慢性乙型肝炎患者肝组织和外周血白细胞中钠离子依赖性维生素C转运体的表达

用免疫组化和Western blot技术检测SVCT1和SVCT2蛋白在慢性乙型肝炎患者及正常人外周血白细胞中的表达.免疫组化结果显示：在慢性乙型肝炎患者肝组织中SVCT1和SVCT2蛋白的阳性表达率和阳性表达强度显著低于正常肝组织（P〈0.01）;Western-blot检测显示,慢性乙型肝炎患者外周血白细胞中SVCT1表达缺失,SVCT2蛋白表达强度为0.481±0.056,均显著低于正常人（分别为0.325±0.085和0.971±0.140）（P〈0.01）.提示慢性乙型肝炎患者体内肝细胞和外周血白细胞存在SVCT1和SVCT2蛋白表达低下或缺失;SVCT1和SVCT2蛋白表达低下或缺失可能是乙型肝炎病毒易感性的主要原因之一.     

MCLA为化学发光探针测定氧化低密度脂蛋白中维生素C诱导的单线态氧产生

以一种海萤荧光素类似物MCLA〔2 methyl 6 (p methoxyphenyl) 3,7 dihydroimidazo [1,2 a]pyrazin 3 one〕作为高灵敏且有选择性的化学发光探针 ,用化学发光的方法直接观测到了少量Cu2 氧化的低密度脂蛋白 (Ox LDL)中维生素C诱导的单线态氧 (1O2 )的产生。实验中通过叠氮化钠 (NaN3 )对MCLA介导的化学发光的猝灭作用进一步证实了上述体系中1O2的形成。根据实验观察的结果 ,分析了这一体系中1O2 形成的可能途径 ,认为首先是维生素C将Cu2 转变为还原态 ,而自身失去一个电子转变为维生素C自由基 ,从而刺激了过氧自由基和烷氧自由基的形成 ,过氧自由基的双分子反应很可能就是体系内1O2 产生的反应机制     

维生素C对万古霉素诱导大鼠肾损伤自噬的作用

摘要 目的：探讨维生素C对万古霉素诱导肾损伤自噬水平的影响。方法：将20只雄性SD大鼠随机分为：对照组、万古霉素组、万古霉素+维生素C组和维生素C组。万古霉素组：连续每天腹腔注射400 mg/kg万古霉素;万古霉素+维生素C组：注射万古霉素之前30 min腹腔注射200 mg/kg维生素C;对照组和维生素C组分别单独注射同体积的生理盐水和200 mg/kg维生素C。连续给药7 d后,通过苏木精-伊红染色（HE）观察大鼠肾组织病理损伤;免疫组化和免疫荧光检测肾组织中LC3B和Beclin 1的表达情况,比较各组之间的表达差异。结果：相对于对照组,万古霉素诱导大鼠肾损伤模型组肾组织出现明显的病理改变,包括肾间质水肿,肾小管细胞质空泡性变化,细胞凋亡坏死等;同时观察到肾组织中LC3B光密度明显升高和Beclin 1的荧光强度显著增强。维生素C处理组,肾组织的病理损伤显著改善并且自噬相关蛋白LC3B和Beclin 1的表达显著降低。相对于对照组,维生素C单独处理组肾组织损伤和自噬相关蛋白的表达无明显变化。结论：维生素C可降低自噬相关蛋白LC3B和Beclin 1的表达,缓解万古霉素诱导的大鼠肾损伤。     

维生素C的钠离子依赖性传输物质

维生素Ｃ是许多酶反应所必需的 ,它不仅能使补偿离子 (如Ｆｅ2 、Ｃｕ2 )保持还原状态[1,2 ] ,并且有助于清除自由基 ,以保护组织不被氧化而损伤[3 ] 。促进糖传输的葡萄糖输运体 (ｇｌｕｃｏｓｅｔｒａｎｓｐｏｒｔｅｒｓ,ＧＬＵＴ)型传输物质能传输氧化型维生素Ｃ[4,5 ] ,但是该传输物质不允许在正常的葡萄糖浓度下吸收有效的生理剂量的维生素Ｃ ,这是因为维生素Ｃ在血浆中仅以还原型存在[6] 。Ｔｓｕｋａｇｕｃｈｉ等[7] 应用大鼠的ｃＤＮＡ文库分离并克隆表达得到两种新的维生素Ｃ传输物质 (ｓｏｄｉｕｍ ｄｅｐｅｎｄｅｎｔｖｉｔ…     

