小鼠FOXP3-siRNA慢病毒载体的构建及其对小鼠动脉粥样硬化的影响

目的:构建小鼠FOXP3基因特异性siRNA慢病毒载体,并对其进行功能性研究.方法:根据GeneBank提供的小鼠FOXP3cDNA序列,设计4条RNA干扰靶点序列,制备双链DNA oligo,与制备好的双酶切慢病毒载体连接,再转入细菌感受态细胞DH-5a,行PCR鉴定出阳性克隆并测序,制备成FOXP3-siRNA慢病毒载体,利用Western-blot方法对构建的载体进行功能性研究;将载体通过尾静脉注入小鼠体内,观察其对小鼠动脉粥样硬化形成的影响.结果:构建的小鼠FOXP3基因siRNA慢病毒载体,经PCR和DNA测序证实与设计完全一致,并对Foxp3+CD4+CD25+调节性T细胞有显著敲减效应;其能显著促进动脉粥样硬化形成.结论:体内外实验表明,成功构建了小鼠FOXP3基因的特异性siRNA慢病毒载体.     

应用载体介导小干扰RNA抑制小鼠巨噬细胞表达肿瘤坏死因子α

目的 研究特异性载体介导的小干扰RNA(siRNA)对小鼠巨噬细胞株表达肿瘤坏死因子(TNF)-α基因的抑制作用.方法 设计两段靶向TNF-α基因的特异性siRNA,合成相应寡核苷酸,插入载体H1启动子下游,构建表达干扰性发夹状RNA(shRNA)载体pHS-A和pHS-B,转染小鼠巨噬细胞株(RAW264.7),用实时荧光定量PCR和酶联免疫吸附法检测siRNA对TNF-α mRNA和蛋白表达的抑制作用.结果 内毒素脂多糖(LPS)刺激后6 h,巨噬细胞表达TNF-α mRNA和合成的TNF-α量均增加,于9～12 h达高峰.pHS-A转染后,LPS刺激巨噬细胞的TNF-α mRNA为0.003 56±0.001 87,TNF-α蛋白表达为(149.93±21.02)pg/ml,比未转染组[分别为0.021 34±0.009 60、(1922.30±149.05)pg/ml]显著减少(P＜0.01),抑制率达83.3%.pHS-B及阴性对照组对基因及蛋白表达均无影响.结论 LPS可刺激小鼠巨噬细胞合成TNF-α.pHS-A可抑制小鼠巨噬细胞TNF-α表达.     

小鼠半胱天冬酶-12特异性小干扰RNA真核表达载体的构建及鉴定

目的 构建小鼠半胱天冬氨酸蛋白酶-12（Caspase-12）基因特异性小干扰RNA（siRNA）表达载体并检测其表达。方法 参照siRNA模板设计原则，设计并合成3对针对caspase-12不同位点的siRNA，分别在脂质体介导下转染小鼠肝癌细胞株Hepal-6，24h收集细胞；RT-PCR分析不同位点siRNA对靶基因mRNA表达的抑制，筛选出抑制效率最高的位点；将此位点siRNA模板序列插入质粒pRNAT-H1．1Neo中，PCR分析及基因测序进行鉴定；构建成功的重组质粒转染Hepal-6细胞48h和72h后，实时荧光定量PCR分析caspase-12 mRNA表达；Western印迹检测Caspase-12的表达。数据行t检验。结果 RT-PCR分析表明，第-对siRNA（siRNA＊1）对caspase-12基因表达的抑制效率最高；重组质粒pRNAT-H1．1Neo-caspasel2（pRNAT-caspl2）经PCR分析及测序表明，siRNA＊1模板序列成功插入预计位点，且序列正确；pRNAT-caspl2转染Hepal-6细胞48h和72h后，与空质粒对照组相比，caspase-12 mRNA水平分别下降72．5％和59．5％（P〈0．05）；pRNAT-caspl2转染后，caspase-12酶原表达量在24、48和72h分别下调17．1％、37．3％和60．1％，48h和72h的抑制率比较，差异有统计学意义（P〈0．05）。结论 成功构建小鼠caspase-12特异性siRNA真核表达载体，它对小鼠肝癌细胞caspase-12基因的表达具有显著和特异的抑制作用。     

