Vitamin A/Retinol and Maintenance of Pluripotency of Stem Cells

Retinol, the alcohol form of vitamin A is a key dietary component that plays a critical role in vertebrate development, cell differentiation, reproduction, vision and immune system. Natural and synthetic analogs of retinol, called retinoids, have generally been associated with the cell differentiation via retinoic acid which is the most potent metabolite of retinol. However, a direct function of retinol has not been fully investigated. New evidence has now emerged that retinol supports the self-renewal of stem cells including embryonic stem cells (ESCs), germ line stem cells (GSCs) and cancer stem cells (CSCs) by activating the endogenous machinery for self-renewal by a retinoic acid independent mechanism. The studies have also revealed that stem cells do not contain enzymes that are responsible for metabolizing retinol into retinoic acid. This new function of retinol may have important implications for stem cell biology which can be exploited for quantitative production of pure population of pluripotent stem cells for regenerative medicine as well as clinical applications for cancer therapeutics.     

全反式视黄酸在骨髓间充质干细胞向神经元分化中的作用研究

目的:探讨全反式视黄酸(ATRA)在骨髓间充质干细胞(MSCs)向神经元分化中的作用及机制。方法:大鼠MSCs在体外培养5～7代后,用0.5μmol/L的ATRA预诱导24h,再换用改良的神经细胞培养基(MNM)作用18h;对照组不用ATRA预诱导,直接用MNM培养。免疫组化检测不同组别巢蛋白(nestin)、神经元特异性烯醇化酶(NSE)、神经元特异性核心抗原(NeuN)和微管相关蛋白-2(MAP-2)的表达情况;ATRA作用前后检测细胞增殖周期和视黄酸受体β(RARβ)的变化。结果:1.ATRA预诱导组巢蛋白、NSE、NeuN和MAP-2的阳性比例明显高于对照组。2.ATRA作用前后,MSCs细胞增殖周期无明显改变。3.MSCs不表达RARβ,用ATRA预诱导细胞后,RARβ表达增加,阳性比例为(20.3±4.2)%。结论:ATRA可能通过RARβ介导靶基因的转录,促进MSCs向神经元分化。     

三氧化二砷联合全反式维甲酸对人卵巢癌细胞的抑制作用

目的:探讨三氧化二砷(AS2O3)联合全反式维甲酸(ATRA)对人浆液性卵巢癌细胞株SKOV3的抑制作用及其机制。方法;四甲基偶氮唑蓝光吸收法(MTT)、流式细胞术及免疫荧光法检测体外培养的SKOV3细胞在AS2O3单独作用及联合ATRA作用下对卵巢癌细胞生长的抑制及促凋亡作用。结果:单用ATRA对SKOV3细胞的生长无影响;单用AS2O3可以抑制SKOV3细胞的生长;联合使用AS2O3和ATRA较单用AS2O3对SKOV3细胞的抑制作用强(P<0.05);流式细胞术证实:5.0μmol/LAS2O3联合1.0μmol/LATRA作用48h时,SKOV3细胞出现G2/M期阻滞增强、S期减低(P<0.05);AnnffxinV染色检测到早期凋亡细胞增多,晚期凋亡及继发性坏死细胞减低(P<0.05)。结论:AS2O3能上调SKOV3细胞中细胞周期抑制蛋白P27的表达,ATRA联合AS2O3可以增强上述效应。故ATRA对诱导SKOV3细胞凋亡有增敏效应,该效应过程可能依赖于细胞周期负性调控因子P27蛋白的过度表达。     

Retinoids induce differentiation of type A spermatogonia in vitro: organ culture of mouse cryptorchid testes

To study the effects of retinoids on testicular germ cell differentiation, especially on stem cells, artificially induced cryptorchid testes of adult mice were cultured in vitro inasmuch as they contain only stem cells, type A spermatogonia. We report here that retinol, retinal, retinol acetate, even retinoic acid, activated cell division in type A spermatogonia and stimulated them to differentiate. These findings are the first demonstration of the in vitro induction of early stage of spermatogenesis by retinoids and also retinoic acid, which is considered to be a biochemically inactive compound for mammalian testicular function.     

