HepG2 hepatoma and primary hepatocellular carcinoma cells by inhibiting their growth and survival. In vivo intratumor administration of MVs induced regression of ectopic tumors developed in SCID mice. We suggest that the mechanism of action is related to the delivery of microRNAs (miRNAs) from HLSC-derived MVs (MV-HLSC) to tumor cells on the basis of the following evidence: (a) the rapid, CD29-mediated internalization of MV-HLSC in HepG2 and the inhibition of tumor cell growth after MV uptake; (b) the transfer by MV-HLSC of miRNAs with potential antitumor activity that was downregulated in HepG2 cells with respect to normal hepatocytes; (c) the abrogation of the MV-HLSC antitumor effect after MV pretreatment with RNase or generation of MVs depleted of miRNAs; (d) the relevance of selected miRNAs was proven by transfecting HepG2 with miRNA mimics. The antitumor effect of MV-HLSC was also observed in tumors other than liver such as lymphoblastoma and glioblastoma. These results suggest that the delivery of selected miRNAs by MVs derived from stem cells may inhibit tumor growth and stimulate apoptosis. Stem Cells2012;30:1985-1998.     

扶正解毒通络方对人肝癌HepG2 细胞MMP-2、MMP-9 表达及细胞侵袭作用的影响

目的:探讨扶正解毒通络方对肝癌HepG2 细胞侵袭作用和基质金属蛋白酶(Matrix metallo proteinases,MMP)-2、MMP鄄9 表达水平的影响及可能的作用机制。方法: CCK8 实验检测扶正解毒通络方对HepG2 细胞活性影响;Transwell 侵袭实验检测扶正解毒通络方对HepG2 侵袭能力的影响;ELISA 法检测扶正解毒通络方干预HepG2 细胞后MMP-2和MMP-9 的表达水平。结果:CCK8 实验结果显示,扶正解毒通络方对HepG2 细胞意志呈剂量时间相关性。Transwell 侵袭实验显示,2.65 mg/ ml 扶正解毒通络方对HepG2 细胞侵袭力抑制率为52.45% (P<0.05)。ELISA 检测结果显示,扶正解毒通络方干预后HepG2 细胞上清液中MMP-2 和MMP-9 表达水平下降(P<0.05)。结论:扶正解毒通络方可以抑制肝癌HepG2 细胞的生长和侵袭,其机制可能是通过下调MMP-2、MMP-9 在肝癌细胞HepG2 的表达。     

重组腺相关病毒介导黑色素瘤分化相关基因7对人肝癌细胞体内外抑制效应的研究

目的观察PEG启动子调控腺相关病毒介导的黑色素瘤分化相关基因7（MDA-7）在体内外抑制肝癌细胞的生物学活性，探讨重组腺相关病毒介导MDA-7基因用于肝癌基因治疗的应用前景。方法构建重组腺相关病毒rAAV-PEG-MDA-7表达系统，体外感染人肝癌细胞株HepG2细胞。Western印迹检测转染细胞内MDA-7蛋白；MTT法检测细胞增殖抑制率；流式细胞术分析细胞周期和细胞凋亡变化。构建肝癌细胞HepG2裸鼠皮下移植瘤模型，尾静脉注射rAAV-PEG-MDA-7，观察其对肝癌生长的抑制作用；ELISA方法检测血浆MDA-7蛋白浓度；TUNEL法分析MDA-7对肿瘤细胞的凋亡诱导情况；免疫组织化学分析MDA-7在肿瘤组织中的表达。结果重组腺相关病毒rAAV-PEG-MDA-7可特异性转染HepG2细胞，MDA-7蛋白在HepG2细胞中高效表达，并呈时间依赖性。重组腺相关病毒rAAV-PEG-MDA-7可抑制HepG2细胞增殖并诱导其凋亡，G0／G1期细胞百分比明显增多，G2／M期的细胞显著减少（P〈0、05）。全身系统性给予rAAV-PEG-MDA-7后，血清中可持续检测到MDA-7蛋白，且注射后2周浓度达高峰（200ng／ml）；肿瘤生长受抑制，抑瘤率为62％（P〈0、05）；免疫组化结果显示MDA-7在肿瘤组织中表达；TUNEL结果显示rAAV-PEG-MDA-7可诱导肿瘤细胞凋亡。结论构建出的重组腺相关病毒rAAV-PEG-MDA-7表达系统具有良好的肿瘤靶向性，通过抑制肝癌细胞增殖和诱导其凋亡发挥抗肝癌作用。     

