体外定向诱导E14小鼠胚胎干细胞为肝细胞

目的探索体外定向诱导胚胎干细胞分化为肝细胞。方法常规方法培养E14小鼠胚胎干细胞(ESC)后，进行悬滴-悬浮培养，形成胚胎体，再进行分阶段定向诱导，在培养第9～12天、第12～18人以及第15～18天分别加入酸性成纤维细胞生长因子、肝细胞生长因子和制瘤素M。利用倒置相差显微镜观察细胞形态变化，逆转录聚合酶链反应(RT-PCR)法分析持异性基因mRNA的表达，并用吲哚氰绿(ICG)摄取和过碘酸雪夫反应(PAS)分析细胞的分化及功能。结果在诱导培养的第13天，细胞出现肝细胞样改变。RTPCR分析可见，在诱导的第6天和第12天分别可以榆测到内胚层或卵黄囊分化标志基因-甲状腺素运载蛋白和α1抗胰蛋白酶的mRNA表达，第6天出现末成熟肝细胞标志基因-甲胎蛋白mRNA表达，第9天、第15天和第18天分别开始出现成熟肝细胞的特异性标志基因-白蛋白、葡萄糖6磷酸酶、酪氨酸氨基转移酶mRNA表达。同时，ESC源性肝细胞表现为ICG摄取和PAS反应阳性，经过诱导，ICG阳性细胞数约占85．1％。结论ESC源性肝细胞具备肝细胞特性，FSC有可能成为肝细胞治疗的替代供体细胞。     

生物反应器内EBs来源细胞的肝细胞分化与成熟

目的 观察微重力生物反应器内拟胚体(EBs)来源细胞的肝细胞分化与成熟.方法 将未分化鼠胚胎干细胞(ES细胞)以1×106/mL移入微重力反应器内,在DMSO、地塞米松、FGF4、HGF等刺激作用下,进行15 d的旋转培养.采用ELISA法动态检测培养液中鼠白蛋白的产生量.培养结束时转移EBs于载玻片和培养板,分别检测EBs来源细胞的糖原储存和对靛青绿(ICG)、荧光素标记低密度脂蛋白(DiI-Ac-LDL)的摄取能力.结果 在生物反应器旋转培养的第5、10 d,未从培养液中检出特异的鼠白蛋白,而在第15 d的培养液中检出一定量的鼠白蛋白.与原代培养的成熟肝细胞一样,转种的EBs来源细胞periodic acid-Schiff(PAS)糖原染色阳性,ICG摄取试验阳性,DiI-Ac-LDL摄取试验阳性.未分化的ES细胞均呈阴性.结论 微重力生物反应器不仅能加快EBs的形成与肝细胞分化,而且能促进分化肝细胞的成熟.     

骨形态发生蛋白2在骨髓间充质干细胞向肝细胞分化中的作用

目的：观察骨形态发生蛋白2（BMP2）在骨髓间充质干细胞（BMSCs）向肝细胞分化中的作用。方法采用贴壁法分离培养大鼠股骨BMSCs ,将体外扩增的第3代BMSCs制作细胞爬片,并行肝细胞定向诱导。根据诱导因子不同分为：肝细胞生长因子（ HGF）组、HGF＋BMP2组、BMP2组及空白对照组。培养10 d左右收集细胞,观察各组细胞形态的变化,并采用ELISA法检测培养液上清中肝细胞特异性标志物甲胎蛋白（ AFP）、白蛋白（ ALB）,免疫细胞化学法检测诱导分化后细胞CK-18的表达。结果 HGF组和HGF＋BMP2组可检测到ALB、AFP及CK-18,且HGF＋BMP2组ALB、AFP及CK-18明显高于HGF组（P均＜0．05）。结论 BMP2不能单独诱导BMSCs向肝细胞分化,但能增强HGF诱导BMSCs向肝细胞分化的作用。     