siRNA腺病毒抑制Survivin基因表达诱导肝癌细胞凋亡

目的为改进质粒siRNA载体的不足,建立siRNA的病毒性载体系统,并探讨凋亡抑制因子Survivin基因在肝癌细胞中的作用。方法通过已建立的Survivin质粒siRNA,构建Survivin的腺病毒siRNA载体系统,筛选重组病毒并感染肝癌细胞HepG2细胞,应用Western blot和RT-PCR检测Survivin基因表达变化,用流式细胞仪观察肿瘤细胞凋亡。结果成功构建Survivin基因的腺病毒siRNA载体及重组腺病毒,感染肝癌细胞明显抑制Survivin蛋白表达,抑制率66.32%;降低Survivin基因的mRNA转录水平达72.04%;应用流式细胞仪观察肿瘤细胞凋亡明显增加。结论腺病毒siRNA载体系统可作为抑制目的基因,进而研究其功能和作用的技术平台,并为其他基因的相关研究打下基础;抑制Survivin基因表达可诱导肿瘤细胞HepG2的凋亡,为今后腺病毒siRNA载体应用于肿瘤动物实验和肿瘤基因治疗提供实验资料。     

血凝素样氧化型低密度脂蛋白受体1特异性小发夹RNA表达载体的构建及其对巨噬细胞源性泡沫细胞氧化应激的影响

目的构建靶向血凝素样氧化型低密度脂蛋白受体1(LOX-1)基因的发卡样siRNA(shRNA)表达载体,并探讨其对巨噬细胞源性泡沫细胞氧化应激的影响。方法(1)采用DNA重组技术,将LOX-1shRNA双链与线性化pGenesil-1质粒表达载体连接,构建LOX-1特异性小发夹RNA表达载体pLOX-1-shRNA1及pLOX-1-shRNA2,用脂质体法转染小鼠单核巨噬细胞(RAW264.7),半定量逆转录聚合酶链反应法检测LOX-1 mRNA的表达,Western blot法检测LOX-1蛋白的表达,选择沉默?...     

靶向肝癌HepG2细胞VEGF基因的siRNA表达载体构建及体外抑制作用

目的: 构建靶向肝癌HepG2细胞VEGF基因的siRNA表达载体并在体外检测其对VEGF基因表达的抑制作用.方法: 设计合成靶向HepG2细胞VEGF基因的siRNA cDNA序列并与pSUPER载体连接, 构建VEGF siRNA表达载体, 经酶切鉴定和测序确认后, 脂质体2000介导VEGF siRNA转染HepG2细胞. RT-PCR及Western blot检测VEGFsiRNA表达载体转染的HepG2细胞中VEGF基因表达.结果: 通过双酶切鉴定和测序分析, 成功构建了靶向HepG2细胞VEGF基因的siRNA表达载体. RT-PCR和Western blot法检测转染VEGFsiRNA表达载体的HepG2细胞VEGF mRNA及VEGF165表达下调, 其抑制率分别为65%和74%. 实验组中的HepG2中VEGF mRNA表达较阴性对照组、空载体组表达量显著下降(0.304±0.062 vs 0.896±0.061, 0.884±0.074,P<0.05).结论: 成功构建了靶向肝癌HepG2细胞VEGF基因的siRNA表达载体, 且该载体在体外能有效抑制HepG2细胞VEGF基因表达.     