视黄酸受体基因在小儿淋巴结的表达与B细胞的发育

目的:研究视黄酸受体基因在小儿淋巴结的表达与B细胞发育的关系,阐明视黄酸促进抗体产生的途径与机制。方法:取5岁以下儿童正常淋巴结作冰冻切片,用地高辛素标记六种视黄酸受体基因(RARα、β、γ、RXRα、β、γ)反义RNA探针作原位杂交,及采用RT-荧光定量PCR,观察视黄酸受体基因在淋巴结的表达与分布,分析其与B细胞发育的关系。结果:原位杂交结果显示:六种视黄酸受体基因在淋巴结中的淋巴细胞和网状细胞中均有表达,分布广泛。RT-荧光定量PCR显示:在不同年龄儿童淋巴结六种视黄酸受体基因表达水平有所差异,1岁以下儿童受体基因表达水平普遍较低,此后随免疫发育而升高。结论:视黄酸受体基因的表达可能参与了B细胞的个体发育和免疫应答。这可能是维生素A增强儿童抗感染免疫力的重要环节和介导视黄酸作用的主要途径。     

两种维甲类药物协同诱导白血病细胞HOXA1基因的表达

本文研究了全反式维甲酸和９－顺维甲酸对ＨＬ－６０细胞增殖及凋亡的影响,并用半定量ＲＴ－ＰＣＲ技术检测了人髓系白血病细胞株ＨＬ－６０细胞中同源盒ＨＯＸＡ１基因的表达,结果表明,全反式到及９－顺到均能抑制ＨＬ－６０细胞增殖并能促进其凋亡,这咱效应在两药联用时更为明显,根据上述结果,我们认为两药联用的抗癌机制之一可能是通过影响ＨＯＸＡ１基因表达的网络调节系统来实现,但在不同的白血病中,其影响程度可能有所     

二十碳五烯酸联合视黄酸对HL-60细胞株N-ras基因表达的影响

目的 :　研究二十碳五烯酸 (Eicosapentaenoicacid,EPA )是否协同视黄酸 (retinoicacid,RA)影响 HL-60细胞的增殖与分化功能及相应分子机制。方法 :　流式细胞仪 (FCM)测定细胞增殖功能 ;NBT还原实验鉴定 HL-60细胞分化 ;Western blot法分析 p2 1 N- ras表达。结果 :　EPA和 RA联合应用能增强 HL-60细胞的增殖抑制和分化效应 ,同时细胞内 p2 1 N- ras表达降低。结论 :　 EPA和 RA可能通过下调节 N-ras基因的蛋白质表达 ,发挥它们对 HL-60细胞增殖与分化功能的联合效应     

Retinoic acid induces Gpx2 gene expression in MCF-7 human breast cancer cells.

We showed previously that the selenium-dependent glutathione peroxidase, GPX-GI, encoded by the Gpx2 gene, is highly expressed in the epithelium of the gastrointestinal (GI) tract and sporadically in breast tissue. To investigate whether Gpx2 gene expression is epithelium specific, we used in situ hybridization to show that Gpx2 mRNA is highly expressed in the crypt epithelium of human intestine. We also used Northern analysis to study human breast cells and found Gpx2 mRNA in human mammary epithelial cell lines as well as freshly isolated normal breast epithelial cells. Because we identified three putative retinoic acid response elements (RARE) in the Gpx2 gene, we examined the regulation of the Gpx2 gene expression by all-trans retinoic acid (RA) in RA-sensitive MCF-7 cells and RA-resistant HT29 cells. Without RA, MCF-7 cells had very low levels of Gpx2 mRNA and a low level of glutathione peroxidase (GPX) activity (17 mU/mg protein), whereas HT29 cells had a high level of Gpx2 mRNA and GPX activity (200 mU/mg protein). RA treatment increased Gpx2 mRNA level 3- to 11-fold and resulted in a fourfold increase of GPX activity (80 mU/mg protein) in MCF-7 cells. Neither Gpx2 mRNA level nor GPX activity was increased in HT29 cells. These results show that the Gpx2 gene is expressed in both breast and intestinal epithelium cells, and suggest that its expression can be highly regulated by retinoic acid, a known differentiation agent.     