体外诱导人脐血间充质干细胞向肝细胞样细胞分化的研究

目的 探讨人脐血间充质干细胞能否在体外诱导分化为肝细胞样细胞,并探索其定向诱导分化为肝细胞样细胞的分化机制。方法 无菌条件下采集正常产妇脐血,用相对密度为1.077的淋巴细胞分离液分离脐血MNC,进而采用贴壁培养法获得MSC,流式细胞仪检测其表面际志。分别用HGF、FGF4、俩者联合、无生长因子四种处理因素,及含2％FBS的DMEM,1×ITS讲行诱导培养,并于诱导前及诱导后的第7、14、21、28天留取细胞,RT～PCR法检测AFP、白蛋白及C-met FGFR2mRNA的表达,免疫细胞化学法检测AFP、白蛋白、抗肝细胞抗体和CK18的表达,PAS法进行糖原染色,分析是否诱导出肝细胞样细胞。结果 MSC强表达CD29、CD44,不表达CD34。诱导后7,21天分别检测出AFP,白蛋白mRNA及蛋白的表达,28天检测出CK18、抗肝细胞抗体的表达,21天、28天均可检测到糖原染色阳性细胞,随诱导时间的延长C-met,FGFR2 mRNA表达增高,HGF FGF4诱导组肝系细胞标志阳性率均高于单独生长因子诱导组(P<0.05);单独生长因子诱导组间无明显差异(P>0.05)。无生长因子诱导组在以上时间点均未检测到上述指标。结论HGF、FGF4均能诱导人脐血MSC分化为具有肝系细胞表型和功能的细胞,且二者在一定程度上有联合作用。生长因子与其相幢受体之间,可能存在正反馈调节机制。     

含淤胆血清的培养体系体外诱导胚胎干细胞表达肝细胞功能的研究

目的探讨病理微环境体外诱导对小鼠胚胎干细胞(ESC)表达肝细胞功能的作用。方法悬滴培养ESC发育5～7d的拟胚体，将其离散细胞种植于不同的分化体系，观察ESC在自主分化、肝细胞生长因子(HGF)或5％淤胆血清 HGF诱导下每天的分化情况(倒置相差显微镜)，以及细胞的糖原、甘油三酯、白蛋白及尿素氮合成功能，细胞吲哚氰绿(ICG)和荧光二乙酯(FDA)染色的情况。结果ESC自主分化难以控制，分化为3个胚层的细胞。HGF促进ESC向内脏内胚层和中胚层(心肌)分化，但两者仅能表达低水平的肝细胞特异性功能。引入淤胆血清的HGF诱导体系中ESC能分化为较为均一的多角形细胞，具有较高水平的糖原、甘油三酯、白蛋白和尿素氮合成能力，ICG和FDA染色阳性。结论自主分化和HGF诱导ESC表达肝细胞代谢的能力有限。体外模拟病理性微环境可诱导ESC定向分化为肝系细胞，并具有较高水平的肝细胞代谢功能。     

低浓度补肾中药血清联合骨形态发生蛋白-2对人脐血间充质干细胞的生物学作用

目的观察低浓度补肾中药血清联合骨形态发生蛋白(BMP)-2对人脐血间充质干细胞(MSCs)体外增殖、成骨分化的生物学作用。方法取正常孕妇脐静脉血培养第3代人脐血MSCs,制备补肾中药大鼠血清,按诱导分化培养基不同,随机分为空白对照组、中药血清组、BMP-2组、中药血清+BMP-2组(联合组)。以MTT比色法检测脐血MSCs增殖活性,观察碱性磷酸酶(ALP)活性、矿化结节及Ⅰ型胶原染色以检测细胞成骨分化能力。结果与空白对照组比较,联合组、BMP-2组在诱导第3、5天细胞增殖活性明显提高,在诱导第3、6、9、12天ALP活性明显提高(P0.05)。与BMP-2组比较,中药血清+BMP-2组在诱导第5天增殖活性明显提高,在诱导第6天ALP活性明显提高(P0.05)。成骨诱导第14天,除空白对照组外各组von Kossa染色均可见细胞间散在黑色颗粒,尤以联合组为明显;而Ⅰ型胶原纤维表达,BMP-2组、联合组与空白对照组相比均明显增强。结论低浓度补肾中药血清联合BMP-2后能诱导人脐血MSCs增殖及定向成骨分化,是复合型诱导因子的一种理想选择。     