猕猴桃(Actinidia Lindl.)叶片与果实维生素C含量的相关性研究

以猕猴桃属中华猕猴桃(Actinidia chinensis)32个品种和1个种间杂交后代群体为研究对象,对猕猴桃属植物叶片与果实维生素C含量的相关性进行了研究。结果表明,在中华猕猴桃种内水平上,幼果与成熟果实的果肉维生素C含量间存在极显著的正相关关系;在种间杂交后代群体中成熟叶片和成熟果实的维生素C含量存在极显著正相关关系,为利用早期相关性状开展猕猴桃育种的可行性提供了理论依据。此外,对15个常见中华猕猴桃品种的果实维生素C含量进行了多重比较,为人工杂交时的亲本选择提供了依据。     

包装方式对不同品种番茄果实贮藏期间维生素C含量的影响

采用国家标准GB 6195 86方法测定5个品种、2种包装方式和室温（22℃）贮藏期间番茄果实中维生素C含量的变化,探讨不同品种、包装方式和贮藏时间对番茄果实中维生素C含量的影响。结果表明：不同品种番茄果实中维生素C含量差异显著,春桃柿子＞丹东409（1）＞丹东409（2）＞小黄妃＞硬粉;保鲜膜包装对保持番茄果实中维生素C含量显著优于散装,随着贮藏时间的延长,5个品种、2种包装方式的番茄果实中维生素C含量均显著降低,贮藏7d,保鲜膜包装降低30%左右,散装降低近50%。     

维生素C对细胞增殖的挽救提高shDNMT1对重编程的促进作用

在利用4个转录因子(Oct4,Sox2,Klf4,c-Myc)获得诱导多能干细胞(iPSC)的过程中,DNA甲基化以及维生素C(Vc)发挥着重要的作用。为了研究DNMT1和维生素C在这一过程中的相互作用,在不使用Vc的情况下,利用shRNA抑制DNMT1 的表达不能有效促进成纤维细胞向iPSC以及pre-iPSC向iPSC的转变。但是,在使用Vc的情况下,shDNMT1可以有效地促进这两种转变。此外,shDNMT1可以抑制成纤维细胞增殖,增大G1期细胞比例。从而在一定程度上抑制shDNMT1对iPSC获得的促进作用。而Vc则可以通过促进细胞增殖,减小G1期细胞比例,挽救 shDNMT1对细胞周期的影响,进而提高shDNMT1对重编程的促进作用。     

维生素C通过调控脂肪形成相关基因的转录促进猪前体脂肪细胞的增殖与分化

探讨维生素C（Vit C）诱导猪前体脂肪细胞增殖分化最佳浓度及在分化过程中,5种脂肪形成相关基因peroxisome proliferator activated receptor gamma(PPARγ)和retinoid X receptor alpha(RXRα),脂肪细胞分化标志基因lipoprotein lipase (LPL),生脂基因phosphoenolpyruvate carboxykinase(PEPCK)、stearoyl CoA desaturase(SCD) mRNA表达时序性的变化. 以3　d龄猪前体脂肪细胞为实验对象,用Vit C诱导猪前体脂肪细胞增殖分化,分别在增殖分化第2、4、6和8 d收获细胞,利用MTT测定其增殖程度;油红O染色提取法检测其脂肪含量;采用SQ RT PCR法检测脂肪生成相关基因PPARγ、RXRα、LPL、PEPCK和SCD mRNA表达的变化. 结果显示,PPARγ mRNA在诱导分化第2 d时有低水平表达,在诱导分化过程中表达量逐步升高,在终末分化阶段仍保持高水平表达;RXRα mRNA在诱导分化第2和4 d表达量很低,诱导分化第6 d时表达增加.在诱导分化第8 d,RXRα mRNA表达与第6 d相比差异不显著,直至终末分化. 脂肪细胞分化标志基因LPL在第2 d开始表达,第4和6 d逐步升高,在终末分化阶段仍保持高水平的表达;生脂基因PEPCK和SCD mRNA在第2和4 d开始表达,第6和8 d仍保持高水平的表达. 研究结果表明,100 μmol/L的Vit C促进猪前体脂肪细胞增殖能力最强;250 μmol/L Vit C能显著促进猪前体脂肪细胞分化. 其作用机制可能是通过对转录因子PPARγ和RXRα及标志基因LPL mRNA时序性表达的调控来进行的,促进生脂基因的表达,从而诱导脂肪细胞的分化.     