苯乙基异硫氰酸盐对小鼠胚胎干细胞分化为神经细胞过程中基因表达影响的研究

目的探讨苯乙基异硫氰酸盐(PEITC)对小鼠胚胎干细胞(mESCs)体外定向诱导分化为神经样细胞中PKCα及其相关发育基因(pax-6、netrin-1)表达的影响。方法mESCs经RA法诱导后,第7天用RT-PCR法检测nestin、glutaminase、Brn-3基因等神经细胞特异性标志物。不同浓度PEITC作用条件下,分别取诱导后1、3、5、7及14天后的细胞,用RT-PCR方法测定细胞的PKCα及其相关发育基因pax-6和netrin-1的mRNA表达情况。结果诱导后第7天,分化的神经样细胞中nestin、glutaminase、Brn-3基因均高表达。正常诱导1天后,PKCα、pax-6和netrin-1基因的mRNA表达急剧下降,3、5、7天逐渐升高,至14天恢复至正常水平。PEITC作用下,PKCα、pax-6、netrin-1的mRNA在诱导后细胞中各阶段的表达水平与正常诱导条件下相比均下降,且随着作用浓度和作用时间的增加,下降趋势明显。结论PEITC对mESCs定向分化为神经样细胞过程中相关发育基因(pax-6、netrin-1)的表达的干扰作用可能与抑制PKCα的表达有关。     

Differentiation of stem cells upon deprivation of exogenous FGF2: a general approach to study spontaneous differentiation of hESCs in vitro

Establishing a model for in vitro differentiation of human embryonic stem cells (hESCs) towards the germ cell lineage could be used to identify molecular mechanisms behind germ cell differentiation that may help in understanding human infertility. Here, we evaluate whether a lack of exogenous fibroblast growth factor 2 (FGF2) is supporting spontaneous differentiation of hESCs cultured on human foreskin fibroblast (hFF) monolayers towards germ cell lineage. Additionally to depriving the hESCs of exogenous FGF2, cells were stimulated with all-trans retinoic acid (ATRA). To get a more comprehensive impression on effects of removal of FGF2 and stimulation with ATRA, we combined the results of three cell lines for each experimental setting. When combining gene expression profiles of three cell lines for 96 genes, only 6 genes showed a significant up-regulation in all cell lines, when no FGF2 was added to the media for 12 weeks. None of these genes are related to the germ lineage, whereas genes for neuronal cells (PAX6 and NR6A1) and endothelial cells (FLT-1 and PTF1A) were up-regulated. To induce and support the differentiation towards the germ lineage we stimulated hESCs with different concentrations of ATRA for 7 and 14 days. We observed no significant difference in gene expression on RNA level when combining all cell lines. Whereas, the overall outcome was negative, one of these cell lines demonstrated an up-regulation of DDX4 on RNA and protein level after 7 days of ATRA stimulation. In summary, our data showed that the lack of exogenous FGF2 results in up-regulation of genes crucial for neuronal and endothelial cell differentiation of hESCs, but not in the up-regulation of genes related to germ cell differentiation when cultured on hFFs. Additionally, we demonstrated that ATRA supplementation did not result in a general specific direction of hESCs towards the germ lineage.     

Carnosic acid inhibits proliferation and augments differentiation of human leukemic cells induced by 1,25-dihydroxyvitamin D3 and retinoic acid

Carnosic acid, the polyphenolic diterpene derived from rosemary, is a strong dietary antioxidant that exhibits antimutagenic properties in bacteria and anticarcinogenic activity in various cell and animal models. In the present study, we show that carnosic acid (2.5-10 microM) inhibits proliferation of HL-60 and U937 human myeloid leukemia cells (half-maximal inhibitory concentration = 6-7 microM) without induction of apoptotic or necrotic cell death. Growth arrest occurred concomitantly with a transient cell cycle block in the G1 phase, which was accompanied by an increase in the immunodetectable levels of the universal cyclin-dependent kinase inhibitors p21WAFI and p27Kipl. Carnosic acid caused only a marginal induction of differentiation, as monitored by the capacity to generate superoxide radicals and the expression of cell surface antigens (CD11b and CD14) and receptors for the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine. However, at low concentrations, this polyphenol substantially augmented (100- to 1,000-fold) the differentiating effects of 1,25-dihydroxyvitamin D3 and all-trans retinoic acid. Furthermore, such combinations of carnosic acid and any of these differentiation inducers synergistically inhibited proliferation and cell cycle progression. These results indicate that carnosic acid is capable of antiproliferative action in leukemic cells and can cooperate with other natural anticancer compounds in growth-inhibitory and differentiating effects.     