抑制MDR1基因表达shRNA RNAi系统的构建

目的构建抑制多药耐药基因(MDR1)表达的短发夹RNA(sh RNA)真核表达载体系统.方法根据MDR1基因已知序列,设计两段21个碱基的MDR1特异性靶序列,合成两对62nt 并含有编码shRNA序列的寡核苷酸,双链退火后,克隆到经过BamH I和Xba I双酶切后线性 PG E-1载体的U6启动子下游,重组构建RNA干扰(RNAi)质粒.结果对重新构建的pshRNA-MDR1载体经P CR扩增后行电泳分析,并对含有插入基因片段行测序分析.结果表明,62个碱基均成功插入到预计位点,并且序列完全一致.结论载体的成功构建为研究其对MDR1 基因表达的抑制作用打下基础,同时使我们发展了在体内合成siRNA的方法.     

针对P基因的siRNA对乙肝病毒S表达稳定性的影响

目的以乙型肝炎病毒（HBV）P基因为靶点，构建表达小干扰RNA（siRNA）的质粒载体psRNA1、psRNA2、psRNA3、psRNA4及对照psiRNA0，体外观察针对P基因的siRNA对HBs表达的影响。方法设计并合成针对HBV-P基因的siRNA寡核苷酸，经退火形成双链后克隆入psiRNA-H1neo质粒中，通过鉴定将构建成功的5个psiRNAs真核表达质粒瞬时转染HepG2．2．15细胞，用半定量RT-PCR对筛选到的位点进行了试验观测抑制效果。结果表明针对P基因的siRNA能下调HBs的表达，其中psiRNA1、psiRNA2的抑制HBs基因表达的效果最强。结论针对P基因的siRNA可以有效的抑制HBs的表达，抑制的效果和siRNA的靶位点有关。     

shRNA干扰Rab9 GTPase表达对麻疹病毒野生株体外增殖的抑制作用

目的构建Rab9 GTPase短发夹RNA（shRNA）表达载体,观察其对Rab9 GTPase基因表达和麻疹病毒野生株体外增殖的抑制作用。方法参照GenBank中Rab9 GTPase基因序列设计合成2对Rab9 GTPase基因特异性shRNA,定向克隆人表达载体,构建重组表达载体,酶切鉴定和序列分析证实后脂质体法转染U937细胞,然后感染麻疹病毒野生株,逆转录聚合酶链反应（RT-PCR）和免疫印迹技术（Western blot）检测转染细胞内Rab9 GTPase mRNA和蛋白质的表达水平;标准蚀斑试验测定病毒滴度;流式细胞仪检测细胞凋亡率的变化;RT-PCR检测转染细胞内双链RNA依赖蛋白激酶（PKR）和2′-5′寡腺甙酸合成酶（OAS-1）的mRNA水平。结果酶切和序列分析证实,成功构建了靶向Rab9 GTPase基因的shRNA表达载体。2个shRNAs均可特异性抑制U937细胞内Rab9 GTPase mRNA和蛋白质的表达,最高抑制率分别为（90.5±0.2）％和（92.1±0.3）％;蚀斑试验结果表明,shRNAs可以有效抑制麻疹病毒野生株体外增殖,其抑制率可达到90％以上;流式细胞仪检测转染后细胞的凋亡率无明显变化;RT-PCR检测PKR和OAs-1的mRNA水平转染前后无明显变化。结论成功构建Rab9 GTPase特异性shRNA表达载体。shRNAs通过特异性抑制Rab9 GTPase基因表达抑制麻疹病毒野生株体外增殖。     

小鼠FGF-21 siRNA腺病毒载体的构建与鉴定

目的构建含FGF-21 siRNA基因的重组腺病毒载体并鉴定。方法首先用BamHⅠ+HindⅢ双酶切本课题组前期构建的质粒pSi-lencer1.0-shFGF-21,得到0.3 bp的目的片段,并将目的片段亚克隆入穿梭质粒(pShuttle-shFGF-21,),用I-CeuI和I-SceI双酶切处理pAdxsi组腺病载体及pShuttle-shFGF-21,连接,转化DH5α感受态细胞,筛选、鉴定、测序后,最后在293细胞内包装扩增为重组腺病毒。结果设计并构建了小鼠FGF-21基因特异性siRNA腺病毒载体,并经酶切和测序鉴定。空斑形成实验测得腺病毒滴度为1×1010PFU/ml。结论成功构建了小鼠FGF-21基因特异性siRNA腺病毒载体。     

