模拟微重力条件下对WB-F344肝干细胞表征分子表达的影响

研究大鼠WB-F344肝干细胞在旋转式细胞培养系统（RCCS）中培养进行细胞大规模扩增并保持干细胞的特性的可能性,为干细胞治疗疾病及肝组织工程提供理想的细胞来源。以WB-F344肝干细胞在RCCS中培养,以平面单层培养为对照,在培养后不同时间分别进行形态观察、流式细胞仪测细胞周期、逆转录-聚合酶链反应（RT-PCR）检测肝干细胞特异性基因甲胎蛋白（AFP）和白蛋白（ALB）的表达, 免疫荧光染色检测 AFP、ALB蛋白的表达。结果表明,RCCS培养的WB-F344细胞粘附在Cytodex-3微载体上状态生长良好,细胞增殖较平面培养有明显增加;RT-PCR和免疫荧光染色检测结果一致：模拟微重力培养组AFP的mRNA表达强度及AFP阳性细胞均显著高于平面培养组,而ALB mRNA表达强度和ALB阳性细胞均低于对照组。说明模拟微重力 培养条件下,能较好的维持肝干细胞特性,进一步证明我们建立的这种培养体系是成功的,是一种理想的肝干细胞培养模式。     

低浓度血清促进胚胎肝祖细胞诱导分化

为了探讨不同血清浓度诱导培养条件下对体外诱导胚胎肝祖细胞成熟分化的影响。本研究选用含有10%FBS、5%FBS、2%FBS的DMEM/HGF/FGF4肝细胞培养基体外诱导胚胎肝祖细胞的成熟分化,分别于诱导后0d、3d、6d、9d、12dALB-GLuc检测细胞白蛋白的合成水平,细胞计数检测细胞的成长曲线。RT-PCR检测肝细胞相关标志DLK、AFP、CK18,免疫荧光检测ALB、UGT1A的表达情况。ICG摄取和尿素氮检测肝细胞成熟功能。诱导后第三天,ALB-GLuc开始增高,于第9天达高峰,2%FBS组ALB-GLuc读数最高。生长曲线显示低血清浓度下细胞的增殖速度明显慢于高血清浓度。RT-PCR和免疫荧光结果显示诱导后第9天,3个诱导组DLK、AFP表达低于无诱导组,CK18、ALB、UGT1A均高于无诱导组,2%FBS组差异最显著。3个诱导组的肝细胞ICG摄取能力明显增高,2%FBS组ICG阳性细胞数为(60.2±9.0)%,明显高于5%FBS(45.0±3.6)%及10%FBS组(35.2±2.9)%,诱导后肝细胞的尿素合成功能增强,尤其是2%FBS组。低浓度血清培养条件能更好的诱导胚胎肝祖细胞的体外成熟分化。     

低浓度CO暴露促进大鼠肝前体细胞增殖的体外研究

目的:观察低浓度一氧化碳暴露对营养相对匮乏情况下大鼠肝前体细胞系生长的影响并探索其作用机制。方法:分别在营养充足(10%胎牛血清,10%FBS)和营养相对匮乏(4%胎牛血清,4%FBS)条件下对大鼠肝脏前体细胞系WB-F344进行体外培养,观察细胞的生长情况;同时,在不同两组4%FBS培养基中分别加入无活性的一氧化碳释放分子iCORM-2和有活性的一氧化碳释放分子CORM-2,两者的浓度均为100μM,观察比较低浓度一氧化碳暴露是否有利于WB-F344的生长和增殖;利用吖啶橙染色观察不同组别培养细胞酸性自噬小体的形成情况,并进行Western-Blot,检测自噬相关蛋白LC3-Ⅰ和LC3-Ⅱ的转化情况,探讨其可能的作用分子机制。结果:在含有10%FBS的培养基中,WB-F344细胞生长良好,呈指数生长,并在第5天达到生长高峰;与其相比,4%FBS培养基对WB-F344的生长有明显抑制,细胞生长相对缓慢,并且相同时间点的细胞增殖数量明显不足。在进行浓度为100μM的一氧化碳分子暴露后,WB-F344细胞生长情况有明显改善,细胞增殖的峰值有显著增加。吖啶橙染色显示,4%FBS培养基条件下培养的WB-F344细胞,细胞内出现橘红色荧光,在暴露于低浓度的一氧化碳分子后,橘红色荧光的强度有明显增加。进一步的Western-Blot检测发现,营养相对匮乏的培养基(4%FBS)能使WB-F344细胞在生长过程产生LC3-Ⅰ向LC3-Ⅱ的转化,在暴露于低浓度的一氧化碳分子后,LC3-Ⅱ的量进一步增加,LC3-Ⅱ/LC3-Ⅰ的比值显著增大(P0.05);进一步的研究显示在此过程中伴随着p-AKT和mTOR蛋白的表达降低,同时,HO-1蛋白的表达增加。结论:低浓度一氧化碳暴露能促进WB-F344细胞产生自噬,有利于大鼠肝前体细胞系的生长和增殖,同时这和AKT/mTOR信号途径的抑制及HO-1的表达增加有关。     