维生素C诱导人宫颈癌Caski细胞凋亡及其分子机制的研究

为了研究维生素C对人宫颈癌Caski细胞体外抑制、诱导凋亡的作用及其分子机制,使用不同剂量维生素C处理人宫颈癌Caski细胞,采用噻唑蓝(MTT)法检测药物对细胞增殖的抑制作用;流式细胞仪检测Cas-ki细胞周期变化;琼脂糖电泳法观察凋亡细胞DNA Ladder现象;Western blot检测凋亡相关蛋白Bcl-2、Bax和E6的表达以及Caspase 3的激活;荧光染色观察细胞线粒体膜电位的改变.分析发现,维生素C可显著抑制人宫颈癌Caski细胞增殖,呈现明显的时间和剂量依赖性;将细胞阻滞于S期;诱导细胞凋亡,下调Bcl-2和E6、上调Bax蛋白表达,促进Caspase3活化,降低线粒体膜电位.表明维生素C在体外可有效抑制人宫颈癌Caski细胞增殖,诱导细胞凋亡.     

维生素C和B_1对铅致小鼠睾丸损伤保护作用研究

目的探讨维生素C、B1联合用药拮抗铅对睾丸的毒性作用。方法 1.30只雄性小鼠随机分为2组(15只/组):空白对照组和铅染毒组。染铅组雄性小鼠每日腹腔注射醋酸铅20 mg/kg,空白对照组给予等容量的生理盐水,一日一次,第42日结束以上实验。将小鼠取血,用试剂盒检测血浆中谷胱甘肽的含量、谷胱甘肽过氧化物酶(GPx)和谷胱甘肽还原酶(GR)的活性。将睾丸组织经过处理后,在光镜及透射电镜下观察睾丸组织结构变化;2.45只雄性小鼠随机分为3组(15只/组):空白对照组、铅染毒组和维生素联合干预组。染铅组雄性小鼠每日腹腔注射醋酸铅20 mg/kg,维生素干预组在染铅后,即时每组小鼠给予维生素C和维生素B1,一日一次,第42日结束。处死小鼠,取血,用试剂盒检测血浆中谷胱甘肽的含量,谷胱甘肽过氧化物酶(GPx)和谷胱甘肽还原酶(GR)的活性。在光镜以及透射电镜下观察睾丸组织结构变化。结果1.与空白对照组比较,醋酸铅染毒组小鼠血浆中还原型谷胱甘肽(GSH)的水平降低,GSH/GSSG的比例降低,GPX活性升高,GR活性降低;光镜下睾丸组织生精小管变薄,各级生精细胞、支持细胞减少,部分细胞核出现核固缩;3.电镜下染毒组小鼠睾丸组织超微结构变化明显,支持细胞溶酶体增多,线粒体肿胀空泡化;2.与醋酸铅染毒组比较,维生素干预组小鼠血浆中还原型谷胱甘肽(GSH)的水平升高,GSH/GSSG的比例升高,GPx活性降低,GR活性升高;光镜观察结果:与染铅组相比,维生素B1和C联合干预组中破坏的睾丸生精上皮有所恢复;电镜观察结果:与染铅组相比,维生素B1和C联合干预组睾丸组织超微结构基本恢复正常,生精细胞和支持细胞结构正常,细胞器丰富。结果 1.醋酸铅使体内GSH的水平降低,GPX活性升高,GR活性降低,从而诱导氧化应激的发生,导致染铅小鼠睾丸的损伤;2.维生素干预组小鼠血浆中还原型谷胱甘肽(GSH)的水平升高,GSH/GSSG的比例升高,GPx活性降低,GR活性升高,维生素干预组小鼠血浆中氧化型谷胱甘肽还原为还原型谷胱甘肽,从而发挥抗氧化作用,对小鼠睾丸起到保护作用。     