Protection on experimental sinus node dysfunction in rabbits with ginsenosides]

This experiment was carried out to evaluate the effect of ginsenosides from stems and leaves (GSL) on the sinus node dysfunction (SND) by observing the changes of the electrophysiological parameters of sinus node in limited period. Anesthetized rabbits were randomly divided into 3 groups. In the acute experiment, GSL, 50 mg/kg, iv, was given to the GSL group (G), n = 12. Normal saline (NS), the same volume, to the control group (C), n = 12. After GSL was administered 30-50 mg/kg.d for 14 days, iv, another data were obtained from chronic experimental group (CE), n = 15. The results show that the parameters, spontaneous cycle length (SCL), maximal sinus node recovery time (SNRTmax), corrected sinus node recovery time (CSNRT) were shorter in G group and CE group than C group respectively (P less than 0.05, P less than 0.01). It was suggested that GSL exerted protective effects on the experimental sinus node dysfunction.     

A vital role for glycosphingolipid synthesis during development and differentiation

Glycosphingolipids (GSLs) are believed to be integral for the dynamics of many cell membrane events, including cellular interactions, signaling, and trafficking. We have investigated their roles in development and differentiation by eliminating the major synthesis pathway of GSLs through targeted disruption of the Ugcg gene encoding glucosylceramide synthase. In the absence of GSL synthesis, embryogenesis proceeded well into gastrulation with differentiation into primitive germ layers and patterning of the embryo but was abruptly halted by a major apoptotic process. In vivo, embryonic stem cells deficient in GSL synthesis were again able to differentiate into endodermal, mesodermal, and ectodermal derivatives but were strikingly deficient in their ability to form well differentiated tissues. In vitro, however, hematopoietic and neuronal differentiation could be induced. The results demonstrate that the synthesis of GSL structures is essential for embryonic development and for the differentiation of some tissues and support the concept that GSLs are involved in crucial cell interactions mediating these processes.     

Alterations in cell surface glycosphingolipids and other lipid classes of fibroblasts in familial hypercholesterolemia.

The glycosphingolipids (GSL) and other major lipid classes were studied in cultured fibroblasts from a family with familial hypercholesterolemia. The GSL content in cells grown in medium containing fetal calf serum was increased 5-fold in the homozygote and 2- to 3-fold in both heterozygous parents. Cell surface labeling experiments, using the membrane probe galactose oxidase followed by reduction with KB3H4, showed an increased incorporation of 3H by the homozygous cells into GL4 (4-fold), GM3 (2- to 3-fold), and GL3a and GD3 (1- to 2-fold); the amount of 3H incorporated by the heterozygous cells was in between that of the homozygous and normal fibroblasts. The specific radioactivity of each of the GSL, except GL2a, was lower in the mutant cells. This unlabeled pool of GSL may be buried in the membrane matrix (less exposure), or located intracellularly, or both. The phospholipids were most markedly elevated (3-fold) in homozygous cells, with a disproportionate increase in phosphatidic acid and sphingomyelin (5- to 6-fold). The content of the GSL, except GL2a, and of the phospholipids was reduced about one-half in the homozygous fibroblasts grown in lipoprotein-deficient medium for 24 hr; by 5 days the GSL content was reduced to only 1.3 times normal and phospholipids to below normal. Incubation of normal fibroblasts in lipoprotein-dificient medium 24 hr had no effect on the GSL or phospholipid content; at 5 days, there was a 50% increase in both GL3a and GL4 with a 25% increase in GM3; there was no change in the phospholipid content. These data suggest that the defective regulation of lipid metabolism in this syndrome may be more extensive than previously realized.     