肝脏细胞条件培养基诱导大鼠骨髓间质细胞分化为肝细胞的作用

目的:探讨肝脏细胞条件培养基诱导大鼠骨髓间质细胞分化为肝细胞的作用.方法:从大鼠骨髓中分离纯化培养间质细胞,诱导前24小时加1μg/L碱性成纤维生长因子(bFGF)入培养液中以促进细胞分裂,再以肝脏细胞条件培养基作诱导剂,分别在诱导培养0、7、14、21、28天时,留取细胞,观察细胞形态的变化,并采用免疫细胞化学方法检测肝细胞功能标志物(AFP、白蛋白、CK18)、用PAS法进行糖元染色试验,以验证诱导分化的结果.结果:诱导后3天间质细胞表现为肝细胞样,随着诱导时间的延长,肝细胞功能标志逐渐出现和成熟.AFP在7、14天时表达较高,21、28天时表达显著减少;白蛋白、CK18和糖元随着诱导时间的延长表达逐渐增多.结论:肝脏细胞条件培养基能诱导骨髓间质细胞分化为肝细胞.     

体外诱导小鼠骨髓间充质干细胞分化为肝样细胞

目的:体外诱导小鼠骨髓间充质干细胞分化(BMSCs)为肝样细胞,提高分化效率.方法:将第一代BMSCs随机分为诱导组和对照组.诱导组加肝细胞生长因子、成纤维生长因子、表皮生长因子、抑瘤素M等进行诱导培养,观察细胞形态,检测甲胎蛋白(AFP)、白蛋白(A1b)、细胞角蛋白18(CK18)、酪氨酸氨基转移酶(TAT)和细胞色素P450 2b9(CYP2b9)的mRNA表达,A1b合成以及A1b和CK18蛋白标记细胞阳性率.结果:诱导组第3天出现多边形细胞,5—7d上皮样细胞呈岛状分布,14 d呈铺路石状.对照组细胞为长梭形.第7,14,21天,诱导组细胞AFP,A1b,CK18 mRNA和A1b蛋白检测阳性;第14,21天,细胞表达TAT和CYP2b9 mRNA.对照组除AFP mRNA呈弱阳性外,其余均为阴性.第7,14,21天,诱导组CK18阳性率分别为71.4%.75.9%,80.6%:A1b阳性率分别为75.0%,79.7%.81.1%.而对照组第7天CK18和A1b阳性率仅2.3%,1.7%,与诱导组相比有显著差异(P_(CK18)=1.97×10~(-5),P_(A11b)=3.08×10~(-6)).结论:BMSCs在体外可以被诱导分化为肝样细胞,诱导率最高可达80%以上.     

人骨髓单个核细胞向肝细胞诱导分化的体外研究

目的 探讨在HGF、FGF_4诱导下骨髓单个核细胞能否向肝细胞分化。方法 找健康忐愿者,年龄4～50岁,胸骨抽取骨髓,用淋巴分离液分离出骨髓单个核细胞,用含2％胎牛血清和1×ITS的DMEM培养,分别用单独HGF、单独FGF_4、HGF和FGF_4联合刺激、无生长因子4种处理因素进行诱导培养,分别于诱导培养0、7、14、21、28天时,留取细胞,用免疫细胞化学法检测CK18、AFP、白蛋白,用PAS法进行糖元染色试验以验证诱导分化的结果。结果 0天时,AFP、白蛋白少数细胞阴性,CK18阴性,糖元未见阳性;加生长因子的三组AFP在7、14天时表达逐渐增高,21、28天减弱;糖元、CK18、白蛋白表达逐渐增高。无生长因子组,在7、14、21、28天时,这此指标均未见表达。结论 在HGF、FGR_4诱导下,骨髓单个核细胞可分化为具肝细胞表型和功能的细胞。     