SVCT1 and SVCT2: key proteins for vitamin C uptake

Summary. Vitamin C is accumulated in mammalian cells by two types of proteins: sodium-ascorbate co-transporters (SVCTs) and hexose transporters (GLUTs); in particular, SVCTs actively import ascorbate, the reduced form of this vitamin. SVCTs are surface glycoproteins encoded by two different genes, very similar in structure. They show distinct tissue distribution and functional characteristics, which indicate different physiological roles. SVCT1 is involved in whole-body homeostasis of vitamin C, while SVCT2 protects metabolically active cells against oxidative stress. Regulation at mRNA or protein level may serve for preferential accumulation of ascorbic acid at sites where it is needed. This review will summarize the present knowledge on structure, function and regulation of the SVCT transporters. Understanding the physiological role of SVCT1 and SVCT2 may lead to develop new therapeutic strategies to control intracellular vitamin C content or to promote tissue-specific delivery of vitamin C-drug conjugates. Authors’ address: Dr. Isabella Savini, Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy     

Glutamate receptors modulate sodium-dependent and calcium-independent vitamin C bidirectional transport in cultured avian retinal cells

Vitamin C is transported in the brain by sodium vitamin C co‐transporter 2 (SVCT‐2) for ascorbate and glucose transporters for dehydroascorbate. Here we have studied the expression of SVCT‐2 and the uptake and release of [¹⁴C] ascorbate in chick retinal cells. SVCT‐2 immunoreactivity was detected in rat and chick retina, specially in amacrine cells and in cells in the ganglion cell layer. Accordingly, SVCT‐2 was expressed in cultured retinal neurons, but not in glial cells. [¹⁴C] ascorbate uptake was saturable and inhibited by sulfinpyrazone or sodium‐free medium, but not by treatments that inhibit dehydroascorbate transport. Glutamate‐stimulated vitamin C release was not inhibited by the glutamate transport inhibitor l ‐β‐threo‐benzylaspartate, indicating that vitamin C release was not mediated by glutamate uptake. Also, ascorbate had no effect on [³H] d ‐aspartate release, ruling out a glutamate/ascorbate exchange mechanism. 2‐Carboxy‐3‐carboxymethyl‐4‐isopropenylpyrrolidine (Kainate) or NMDA stimulated the release, effects blocked by their respective antagonists 6,7‐initroquinoxaline‐2,3‐dione (DNQX) or (5R,2S)‐(1)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine hydrogen maleate (MK‐801). However, DNQX, but not MK‐801 or 2‐amino‐5‐phosphonopentanoic acid (APV), blocked the stimulation by glutamate. Interestingly, DNQX prevented the stimulation by NMDA, suggesting that the effect of NMDA was mediated by glutamate release and stimulation of non‐NMDA receptors. The effect of glutamate was neither dependent on external calcium nor inhibited by 1,2‐bis (2‐aminophenoxy) ethane‐N′,N′,N′,N′,‐tetraacetic acid tetrakis (acetoxy‐methyl ester) (BAPTA‐AM), an internal calcium chelator, but was inhibited by sulfinpyrazone or by the absence of sodium. In conclusion, retinal cells take up and release vitamin C, probably through SVCT‐2, and the release can be stimulated by NMDA or non‐NMDA glutamate receptors.     