放射性碘标记特异性溶瘤重组腺病毒KH901在荷人肝癌裸鼠体内的抑瘤作用

研究131I标记的特异性溶瘤重组腺病毒KH901的生物活性及131I-KH901对荷人肝细胞肝癌裸鼠肿瘤的抑制作用。采用N-溴代琥珀酰亚胺(NBS)法用131I标记KH901,并采用ELISA法测定粒细胞-巨噬细胞集落刺激因子(GM-CSF)的生物活性;通过给药后荷人肝细胞肝癌裸鼠肿瘤的生长实验观察131I-KH901对肿瘤生长的影响,组织切片观察肿瘤组织形态学变化。结果显示:131I-KH901在肿瘤细胞中表达大量的GM-CSF因子,24 h后,在肿瘤细胞和正常细胞中产生的GM-CSF因子分别为183.27±6.90 pg/ml和20.44±0.77 pg/ml;肿瘤生长观察结果显示,131I-KH901瘤内给药能明显抑制肿瘤生长,抑瘤率为71.3%,显著大于131I组(22.7%)及KH901瘤内给药组(52.7%)(P<0.05),组织形态学观察示,注射131I-KH901后,肿瘤出现大片坏死区。因此,131I-KH901能明显抑制裸鼠体内人肝癌细胞的生长,可望成为腺病毒溶瘤和放射性核素联合治疗肿瘤的新型药物。     

The proliferation and function of human mononuclear leukocytes and natural killer cells in serum-free medium

We recently developed a serum-free (SF) culture medium that supports the growth of several established lymphoid cell lines. In an effort to develop a standardized medium for assay of human natural killer (NK) cell activity, we compared the cytotoxic activity of peripheral blood mononuclear leukocytes (PBL) and purified large granular lymphocytes (LGL) cultured in SF medium containing interleukin 2 (IL-2) or medium containing 10% fetal bovine serum (FBS) plus IL-2. The results indicated that PBL had a 30% increase in cumulative net cell growth and had as high or higher cytotoxic activity after growth in SF medium than in medium containing FBS. Purified LGL had a 50% increase in cumulative net cell growth and persisted approximately 2 weeks longer in culture in medium containing FBS than in SF medium. However, the cytotoxic activity of cells grown in SF medium persisted during the initial 3 weeks of culture. Purified LGL that were maintained and were subcultured at cell densities of 10(6) cells or greater per milliliter of either SF or FBS-containing medium had equivalent levels of cytotoxicity over a 44-day period in either medium compared with cells subcultured at a density of 5 X 10(5) cells per milliliter of medium. NK cells produced a cytotoxic factor (NKCF) in SF medium, and its cytotoxic activity was blocked by 10% FBS. We conclude that the SF medium supplemented with IL-2 can be used as an alternative to FBS-containing medium with IL-2 for the growth of NK cells and is advantageous for the production of NKCF.     

Hydrogel-coated textile scaffolds as candidate in liver tissue engineering: II. Evaluation of spheroid formation and viability of hepatocytes

In this study we evaluate the performance of primary rat hepatocytes and HepG2 cells on chitosan-collagen hydrogel-coated textile scaffolds. Light microscopy and electron microscopic observations showed attachment and aggregate formation tendency of hepatocytes on the scaffolds. As tested by the tetrazolium reduction (MTT) assay it was evident that cells had preserved mitochondrial functionality. It was also observed that pure collagen and collagen blended scaffolds allowed higher cell growth than pure chitosan scaffold. Fluorescent live/dead staining showed a metabolically active, viable cell population on all scaffold compositions with occurrence of few dead cells. Cell functionality was confirmed by secretion of albumin, which was maintained throughout culture period. Take collectively our results suggests that hydrogel-coated textile scaffolds could be promising for tissue-engineering applications, as they allow favorable hepatocyte attachment, spheroid formation and maintenance of function. These scaffolds could be useful for co-culturing hepatocytes and nonparenchymal endothelial cells in bioartificial liver support systems.     

人着色性干皮病基因D对HepG2细胞Mdm2和Mdm4表达的影响

 目的：探讨人剪切修复基因着色性干皮病基因D（xeroderma pigmentosum D, XPD）对肝癌细胞鼠双微体2（murine double minute 2，Mdm2）和鼠双微体4（murine double minute 4，Mdm4）表达的影响。方法：用脂质体转染法将重组质粒pEGFP-N2/XPD和空载质粒pEGFP-N2转染进入人肝癌细胞株HepG2，实验分为3组：（1）空白对照组；（2）pEGFP-N2组；（3）pEGFP-N2/XPD组。用MTT法观察细胞的增殖活力；用流式细胞术检测细胞周期和凋亡率；用RT-PCR和Western blotting检测XPD、Mdm2、Mdm4和P53表达量的变化。结果：MTT结果显示，重组质粒pEGFP-N2/XPD的转染抑制了肝癌细胞的增殖活力；流式细胞术结果显示，重组质粒pEGFP-N2/XPD的转染引起了G 1期细胞增加，S期减少，凋亡率增加。RT-PCR和Western blotting检测发现，重组质粒pEGFP-N2/XPD的转染使得XPD表达增高，同时使得Mdm2和Mdm4表达降低，P53表达增高。结论：XPD能下调肝癌细胞中Mdm2和Mdm4的表达，上调P53表达，并能抑制肝癌细胞增殖及诱导其凋亡。