金雀异黄素对SGC-7901胃癌细胞超微结构及极光激酶A基因表达的影响

应用原子力显微镜(Atomic Force Microscope,AFM)技术、Real-time PCR及Western blotting方法,分别检测金雀异黄素(genistein)对SGC-7901胃癌细胞超微结构、Aurora-A基因mRNA和蛋白表达的影响.AFM扫描显示,经20μg/mLgenistein处理SGC-7901胃癌细胞48 h,细胞的超微结构呈现凋亡形态特征改变;Aurora-A表达分析,5μg/mL组、10μg/mL组和20μg/mL组与对照组比较,其mRNA表达量分别下降(31.6±0.02)%、(38.8±0.04)%和(59.9±0.02)%,其中5μg/mL组与10μg/mL组间比较无显著性差异(P＞0.05),其余组间比较均有显著性差异(P＜0.05);其蛋白表达量分别下降(26.8±0.04)%、(33.0±0.10)%和(48.5±0.09)%,组间比较均有显著性差异(P＜0.05).由此推断金雀异黄素诱导SGC-7901胃癌细胞凋亡的分子机制可能涉及极光激酶A mRNA及蛋白表达下调.     

蒙药沙参四味汤抑制人肺癌H460细胞生长作用的研究

目的:探讨蒙药沙参四味汤对人肺癌NCI-H460细胞的体外增殖抑制作用。方法:实验分为空白对照组及用药组。将人肺癌NCI-H460细胞与不同浓度的沙参四味汤(5μg/mL、10μg/mL、15μg/mL、20μg/mL、25μg/mL、30μg/mL)进行联合体外培养,采用四甲基偶氮唑蓝法(MTT法)测定不同浓度的沙参四味汤对肺癌NCI-H460细胞增殖的影响。结果:沙参四味汤对人肺癌NCI-H460细胞具有潜在的体外增殖抑制作用:不同浓度的沙参四味汤均能抑制人肺癌NCI-H460细胞的生长,分别作用24 h、48h、72 h后其最高细胞抑制率(IR)分表达到60.36%、69.31%和86.87%,24 h、48 h、72 h后其半数致死量(IC50)分别为30.60μg/mL、19.36μg/mL和14.93μg/mL。结论:沙参四味汤对人肺癌H460细胞具有体外增殖抑制作用,呈现浓度依赖性。     

小鼠成体肝脏前体细胞最佳培养条件探索

目的建立一种专门用于小鼠肝脏前体细胞的培养条件,并鉴定该条件在连续传代培养的过程中是否能够维持前体细胞良好的生长状态,并减少分化的发生。方法配制4种不同配方的培养基,在连续传代过程中通过观察细胞形态学变化而选择适用的配方,并经Western blot检测细胞内肝细胞标志物白蛋白(albumin,ALB)、肝脏前体细胞标志物甲胎蛋白(α-fetoprotein,AFP)和胆管细胞标志物细胞角蛋白-19(cytokeratin-19,CK-19)表达量的变化,流式细胞术分析细胞周期,以及免疫荧光染色鉴定前体细胞标志物的表达以进一步验证选择配方的有效性。结果使用第4种培养基进行培养时,细胞形状规则,边界清晰,生长活力较好,连续传代培养后细胞的形态未发生明显改变。Western blot检测显示,ALB、AFP和CK-19在P0代、P3代和P8代中的表达量没有显著改变。流式细胞术结果显示,经连续多次传代的细胞约76%的细胞处于G0/G1期,近24%的细胞处于S+G2/M期,而且约89%的细胞表达肝脏前体细胞标志物上皮细胞粘附分子(epithelial cell adhesion molecule,EpCAM)。免疫荧光染色进一步证实了大多数细胞表达EpCAM。结论建立了合适的培养基配方可用于小鼠成体肝脏前体细胞的连续传代培养,为更加深入地研究肝脏前体细胞的机制与再生医学方面创造了前提条件。     