骨形态发生蛋白7诱导骨髓间充质细胞向软骨细胞分化的作用

目的 观察转染骨形态发生蛋白7(BMP7)基因的骨髓间充质细胞(BMSCs)所表达的BMP7蛋白在体外诱导该细胞向软骨细胞方向分化的作用.方法 分别取诱导液诱导的BMSCs(诱导组)、转染BMP7基因的BMSCs(转染组)以及未处理的BMSCs(对照组),在6孔板中细胞爬片,经HE、阿尔新蓝染色后,在倒置显微镜下观察形态.3组BMSCs培养7、14、21 d后,以半乳糖醛酸为对照品绘制标准曲线法测定培养液中糖胺多糖(GAG),ELISA法测定胶原蛋白Ⅱ.结果 HE、阿尔新蓝染色后,转染组和诱导组BMSCs分别呈红色和蓝色,具有软骨细胞特性.BMP7诱导后的细胞团表达了GAG和胶原蛋白Ⅱ.第21天,转染组GAG为(39.5±5.4)mg/L,胶原蛋白Ⅱ为(152.8±14.5)μg/L;诱导组GAG为(40.8±6.1)mg/L,胶原蛋白Ⅱ为(155.5±19.3)μg/L;对照组GAG为(17.1±3.4)mg/L,胶原蛋白Ⅱ为(89.7±14.3)μg/L;与对照组比较,转染组与诱导组GAG、胶原蛋白Ⅱ水平明显增高(P均<0.05),转染组与诱导组比较,则未见明显改变(P>0.05).结论 转染BMP7基因的BMSCs表达的活性BMP7能够诱导BMSCs向软骨细胞方向分化,且其诱导水平达到了诱导液的诱导水平.     

Analysis of the temporal and concentration-dependent effects of BMP-4, VEGF, and TPO on development of embryonic stem cell-derived mesoderm and blood progenitors in a defined, serum-free media

OBJECTIVE: To develop a robust serum-free (SF) system for generation of hemogenic mesoderm and blood progenitors from pluripotent cells. MATERIALS AND METHODS: Embryonic stem cells (ESCs) maintained in N2B27 supplemented with leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP)-4 were induced to differentiate into Brachyrury/T-expressing cells (measured using a green fluorescent protein reporter) and myeloid-erythroid colony-forming cells (ME-CFCs), by removing LIF, changing the base media formulation, and via the time- and concentration-dependent addition of other factors. RESULTS: Presence of 10 ng/mL BMP-4 permitted the emergence of cells expressing T and the vascular endothelial growth factor receptor (VEGFR)-2, however, <5% of the cells were double-positive on day 4. Adjusting the SF media formulation allowed only 5 ng/mL BMP-4 to yield 24% +/- 4% Brachyury-green fluorescent protein VEGFR-2(+) cells by day 4. These cells could develop into ME-CFC, producing 4.4 +/- 0.8 CFC per 1000 cells at day 8. We also examined the timing and concentration sensitivity of BMP-4, VEGF, and thrombopoietin (TPO) during differentiation. BMP-4 with 50 ng/mL TPO generated 232 +/- 48 CFC per 5 x 10(4) cells, similar to the serum-control, and this response could be enhanced to 292 +/- 42 CFC per 5 x 10(4) cells by early (between day 0-5), but not late (after day 5) VEGF treatment. CONCLUSION: Moving to SF systems facilitates directed differentiation by eliminating confounding signals. This article describes modifications to the N2B27 media that amplify mesoderm induction and extends earlier work defining blood progenitor cell induction from ESC with BMP-4, VEGF, and TPO.     

Alterations in Epithelial and Mesenchymal Intestinal Gene Expression During Doxorubicin-Induced Mucositis in Mice

In the current study we aimed to gain insight into epithelial-mesenchymal cross-talk and progenitor compartment modulation during doxorubicin (DOX)-induced mucositis in mice. Intestinal segments were collected on various days after DOX treatment. DOX-induced damage at day 1–2 was characterized by increased epithelial proliferation and apoptosis and a decrease in the expression of epithelial differentiation markers. Concurrently, T-cell factor-4 (TCF4) levels increased and the epithelial differentiation enhancing factor, bone morphogenic protein-4 (BMP4), decreased. During severe damage (day 3), BMP4 levels were significantly increased, which inversely correlated with epithelial proliferation. At the same time, the expression of the epithelial differentiation markers was increasing again. At day 7, BMP4 levels were down-regulated, while the levels of the epithelial differentiation markers and TCF4 were normalized again. These data suggest that in response to DOX-induced damage, BMP4 and TCF4 are modulated in such a way that homeostasis of the progenitor compartment is partly preserved.     