Characteristic expression of glycosphingolipid profiles in the bipotential cell differentiation of human promyelocytic leukemia cell line HL-60

Changes of glycosphingolipids (GSLs) in the bipotential cell differentiation of human promyelocytic leukemia cell line HL-60 cells were investigated by high-performance thin-layer chromatography (HPTLC), with special reference to morphological and functional changes, such as phagocytosis and nitroblue tetrazolium (NBT) reduction. Nine molecular species of neutral GSLs and 13 or more species of sialo-GSLs, ie, gangliosides, were detected on the HPTLC chromatograms for untreated HL-60 cells. The major components were ceramide dihexoside (CDH), GM3, and sialo-paragloboside (SPG). When HL- 60 cells were induced to differentiate into both myeloid mature cells and macrophage-like cells in vitro, no new molecular species of GSLs specific for one of the cell differentiations was induced, but distinctive quantitative changes in the GSL composition were definitely observed between the two cell differentiations. During the myeloid differentiation induced by either dimethylsulfoxide (DMSO) or retinoic acid (RA), CDH, paragloboside (PG), and gangliosides having longer sugar moieties characteristically increased with a concomitant decrease of GSLs with shorter sugar chains, such as ceramide monohexoside (CMH) and GM3, and the GSL composition profile of myeloid differentiation- induced HL-60 cells became more similar to that of normal human granulocytes. However, some marked differences were noted between the induced HL-60 cells and the normal granulocytes, especially in the ganglioside compositions. These differences might reflect either some deficiency in the in vitro myeloid differentiation or some leukemic properties of HL-60 cells. In marked contrast to the change of GSL composition during myeloid differentiation, a remarkable increase of GM3, with a concurrent marked decrease of CDH, was observed in the process of cell differentiation into macrophage-like cells with 12-O- tetradecanoyl-phorbol-13-acetate (TPA), which suggested an increase in the biosynthesis of GM3. These results demonstrate that HL-60 cells express distinct GSL profiles, depending not only on maturation stages but also on differentiation directions.     

Inhibition of substrate synthesis as a strategy for glycolipid lysosomal storage disease therapy

The glycosphingolipid (GSL) lysosomal storage diseases are caused by mutations in the genes encoding the glycohydrolases that catabolize GSLs within lysosomes. In these diseases the substrate for the defective enzyme accumulates in the lysosome and the stored GSL leads to cellular dysfunction and disease. The diseases frequently have a progressive neurodegenerative course. The therapeutic options for treating these diseases are relatively limited, and for the majority there are no effective therapies. The problem is further compounded by difficulties in delivering therapeutic agents to the brain. Most research effort to date has focused on strategies for augmenting enzyme levels to compensate for the underlying defect. These include bone marrow transplantation (BMT), enzyme replacement and gene therapy. An alternative strategy that we have been exploring is substrate deprivation. This approach aims to balance the rate of GSL synthesis with the impaired rate of GSL breakdown. The imino sugar N-butyldeoxynojirimycin (NB-DNJ) inhibits the first step in GSL biosynthesis and has been used to evaluate this approach. Studies in an asymptomatic mouse model of Tay–Sachs disease have shown that substrate deprivation prevents GSL storage in the CNS. In a severe neurodegenerative mouse model of Sandhoff disease, substrate deprivation delayed the onset of symptoms and disease progression and significantly increased life expectancy. Combining NB-DNJ and BMT was found to be synergistic in the Sandhoff mouse model. A clinical trial in type I Gaucher disease has been undertaken and has shown beneficial effects. Efficacy was demonstrated on the basis of significant decreases in liver and spleen volumes, gradual but significant improvement in haematological parameters and disease activity markers, together with diminished GSL biosynthesis and storage as determined by independent biochemical assays. Further trials in type I Gaucher disease are in progress; studies are planned in patients with GSL storage in the CNS.     