SIRT1对TGF-β1活化肾脏成纤维细胞的抑制机制

[目的]探讨沉默信息调节因子1(SIRT1)对转化生长因子β1(TGF-β1)活化肾脏成纤维细胞的抑制作用及机制。[方法]设立成纤维细胞NRK-49F组、TGF-β1刺激组(10 ngl/mL)、SIRT1蛋白低、中、高剂量组(10.0μg/mL、100.0μg/mL、1 000.0μg/mL),以上各组每孔设6个平行样,培养72 h。采用MTT法测定各组细胞活力,流式细胞术测定细胞凋亡水平,ELISA法测定细胞纤维化程度α-SMA、FN、Vimentin蛋白水平,RT-PCR法及蛋白印迹法测定Smad3、SOSC1 mRNA和蛋白水平。[结果] TGF-β1刺激组OD值、存活率、α-SMA、FN、Vimentin蛋白、Smad3、SOSC1 mRNA和蛋白表达水平高于成纤维细胞NRK-49F组,凋亡率低于成纤维细胞NRK-49F组(P<0.05)。SIRT1蛋白低、中、高剂量组OD值、存活率、α-SMA、FN、Vimentin蛋白、Smad3、SOSC1 mRNA、Smad3、SOSC1蛋白表达水平低于TGF-β1刺激组,凋亡率高于TGF-β1刺激组,随着SIRT1蛋白剂量逐渐...     

ES细胞体外定向分化为成熟肝细胞的实验研究

探讨了肝细胞在胚胎干细胞（ES cell）体外诱导分化系统中成熟分化的条件、机制及其鉴定方法.利用TGF, bFGF、HGF等细胞生长因子进行BALB/c小鼠ES细胞向肝细胞方向的定向诱导.利用反转录聚合酶链反应（RT-PCR）、免疫细胞化学（ICC）和放射免疫法（RIA）动态检测肝细胞特异性基因和蛋白AFP,ALB,G6P,TAT,CK8, CK18等在培养体系中的表达,并测定肝细胞的尿素合成功能,最后测定肝细胞分化率.结果,肝细胞特异基因AFP, ALB,G6P和TAT最早分别于第3、9、11、13天表达,肝细胞特异蛋白AFP,CK8,CK18和ALB最早分别于第7、9、9和11天开始表达.第12天开始检测到尿素出现,浓度为8.3 μmol/L,并随培养时间延长而浓度逐渐增加.最后,测得生长因子诱导组肝细胞的分化率为32％,对照组肝细胞分化率为8%.说明肝细胞可以在ES细胞体外诱导分化系统中出现并成熟分化,bFGF、HGF、OSM等可以明显提高细胞分化率和成熟度,有望成为解决肝功能替代疗法中细胞来源问题的新希望.     

紫花牡荆素对胃癌SGC-7901增殖的影响及分子机制

目的:探讨紫花牡荆素(casticin)对胃癌SGC-7901细胞增殖的影响,以及对Bcl-2表达的调节作用.方法:用紫花牡荆素预处理胃癌SGC-7901细胞,MTT法分别检测紫花牡荆素不同浓度(1 g/mL、2g/mL、4g/mL、8g/mL)和不同处理时间(6h、12h、24h、48h)细胞增殖情况;IC50浓度处理胃癌SGC-7901细胞之后,Western blot和RT-PCR检测Bel-2蛋白和mRNA表达水平.结果:5.6g/mL紫花牡荆素处理SG-C-7901细胞12小时后,对细胞增殖抑制作用显著,Westernblot和RT-PCR结果显示Bcl-2表达显著下调.结论:花牡荆素可显著抑制SGC-7901细胞增殖,而这一作用可能与Bel-2表达下调有关.     