Human choroid plexus papilloma cells efficiently transport glucose and vitamin C

In vitro and in vivo studies suggest that the basolateral membrane of choroid plexus cells, which is in contact with blood vessels, is involved in the uptake of the reduced form of vitamin C, ascorbic acid (AA), through the sodium‐vitamin C cotransporter, (SVCT2). Moreover, very low levels of vitamin C were observed in the brains of SVCT2‐null mice. The oxidized form of vitamin C, dehydroascorbic acid (DHA), is incorporated through the facilitative glucose transporters (GLUTs). In this study, the contribution of SVCT2 and GLUT1 to vitamin C uptake in human choroid plexus papilloma (HCPP) cells in culture was examined. Both the functional activity and the kinetic parameters of GLUT1 and SVCT2 in cells isolated from HCPP were observed. Finally, DHA uptake by GLUT1 in choroid plexus cells was assessed in the presence of phorbol‐12‐myristate‐13‐acetate (PMA)‐activated human neutrophils. A marked increase in vitamin C uptake by choroid plexus cells was observed that was associated with superoxide generation and vitamin C oxidation (bystander effect). Thus, vitamin C can be incorporated by epithelial choroid plexus papilloma cells using the basolateral polarization of SVCT2 and GLUT1. This mechanism may be amplified with neutrophil infiltration (inflammation) of choroid plexus tumors.

     

Retracted: Superoxide‐dependent uptake of vitamin C in human glioma cells

Glioblastomas are lethal brain tumors that resist current cytostatic therapies. Vitamin C may antagonize the effects of reactive oxygen species (ROS) generating therapies; however, it is often used to reduce therapy‐related side effects despite its effects on therapy or tumor growth. Because the mechanisms of vitamin C uptake in gliomas are currently unknown, we evaluated the expression of the sodium‐vitamin C cotransporter (SVCT) and facilitative hexose transporter (GLUT) families in human glioma cells. In addition, as microglial cells can greatly infiltrate high‐grade gliomas (constituting up to 45% of cells in glioblastomas), the effect of TC620 glioma cell interactions with microglial‐like HL60 cells on vitamin C uptake (Bystander effect) was determined. Although glioma cells expressed high levels of the SVCT isoform‐2 (SVCT2), low functional activity, intracellular localization and the expression of the dominant‐negative isoform (dnSVCT2) were observed. The increased glucose metabolic activity of glioma cells was evident by the high 2‐Deoxy‐d ‐glucose and dehydroascorbic acid (DHA) uptake rates through the GLUT isoform‐1 (GLUT1), the main DHA transporter in glioblastoma. Co‐culture of glioma cells and activated microglial‐like HL60 cells resulted in extracellular ascorbic acid oxidation and high DHA uptake by glioma cells. This Bystander effect may explain the high antioxidative potential observed in high‐grade gliomas.

     

Maturational loss of the vitamin C transporter in erythrocytes

Erythrocytes have the same intracellular concentration of ascorbate as plasma, which is much lower than that of nucleated cells. To determine why erythrocytes are unable to concentrate ascorbate, we tested for the presence of ascorbate transporters in these cells. Human erythrocytes had very low rates of uptake of radiolabeled ascorbate, which was accounted for by the lack of ascorbate transporter SVCT2 in immunoblots. Using a cell culture model of Friend virus-infected mouse erythroblasts, immunoblots showed that the SVCT2 was present in the erythroblast stages, but was lost following extrusion of the nucleus in the formation of the reticulocyte stage. Rates of specific ascorbate transport correlated with the presence of the SVCT2. These results show that mature erythrocytes fail to concentrate ascorbate due to the loss of SVCT2 during maturation in the bone marrow.     

Stimulation of differentiation in sodium-dependent vitamin C transporter 2 overexpressing MC3T3-E1 osteoblasts

Sodium-dependent vitamin C transporter (SVCT) 2 facilitates reduced ascorbic acid (AA) transport in MC3T3-E1 osteoblasts. Our previous studies suggested that Zn-induced osteoblast differentiation and Ca2+-, PO4(3-)-stimulated osteopontin (OPN) expression might result from their up-regulation effect on SVCT2 expression and AA uptake. Here, we investigated the role of SVCT2 on osteoblast differentiation by using SVCT2-overexpressing cells. Two clones of SVCT2-introduced cells overexpressed SVCT2 mRNA by 2.8- and 3.1-fold those of control cells, which resulted in obvious increase of AA uptake by 2.1- and 2.4-fold in Vmax with no change in Km. Alkaline phosphatase activity, hydroxyproline content significantly increased in SVCT2-overexpressing cells, and the induction of OPN mRNA was through up-regulation of OPN promoter activity by SVCT2 overexpression. Moreover, SVCT2-overexpressing cells exhibited more ability to promote mineralization and increase calcium deposition under the stimulation of 5 mM beta-glycerophosphate. These findings indicate that SVCT2 stimulates osteoblast differentiation and mineralization.     