The neutral glycosphingolipid globotriaosylceramide promotes fusion mediated by a CD4-dependent CXCR4-utilizing HIV type 1 envelope glycoprotein

Previously, we showed that the addition of human erythrocyte glycosphingolipids (GSLs) to nonhuman CD4⁺ or GSL-depleted human CD4⁺ cells rendered those cells susceptible to HIV-1 envelope glycoprotein-mediated cell fusion. Individual components in the GSL mixture were isolated by fractionation on a silica-gel column and incorporated into the membranes of CD4⁺ cells. GSL-supplemented target cells were then examined for their ability to fuse with TF228 cells expressing HIV-1_LAI envelope glycoprotein. We found that one GSL fraction, fraction 3, exhibited the highest recovery of fusion after incorporation into CD4⁺ nonhuman and GSL-depleted HeLa-CD4 cells and that fraction 3 contained a single GSL fraction. Fraction 3 was characterized by MS, NMR spectroscopy, enzymatic analysis, and immunostaining with an antiglobotriaosylceramide (Gb3) antibody and was found to be Gal(α1→4)Gal(β1→4)Glc-Cer (Gb3). The addition of fraction 3 or Gb3 to GSL-depleted HeLa-CD4 cells recovered fusion, but the addition of galactosylceramide, glucosylceramide, the monosialoganglioside, GM3, lactosylceramide, globoside, the disialoganglioside, GD3, or α-galactosidase A-digested fraction 3 had no effect. Our findings show that the neutral GSL, Gb3, is required for CD4/CXCR4-dependent HIV-1 fusion.     

Activation state and intracellular trafficking contribute to the repertoire of endogenous glycosphingolipids presented by CD1d

Myeloid antigen-presenting cells (APC) express CD1d molecules that present exogenous and endogenous lipid antigens that activate CD1d-restricted T cells, natural killer T (NKT) cells. NKT cell activation has been shown to mediate the potent downstream activation of other immune cells through cell–cell interactions and rapid, prolific cytokine production. Foreign antigens are not required for NKT cell activation. The endogenous lipids bound to CD1d are sufficient for activation of NKT cells in the setting of Toll-like receptor-induced cytokines. The most potent NKT cell antigens identified are glycosphingolipids (GSL). The GSL repertoire of endogenous ligands bound to CD1d molecules that are expressed in myeloid APC at steady state and in the setting of activation has not been delineated. This report identifies the range of GSL bound to soluble murine CD1d (mCD1d) molecules that sample the endoplasmic reticulum/secretory routes and cell surface-cleaved mCD1d that also samples the endocytic system. Specific GSL species are preferentially bound by mCD1d and do not solely reflect cellular GSL. GM1a and GD1a are prominent CD1d ligands for molecules following both the ER/secretory and lysosomal trafficking routes, whereas GM2 was eluted from soluble CD1d but not lysosomal trafficking CD1d. Further, after LPS activation, the quantities of soluble CD1d-bound GM3 and GM1a markedly increased. A unique α-galactose-terminating GSL was also found to be preferentially bound to mCD1d at steady state, and it increased with APC activation. Together, these studies identify the range of GSL presented by CD1d and how presentation varies based on CD1d intracellular trafficking and microbial activation.     

Alteration of the carbohydrate binding specificity of verotoxins from Gal alpha 1-4Gal to GalNAc beta 1-3Gal alpha 1-4Gal and vice versa by site-directed mutagenesis of the binding subunit.

Verotoxin 1 (VT-1) and Shiga-like toxin II (SLT-II) bind to the glycosphingolipid (GSL), globotriaosylceramide (Gb3), whereas pig edema disease toxin (VTE) binds to globotetraosylceramide (Gb4) and to a lesser degree Gb3. Amino acids important in the GSL binding specificity of VT-1 and VTE have been identified by site-directed mutagenesis. One mutation, Asp-18----Asn, in VT-1 resulted in binding to Gb4 in addition to Gb3 in a manner similar to VTE. Several mutations in VTE resulted in the complete loss of GSL binding; however, one mutation resulted in a change in the GSL binding specificity of the VTE B subunit. The double mutation Gln-64----Glu and Lys-66----Gln (designated GT3) caused a selective loss of Gb4 binding, effectively changing the binding phenotype from VTE to VT-1. Both wild-type VTE and GT3 were purified to homogeneity and binding kinetics in vitro were determined with purified GSLs from human kidney. The cell cytotoxicity spectrum of the mutant toxin was also found to be altered in comparison with VTE. These changes were consistent with the GSL content of the target cells.     