盐酸小檗碱对白色念珠菌菌态转换的影响研究

为探讨盐酸小檗碱对白色念珠菌酵母相向菌丝相转换的抑制作用和分子机制,采用微量稀释法测定盐酸小檗碱抑制白色念珠菌生长的MIC值,倒置荧光显微镜观察菌丝动态形成过程;hochest染色法观察不同浓度盐酸小檗碱对菌丝形成的影响;RT-PCR检测在不同浓度盐酸小檗碱作用下白色念珠菌菌丝形成关键基因EFG1、HWP1、ECE1、ALS1表达水平的变化。结果显示,盐酸小檗碱对白色念珠菌的MIC值为32μg/mL,同时白色念珠菌在4 h芽管形成明显,8 h转化为菌丝;hochest染色结果显示:128和32μg/mL的盐酸小檗碱可以通过抑制菌丝的形成以及降低细胞密度,从而达到抑制白色念珠菌发生菌态转换的目的,并且128μg/mL盐酸小檗碱的抑制作用明显优于32μg/mL盐酸小檗碱和4μg/mL氟康唑,而8μg/mL盐酸小檗碱抑制作用不明显;RT-PCR结果表明,128μg/mL盐酸小檗碱可以抑制白色念珠菌4 h干预组菌丝形成关键基因HWP1和ECE1的表达、6 h干预组HWP1、ECE1和ALS1的表达,差异有统计学意义(P0.05);32μg/mL盐酸小檗碱可以抑制白色念珠菌4 h干预组菌丝形成关键基因EFG1和ALS1的表达,6 h干预组EFG1、HWP1和ALS1的表达,差异具有统计学意义(P0.05),但8μg/mL盐酸小檗碱无明显抑制作用。结果表明,盐酸小檗碱可以明显抑制白色念珠菌的菌态转换,其作用机制可能与菌丝形成关键基因EFG1、HWP1、ECE1、ALS1的表达下调相关。     

Let-7f microRNA negatively regulates hepatic differentiation of human adipose tissue-derived stem cells

MicroRNAs (miRNAs) are noncoding RNAs involved in the regulation of the diverse biological processes such as metabolism, proliferation, and cell cycle, in addition to regulation of differentiation. So far, some miRNAs have been recognized to have important role in regulating hepatic functions. Statistically, let-7f has been revealed as a negative regulator of hepatic differentiation. In the present study, we investigated the effect of let-7f on hepatic differentiation of human adipose tissue-derived stem cells (hADSCs). hADSCs were transduced with recombinant lentivirus containing human inhibitor let-7 f. The expression of hepatocyte nuclear factors alpha (HNF4a), albumin (ALB), alpha fetoprotein (AFP), cytokeratin 18 (CK18), and cytokeratin 19 (CK19) was evaluated using quantitative real-time PCR (qRT-PCR). Immunocytochemistry was used to investigate the expression levels of the hepatocyte markers including ALB, AFP, and HNF4a, and biochemical analysis was implemented for hepatic function, glycogen deposition, and urea secretion. qRT-PCR showed significant upregulation in HNF4a, ALB, AFP, CK18, and CK19 expression in cells transduced with let-7f inhibitor lentiviruses. Moreover, positive staining was detected for ALB, AFP, and HNF4a using immunocytochemistry. Urea production and glycogen deposits were also found in the treated cells, the two specific features of the hepatic cells. Therefore, let-7f silencing led to the increased expression of the hepatocyte-specific factors and the accelerated hADSCs hepatic differentiation. Summing all these finding together, our present report has provided evidences that inhibition of let-7f would facilitate induction of hADSCs into hepatocyte-like cells and possibly in regenerative therapy of the liver disease in a wider spectrum.     