PI3K／Akt信号通路在骨髓间充质干细胞增殖及成骨分化调控中的作用

目的：研究PI3K/Akt信号通路抑制剂LY294002对骨髓间充质干细胞（mesenchymal stem cells,MSCs）增殖及分化的影响。方法采用贴壁法体外分离人骨髓间充质干细胞（hMSCs）,加入PI3K抑制剂LY294002（1、10μmol/L）,应用MTT法测定细胞增殖,常规成骨诱导分化培养3或7d,采用碱性磷酸酶（ALP）染色观察成骨分化水平,化学比色法测定ALP活性,茜素红染色后观察矿化钙结节数量并定量分析,Westernblot检测磷酸化Akt蛋白表达,应用Realtime-PCR检测各组细胞BMP2、Runx2、OPN及Osterix等成骨分化标记物的基因表达水平。结果从24至72h,LY294002对hMSCs增殖均产生显著抑制,随时间推延,可见抑制增殖效果增强（P＜0.05）。ALP染色和定量测定提示10μmol/L的ALP活性最强,在不同时间显著高于对照组和1μmol/L组（P＜0.05）。成骨诱导培养3和7d,1、10μmol/L组矿化量都显著高于对照组（P＜0.05）。10μmol/L组矿化量在成骨诱导7d也显著高于1μmol/L组（P＜0.05）。Westernblot检测结果证实成骨诱导可激活Akt磷酸化蛋白表达,但LY294002可抑制该蛋白磷酸化。成骨诱导分化7d,1、10μmol/L均明显促进BMP2、Runx2、OPN、Osterix4种基因mRNA表达（均P＜0.05）。结论PI3K/Akt信号通路参与hMSCs增殖和分化过程。成骨分化伴随下游Akt蛋白表达。PI3K抑制剂可抑制hMSCs增殖,但同时促进其向成骨分化和矿化。     

Erythroid progenitor cells and stimulating factors during murine embryonic and fetal development

Murine embryonic and fetal yolk-sacs, peripheral blood, and livers were assayed for hemopoietic multipotential and progenitor cell content between days 6 and 13 of gestation. Multipotential cells (Mix-CFC), erythroid-committed progenitor cells (BFU-E), and nonerythroid progenitor cells (predominantly GM-CFC) were assayed by their ability to form hemopoietic colonies in vitro when stimulated by pokeweed-mitogen-stimulated spleen-cell-conditioned media (as a source of Multi-CSF) and either human or murine erythropoietin. Late erythroid progenitor cells (CFU-E) were stimulated to form colonies by erythropoietin. Mix-CFC, BFU-E, and nonerythroid cells were first detected on day 8 in yolk-sacs, day 9 in peripheral blood, and day 11 in liver. Maximum absolute numbers of yolk-sac Mix-CFC (182), BFU-E (331), and non-erythroid CFC (1358) occurred at 11 days of gestation. The maximum frequency of peripheral blood mix-CFC (24/10(5) cells) and BFU-E (55/10(5) cells) occurred at ten days of gestation. The absolute numbers of hepatic Mix-CFC, BFU-E, nonerythroid CFC, and CFU-E increased exponentially from 11 to 13 days' gestation. CFU-E were first detected at nine days in peripheral blood, at ten days in yolk-sac, and 11 days in liver and at all ages were equally responsive to erythropoietin. The maximum frequency (151/10(5) cells) of CFU-E in the peripheral blood and the maximum number per yolk-sac (1699) both occurred on day 11 of gestation. In confirmation of previous studies, yolk-sac fluid was found to contain a macrophage colony-stimulating activity. In addition, an activity capable of stimulating fetal liver CFU-E was also detected in yolk-sac fluid. However, no activity (Multi-CSF) capable of stimulating Mix-CFC or BFU-E was detected in either yolk-sac fluid or fetal plasma.     