三邻甲苯基磷酸酯对人成神经瘤SH-SY5Y细胞分化的影响

目的观察三邻甲苯基磷酸酯（Tri-ortho-cresyl phosphate,TOCP）对人成神经瘤SH-SY5Y细胞形态分化的影响,为更全面的了解TOCP的毒性提供依据。方法选用人成神经瘤细胞SH-SY5Y为细胞模型,以全反式维甲酸（ATRA）为诱导分化工具药物,在诱导中以及诱导后以不同浓度TOCP作用,动态观察SH-SY5Y细胞形态学变化,台盼蓝拒染法检测死亡细胞百分比。结果 ATRA对SH-SY5Y细胞作用7 d可导致细胞出现长出较长突起的分化特征,在诱导分化过程中及诱导分化后以不同剂量TOCP作用于细胞,各剂量组与对照组相比,无论是突起长度还是分化细胞比例差异均有统计学意义（P〈0.05）,其中,0.6 mmol/L组抑制效果最强。结论 TOCP可抑制SH-SY5Y细胞的分化,并呈现一定时间和剂量依赖关系。     

Dexamethasone induces sodium-dependant vitamin C transporter in a mouse osteoblastic cell line MC3T3-E1

The regulation of intracellular ascorbic acid (AsA) levels may be under the control of an AsA-specific membrane transporter. The present study investigates AsA uptake and expression of Na-dependent vitamin C transporter (SVCT) mRNA in the mouse osteoblastic cell line, MC3T3-E1. Among eight compounds tested, dexamethasone (Dex) all-trans retinoic acid, transforming growth factor beta, prostaglandin E2 and transferrin significantly and respectively) stimulated the update of AsA into MC3T3-E1 cells. Among these five, Dex was the most active, inducing mSVCT2 mRNA and the uptake of AsA in a time- and concentration-dependant manner. Dex did not induce mSVCT1 mRNA. These results suggest that the Dex-induced stimulation of AsA incorporation into osteoblastic cells is mediated by the induction of mSVCT2. Since Dex reduced alkaline phosphatase activity in MC3T3-E1 cells in our culture conditions, Dex-induced stimulation of AsA incorporation might not be the result of differentiation. Hormone-regulated changes of SVCT expression may have an important role in cell functions.     

视黄酸对幼儿淋巴结B细胞发育的影响

目的:研究视黄酸在B细胞分化发育中的作用,探讨维生素A增加抗体产生的机制。方法:取幼儿正常淋巴结细胞体外培养,加入视黄酸或视黄酸受体拮抗剂干预前后,采用流式细胞术检测分析细胞表面标志的变化,观察B细胞的成熟分化。结果:体外培养的幼儿淋巴结B细胞在无刺激剂存在时,CD19+IgM+的成熟B细胞百分比随培养时间逐渐增加,而相对不成熟的CD19+IgM-B细胞百分比逐渐减少,以48h明显(P<0.05);体外培养加入视黄酸可使CD19+IgM+B细胞百分比进一步升高,在培养24h和48h均显著高于对照组(P均<0.05);同时加入视黄酸拮抗剂则能完全拮抗该促进作用。结论:视黄酸通过视黄酸受体介导促进体外培养的淋巴结B细胞的分化发育。     

基因Ercc6l在P19胚胎癌细胞内的表达研究

[目的]探讨基因Ercc6l(excision repair cross-complementing rodent repair deficiency,complementation group6-like)在P19胚胎癌细胞分化过程中的表达情况。[方法]用二甲亚砜(DMSO)和视黄酸(RA)分别诱导P19细胞向心肌细胞方向和神经细胞方向分化,RT-PCR检测P19细胞诱导分化过程中Ercc6l mRNA的表达变化。[结果]RT-PCR检测表明,基因Ercc6l在未分化的P19细胞中不表达,但当P19细胞诱导分化为心肌细胞或神经细胞后,Ercc6l表达显著上调。[结论]Ercc6l在调控胚胎细胞分化和胚胎发育过程中具有重要作用。     

Selective isolation of human breast carcinoma cells resistant to the growth-inhibitory effects of retinol