Role of epimorphin in bile duct formation of rat liver epithelial stem-like cells: involvement of small G protein RhoA and C/EBPβ

Epimorphin/syntaxin 2 is a high conserved and very abundant protein involved in epithelial morphogenesis in various organs. We have shown recently that epimorphin (EPM), a protein exclusively expressed on the surface of hepatic stellate cells and myofibroblasts of the liver, induces bile duct formation of hepatic stem-like cells (WB-F344 cells) in a putative biophysical way. Therefore, the aim of this study was to present some of the molecular mechanisms by which EPM mediates bile duct formation. We established a biliary differentiation model by co-culture of EPM-overexpressed mesenchymal cells (PT67(EPM)) with WB-F344 cells. Here, we showed that EPM could promote WB-F344 cells differentiation into bile duct-like structures. Biliary differentiation markers were also elevated by EPM including Yp, Cx43, aquaporin-1, CK19, and gamma glutamyl transpeptidase (GGT). Moreover, the signaling pathway of EPM was analyzed by focal adhesion kinase (FAK), extracellular regulated kinase 1/2 (ERK1/2), and RhoA Western blot. Also, a dominant negative (DN) RhoA-WB-F344 cell line (WB(RhoA-DN)) was constructed. We found that the levels of phosphorylation (p) of FAK and ERK1/2 were up-regulated by EPM. Most importantly, we also showed that RhoA is necessary for EPM-induced activation of FAK and ERK1/2 and bile duct formation. In addition, a dual luciferase-reporter assay and CHIP assay was performed to reveal that EPM regulates GGT IV and GGT V expression differentially, possibly mediated by C/EBPβ. Taken together, these data demonstrated that EPM regulates bile duct formation of WB-F344 cells through effects on RhoA and C/EBPβ, implicating a dual aspect of this morphoregulator in bile duct epithelial morphogenesis.     

小分子化合物体外诱导肝上皮样干细胞-WB细胞表达PDX1

目的：近年来干细胞治疗糖尿病一直是国内外研究人员关注的焦点,而肝细胞向胰岛样细胞转变也是热点之一。本实验应用小分子化合物在体外诱导WB-F344大鼠来源肝上皮样干细胞（简写WB细胞）表达胰腺内分泌前体细胞基因PDX1,建立一种体外诱导WB细胞分化为胰腺内分泌前体细胞的实验方法。方法：选用5-AZA TSA,RA,ITS等小分子化合物,分两步法直接诱导WB细胞分化为表达PDX1的胰腺内分泌前体细胞,用含有不同浓度5-AZA分化培养基诱导WB分化,摸索诱导分化的最佳条件。观察细胞形态变化,RT-PCR及实时定量PCR检测部分基因表达情况,免疫荧光检测PDX1的表达。结果：5AZA 5 uM处理2 d,TSA 1 d,RA联合ITS诱导7天,诱导的WB细胞表达PDX1较1-4 uM 5-AZA诱导强,并表达胰腺内分泌前体细胞的相关基因,NGN3,Neurod,NKX2.2,WB表达的Nestin仍持续表达,Insulin1有少量表达。WB表达的肝干细胞基因如ALB,AFP大量下调,标志分化的基因C/EBP下调。结论：5-AZA,TSA,RA,ITS等小分子化合物能够诱导肝上皮样细胞WB表达PDX1,并且这种诱导分化的细胞具有胰腺内分泌前体细胞特征。本实验进一步证明在体外微环境中,肝干细胞能向胰腺内分泌细胞转化,而肝细胞增极强,为将来干细胞治疗糖尿病提供充足的细胞来源     

利用人脐血单个核细胞重建急性肝损伤小鼠肝组织的实验研究

利用人脐血单个核细胞重建急性肝损伤小鼠肝组织,探索建立人-小鼠嵌合肝模型方法。15只SCID小鼠,以四氯化碳(CCL4)制备急性肝损伤模型,24h后行2/3肝切除,然后分为三个实验组细胞移植组(7只)、阴性对照组(3只)及空白对照组(5只);将人脐血单个核细胞悬液注入细胞移植组小鼠脾脏内,阴性对照组小鼠脾脏内注入等量磷酸盐缓冲液(PBS),空白对照组不注射细胞悬液和PBS。术后7d、14d及21d取小鼠肝组织观察病理变化、检测人白蛋白(ALB)及细胞角蛋白19(CK19),同时检测小鼠血清及肝组织匀浆中人ALB含量。全部小鼠表现出急性肝损伤组织学特征;细胞移植组小鼠术后7d、14d、21d肝组织内均见大量人ALB及CK19阳性表达细胞,血清及肝组织匀浆可检测出人ALB;阴性对照组小鼠肝组织未见人ALB及CK19阳性表达,血清及肝组织匀浆中未检测出人ALB。人脐血单个核细胞在部分肝切除的急性肝损伤小鼠肝组织内可大量分化为人肝细胞及胆管细胞,在建立模型方面已取得关键突破。     