The vitamin C transporter SVCT2 is down-regulated during postnatal development of slow skeletal muscles

Vitamin C plays key roles in cell homeostasis, acting as a potent antioxidant as well as a positive modulator of cell differentiation. In skeletal muscle, the vitamin C/sodium co-transporter SVCT2 is preferentially expressed in oxidative slow fibers. Besides, SVCT2 is up-regulated upon the early fusion of primary myoblasts. However, our knowledge of the postnatal expression profile of SVCT2 remains scarce. Here we have analyzed the expression of SVCT2 during postnatal development of the chicken slow anterior and fast posterior latissimus dorsi muscles, ranging from day 7 to adulthood. SVCT2 expression is consistently higher in the slow than in the fast muscle at all stages. After hatching, SVCT2 expression is significantly down-regulated in the anterior latissimus dorsi, which nevertheless maintains a robust slow phenotype. Taking advantage of the C2C12 cell line to recapitulate myogenesis, we confirmed that SVCT2 is expressed in a biphasic fashion, reaching maximal levels upon early myoblasts fusion and decreasing during myotube growth. Together, these findings suggest that the dynamic expression levels of SVCT2 could be relevant for different features of skeletal muscle physiology, such as muscle cell formation, growth and activity.     

Polarized localization of vitamin C transporters, SVCT1 and SVCT2, in epithelial cells

Messenger RNA of homologous sodium-vitamin C cotransporters, SVCT1 and SVCT2, were found in the intestine. Studies using cultured intestinal cells suggested an apical presence of SVCT1 but the function of SVCT2 was unknown. Here, we showed that enterocytes from heterozygous SVCT2-knockout mice had lower sodium-dependent vitamin C accumulation compared to those from the wildtype. Thus, SVCT2 appears to be functional in enterocytes. We then tested whether SVCT2 could have a redundant function as SVCT1 by constructing and expressing EGFP-tagged SVCTs in intestinal Caco-2 and kidney MDCK cells. In confluent epithelial cells, SVCT1 protein expressed predominantly on the apical membrane. SVCT2, in contrast, accumulated at the basolateral surface. Functionally, SVCT1 expression led to more transport activity from the apical membrane, while SVCT2 expression only increased the uptake under the condition when basolateral membrane was exposed. This differential epithelial membrane distribution and function suggests non-redundant functions of these two isoforms.     

Ascorbic acid depletion enhances expression of the sodium-dependent vitamin C transporters, SVCT1 and SVCT2, and uptake of ascorbic acid in livers of SMP30/GNL knockout mice

In this study, we examined whether ascorbic acid (AA) and dehydroascorbic acid (DHA), the oxidized form of AA, levels in tissues regulate the AA transporters, sodium-dependent vitamin C transporters (SVCT) 1 and SVCT2 and DHA transporters, glucose transporter (GLUT) 1, GLUT3, GLUT4 mRNA by using senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice. These mice are incapable of synthesizing AA in vivo. AA depletion enhanced SVCT1 and SVCT2 mRNA expression in the liver and SVCT1 and GLUT4 mRNA expression in the small intestine, but not in the cerebrum or kidney. Next, we examined the actual impact of AA uptake by using primary cultured hepatocytes from SMP30/GNL KO mice. In the AA-depleted hepatocytes from SMP30/GNL KO mice, AA uptake was significantly greater than in matched cultures from wild-type mice. These results strongly affirm that intracellular AA is an important regulator of SVCT1 and SVCT2 expression in the liver.