Melatonin promotes hepatic differentiation of human dental pulp stem cells: clinical implications for the prevention of liver fibrosis

Melatonin's effect on hepatic differentiation of stem cells remains unclear. The aim of this study was to investigate the action of melatonin on hepatic differentiation as well as its related signaling pathways of human dental pulp stem cells (hDPSCs) and to examine the therapeutic effects of a combination of melatonin and hDPSC transplantation on carbon tetrachloride (CCl₄)‐induced liver fibrosis in mice. In vitro hepatic differentiation was assessed by periodic acid‐Schiff (PAS) staining and mRNA expression for hepatocyte markers. Liver fibrosis model was established by injecting 0.5 mL/kg CCl₄ followed by treatment with melatonin (5 mg/kg, twice a week) and hDPSCs. In vivo therapeutic effects were evaluated by histopathology and by means of liver function tests including measurement of alanine transaminase (ALT), aspartate transaminase (AST), and ammonia levels. Melatonin promoted hepatic differentiation based on mRNA expression of differentiation markers and PAS‐stained glycogen‐laden cells. In addition, melatonin increased bone morphogenic protein (BMP)‐2 expression and Smad1/5/8 phosphorylation, which was blocked by the BMP antagonist noggin. Furthermore, melatonin activated p38, extracellular signal‐regulated kinase (ERK), and nuclear factor‐κB (NF‐κB) in hDPSCs. Melatonin‐induced hepatic differentiation was attenuated by inhibitors of BMP, p38, ERK, and NF‐κB. Compared to treatment of CCl₄‐injured mice with either melatonin or hDPSC transplantation alone, the combination of melatonin and hDPSC significantly suppressed liver fibrosis and restored ALT, AST, and ammonia levels. For the first time, this study demonstrates that melatonin promotes hepatic differentiation of hDPSCs by modulating the BMP, p38, ERK, and NF‐κB pathway. Combined treatment of grafted hDPSCs and melatonin could be a viable approach for the treatment of liver cirrhosis.     

肝细胞条件培养液对人脂肪问充质干细胞向肝细胞分化和增殖的作用

目的 探讨人脂肪间充质干细胞在改良的诱导体系下向肝细胞的分化和增殖情况,为肝组织工程提供新的种子细胞来源.方法 从人脂肪组织分离出脂肪间充质干细胞,用含有碱性成纤维细胞生长因子、肝细胞生长因子、成纤维细胞生长因子-4的肝细胞诱导液进行诱导,并于诱导7 d后加入抑瘤素M.用细胞计数试剂盒-8法检测整个诱导过程细胞的增殖情况;通过光学显微镜观察诱导细胞的形态变化;用RT-PCR法和免疫荧光法分别检测肝细胞特异性基因和蛋白的表达;并对多种肝细胞特异性功能进行检测.组间比较采用t-test检验.结果 用改良肝细胞诱导液培养的人脂肪间充质干细胞在培养第5、7、14、21天时,细胞数均明显多于用对照培养液培养的细胞(f值分别为6.59、8.69、15.94和24.64,P值均＜0.05).诱导细胞表现出上皮样肝细胞形态,表达肝细胞特异性基因和蛋白;具有多种肝细胞特异性功能,如靛青绿摄取/排泌、糖原合成以及白蛋白分泌功能.结论 人脂肪间充质干细胞在含碱性成纤维细胞生长因子、肝细胞生长因子、成纤维细胞生长因子-4和抑瘤索M的诱导体系中能够分化为更加成熟的具有多种肝细胞特异性功能的细胞,且此诱导体系同时具有促进细胞增殖的作用.     

BMP-4 induced proliferation and oriented differentiation of rat hepatic oval cells into hepatocytes

Objective:To explore the role of bone morphogenetic protein 4(BMP-4) in hepatic progenitor cells(HPCs).Methods:The effect of BMP-4 on rat hepatic oval cells was examined by using the WB-F344 rat hepatocytic epithelial stem-cell-like cell line.This hepatocytic cell line could exert various hepatocytc functions including the secretion of albumin and urea.Immunohistochemistry was used to examine the effects of BMP-4 and its antagonist,Noggin,on the proliferation and differentiation of these cells,cellular uptake and excretion of indocyanine green,the periodic acid-schiff(PAS) assay for glycogen storage and the expression of hepatic markers.Results:Our results showed for the first time that BMP-4 may acted as a potential inducer of hepatic differentiation in rat hepatic oval cells.Conclusions:This cell source offers a much-needed attractive and expandable source for future investigations of drug screening,stem cell technologies and cellular transplantation,in a society with increasing levels of liver disease and damage.