RhoA promotes differentiation of WB-F344 cells into the biliary lineage

When cultured on Matrigel, liver precursor epithelium WB-F344 cells could be induced to differentiate into biliary cells in which RhoA expression was upregulated. To further investigate the role of RhoA in WB cell differentiation initiated by Matrigel treatment, we constructed constitutively active RhoA-expressing vectors and stably transfected them into WB-F344 cells. Accompanying upregulation of biliary lineage markers and morphological changes, cells with ectopically active RhoA expression were found to form bile-duct-like structures even without Matrigel treatment. Besides, ROCK inhibitor Y27632 treatment eliminated luminal morphogenesis. F-actin cytoplasmic staining further verified that the RhoA–ROCK signal pathway was involved in differentiation of WB cells into the biliary lineage. In conclusion, our results suggested that the RhoA–ROCK–stress fibre system plays an obligatory role in Matrigel-induced WB-F344 cell luminal morphogenesis and further differentiation.     

Effects of bisphenol A and 4-nonylphenol on cellular responses through the different induction of LPA receptors in liver epithelial WB-F344 cells

Abstract

Lysophosphatidic acid (LPA) signaling via G protein-coupled transmembrane LPA receptors (LPA₁ to LPA₆) mediates a variety of cellular functions, including cell proliferation, migration, morphogenesis, and differentiation. Recently, we demonstrated that the different induction of LPA receptors by estrogens regulates cell motile activity of rat liver epithelial WB-F344 cells. In the present study, to assess whether endocrine disruptors (EDs) are involved in cellular functions through LPA signaling, we measured cell motile activity and LPA receptor expressions in WB-F344 cells treated with bisphenol A (BPA) and 4-nonylphenol (4-NP). Using quantitative real time RT-PCR analysis, the Lpar1 expression was elevated in BPA-treated cells, whereas the Lpar3 expression was decreased. In contrast, 4-NP increased the Lpar3 expression, but not the Lpar1 and Lpar2. For cell motility assay with a Cell Culture Insert, cell motile activity of BPA-treated cells was significantly lower than that of untreated cells. In contrast, 4-NP markedly enhanced cell motile activity. The effects of BPA and 4-NP on cell motility were inhibited by the Lpar1 or Lpar3 knockdown. These results suggest that BPA and 4-NP may regulate cell motile activity through the different induction of LPA receptors in WB-F344 cells.     

茵陈四苓颗粒对肝衰竭环境下骨髓间充质干细胞分化及IDO蛋白表达的影响

目的:研究肝衰竭体外模型环境下茵陈四苓颗粒对骨髓间充质干细胞的肝向分化及IDO蛋白表达的影响。方法:通过建立肝衰竭血清、中药干预血清、健康血清分别与骨髓间充质干细胞(BMSCs)共培养体系,检测BMSCs中CK18免疫荧光表达,Western-blot法检测甲胎蛋白(AFP)、肝组织白蛋白(ALB)、吲哚胺2,3—双加氧酶(IDO)蛋白表达。结果:1、CK18免疫荧光结果显示肝衰竭组及中药干预组染色均为弱阳性,但后者染色更强,而健康组无阳性染色;2、AFP、ALB在健康组、肝衰竭组、中药干预组均有表达,且各组间ALB比较均有差异(P0.05),三组AFP表达水平逐渐增高,各组间差异有统计学意义。IDO蛋白在健康组无表达,肝衰竭组表达低于中药干预组,且两组间差异具有统计学意义。结论:茵陈四苓颗粒能增强BMSCs在肝衰竭环境下的肝向分化趋势及免疫调节活性。