The anchorage-dependent and anchorage-independent growth of the human mammary carcinoma cell line MDA-MB-231 is inhibited by vitamin A (retinol). Clones resistant to growth inhibition by retinol were isolated from this cell line in soft agar without the use of mutagens. This paper describes the isolation and characterization of the resistant lines. The clones were selectively resistant to retinol. There was significant growth inhibition after treatment with retinoic acid and 13-cis-retinoic acid. The resistant clones maintain their resistance to retinol through multiple passages. Resistance is specific for inhibition of growth, because treatment of the resistant clones results in stimulation of plasminogen activator activity without alteration of proliferation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows no significant qualitative or quantitative difference in the clones when compared with the MDA-MB-231 parent line. Although the clones do not regrow in soft agar, they are tumorigenic in athymic mice. Tumors are produced at a rate similar to the parent line. The advantage of this isolation method is that sensitive and resistant malignant cells derived from the same parent cell line are now available to study the molecular events involved in the inhibition of cellular proliferation after treatment with retinol.     

生长分化因子9对卵巢功能的影响和调控

生长分化因子9(growth differentiation factor 9,GDF-9)是转化生长因子β(transforming growth factor beta,TGF-β)超家族的新成员。作为卵母细胞分泌的因子,GDF-9在女性生殖功能中的作用近年得到广泛讨论。研究发现GDF-9可参与调节颗粒细胞分化发育,相关功能蛋白、因子产生,生殖系统相关基因表达以及卵巢细胞代谢、凋亡等过程,从而在卵巢卵泡生长及卵母细胞成熟、排卵和黄素化等方面发挥重要作用。骨形态发生蛋白15(BMP-15)是一种与GDF-9具有高度氨基酸同源性和相似蛋白结构的卵母细胞源性因子,研究发现GDF-9可与BMP-15形成异源二聚体并发挥协同作用。目前认为,GDF-9是卵母细胞与颗粒细胞完成双向通讯重要的影响因子,参与两种细胞的同步化通讯和协调生长过程,其异常表达可能与女性生殖内分泌失调及随之引发的不孕存在一定关系。本文从GDF-9对卵母细胞发育的影响及在卵泡形成过程中的调控等方面,对其影响和调节卵巢功能的作用进行综述。     

Effect of retinoic acid on ferritin H expression during brain development and neuronal differentiation

We have previously shown that brain ferritin H expression, which has been associated with iron utilization, is developmentally regulated. Because retinoic acid (RA) regulates gene expression and is involved in cellular differentiation, we tested the hypothesis that RA regulates ferritin H during brain development and neuronal differentiation. RA, administered to rats on postnatal day 1, produced a 4-fold increase in brain ferritin H mRNA (p < 0.01) after 24 h. To examine whether RA-stimulated neuronal differentiation contributed to this up-regulation, ferritin and ferritin H mRNA were measured in human neuronal precursor cells (NTera-2, NT2) before and after 4-weeks of RA-stimulated differentiation into post-mitotic neurons. Differentiation resulted in a 2-fold increase in both ferritin and ferritin H mRNA (p < 0.05). Immunocytochemistry and Northern analysis showed significant elevations in ferritin expression that began as early as 24 h after RA treatment. While there was also a significant increase in the labile iron pool after RA treatment, this did not occur until 72 h. These data show that RA regulates ferritin H expression during rat brain development and neuronal differentiation and suggests a new role for RA in brain iron metabolism.     

体外诱导胚胎干细胞分化为神经细胞特性的实验研究

目的研究体外利用全反视黄酸(RA)诱导小鼠胚胎干细胞(mESC)分化为神经细胞的培养方法。方法悬滴3天转悬浮1天两者结合的培养方法得到胚体(EBs),再利用RA对其处理4天后,进行免疫组织化学、RT-PCR以及兴奋性功能的检测,观察神经细胞获得的效率及其生理特性。结果利用RA诱导方法得到的神经细胞,免疫组化表明nestin蛋白表达的阳性率为(53.49±6.02)%,且RT-PCR法分析其能表达nestin、glutaminase和Brn-3等神经细胞特异基因,同时在Glu刺激下具有与脑来源神经元相似的特性和功能。结论利用本实验诱导方法,获得神经细胞的比例较高,且具有与神经元相似的生物学特性。