An inhibitor of glycosphingolipid metabolism blocks HIV-1 infection of primary T-cells

OBJECTIVE: HIV-1 uses CD4 and chemokine receptors to enter cells. However, other target membrane components may also be involved. This study examines the role of glycosphingolipids (GSL) in HIV-1 entry into primary lymphocytes and its modulation by an inhibitor of GSL biosynthesis. METHODS: CD4 lymphocytes purified from normal or the p-group subtype individuals that were defective in Gb3 synthesis were treated with a GSL biosynthesis inhibitor, 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP). The PPMP-treated cells were tested for HIV-1 replication by measuring p24 antigen production for 7-14 days post-infection and for susceptibility to HIV-1 Env-mediated fusion monitored by a fluorescent dye transfer assay. The effects of PPMP treatment on HIV-1 binding to CD4 lymphocytes were also examined by measuring HIV-1 p24. RESULTS: CD4 lymphocytes from p donors that are devoid of Gb3, but have elevated levels of GM3 were highly susceptible to HIV-1 fusion/entry. Pre-treatment of primary human CD4 lymphocytes from normal or p-sub-group type with PPMP, significantly reduced HIV-1 replication with no change in CD4 and CXCR4 levels. Inhibition of HIV-1 infection was due to the block in HIV-1 Env-mediated plasma membrane fusion. Binding of HIV-1 to CD4 lymphocytes was not affected by PPMP treatment. CONCLUSION: Manipulation of glycosphingolipid metabolic pathways may alter susceptibility of CD4 lymphocytes to HIV-1 entry.     

Neutral and sialosyl glycosphingolipid composition and metabolism of human T-lymphoblastic cell line MOLT-3 cells: distinctive changes as markers specific for their differentiation

Changes in the composition and metabolism of glycosphingolipid (GSL), which is one of the cell surface constituents, during cell differentiation of human T-lymphoblastic leukemia cell line MOLT-3 cells were examined with special reference to their alterations in E rosette-forming capacity and expression of surface antigens specific for T-cell lineage. Three molecular species of neutral GSL and greater than or equal to 13 molecular species of acidic sialosyl-GSL (ganglioside) were detectable on high-performance thin-layer chromatography (HPTLC) in untreated MOLT-3 cells. The major components were ceramide monohexoside and gangliosides GM3 and GD1a. When the cells were induced by 12-O-tetradecanoyl phorbol 13-acetate (TPA) to differentiate into more mature T cells, the ganglioside composition changed distinctively, and the total ganglioside content increased considerably; mono-, di-, and tri-sialosyl gangliosides concomitantly showed significant increase, but no new molecular species of GSL specific for the differentiation were detected. The activity of one sialyltransferases, CMP-sialic acid:CDH sialyltransferase, which synthesizes ganglioside GM3 and the total sialic acid content of the cell surface, parallelled the extent of cell differentiation. Examination of another human T-lymphoblastic leukemia cell line, HPB- ALL, indicated that TPA could also induce the cells to differentiate along T-cell lineage and that changes in the ganglioside pattern during differentiation are similar to those of MOLT-3 cells. The results indicate that human T-lymphoid cell differentiation intimately involves elongation of neutral oligosaccharide-moieties and the addition of sialic acid residues to gangliosides, resulting in more mature T cells containing higher gangliosides. Both the sialyltransferase activity and the sialic acid content, as well as the ganglioside pattern, might be new biochemical markers specific for human T-lymphoblastic cell differentiation.