鞘氨醇激酶在免疫细胞中的作用

近年来,专家们越来越清楚地认识到神经鞘脂类是重要的信号传递分子。尤其是神经鞘脂类的代谢产物,比如神经酰胺和1-磷酸鞘氨醇(S1P),已经被认为是一类重要的生物活性分子,参与细胞内的各种生[1-2]     

鞘氨醇激酶调节细胞凋亡的研究进展

鞘磷脂衍生物神经酰胺(Cer)、鞘氨醇(Sp)及1-磷酸鞘氨醇(SIP)在调控细胞增殖、存活及凋亡中发挥着重要作用。鞘氨醇激酶(SPK1)是调控细胞内Cer、Sp、SIP代谢平衡的关键酶。SPK1磷酸化Sp生成SIP，SIP通过细胞内和细胞外作用机制调节细胞生长和凋亡。SPK1参与细胞因子的信号传递。高表达SPK1抑制半胱天冬酶的裂解并上调Bcl-2基因的表达。本文综述了SPK的结构、调控及对细胞凋亡的调节作用。     

IL-6对人多发性骨髓瘤细胞鞘氨醇激酶的激活作用

目的：鞘氨醇激酶是细胞内合成磷酸鞘氨醇的激酶。该酶是细胞迁移、增殖及凋亡调节中发挥重要作用的信号分子。本研究拟确定鞘氨醇激酶在人多发性骨髓瘤细胞的表达及在IL-6信号途径中的作用。方法：采用RT-PCR方法鉴定了鞘氨醇激酶在骨髓瘤细胞的表达情况，通过鞘氨醇激酶活性测定确定IL-6对多发性骨髓瘤细胞鞘氨醇激酶的激活作用。结果：人多发性骨髓瘤细胞表达鞘氨醇激酶及S1P受体EDG1，3，5。IL-6通过PI-3K和MAPK激活鞘氨醇激酶。结论：在多发性骨髓瘤细胞中，鞘氨醇激酶参与IL-6的信号转导。鞘氨醇激酶有可能成为多发性骨髓瘤治疗的新靶点。     

鞘氨醇激酶——运动改善胰岛素抵抗的新靶点

随着生活水平的日益提高,肥胖、2型糖尿病(Type 2 Diabetes Mellitus,T2DM)等代谢性疾病发病率逐年上升,胰岛素抵抗(Insulin Resistance,IR)是诸多代谢性疾病的共同病理生理基础。运动通过提高机体外周组织代谢水平而显著改善IR已被广泛应用于代谢性疾病的防治。近年来,鞘氨醇激酶(Sphingosine kinases,SK)作为机体脂代谢的重要调控蛋白受到了广泛关注,其催化鞘氨醇(Sphingosine)合成鞘氨醇-1-磷酸(Sphingosine-1-Phosphate,S1P),SK及其S1P在运动调控机体脂代谢从而改善IR的过程中发挥举足轻重的作用。本文对近年来有关SK及其催化产物S1P在运动改善机体IR过程中的作用研究加以综述,为揭示运动防治IR机制提供新的研究靶点。     

鞘氨醇-1-磷酸受体-1促进肿瘤转移与放疗抵抗的研究进展

远处转移是癌症治疗的主要障碍之一。鞘氨醇-1-磷酸受体-1（sphingosine-1-phosphate receptor 1,S1PR1）在恶性肿瘤中过度表达,通过激活下游信号通路增强细胞侵袭和迁移活性、调控上皮间质转化（EMT）以及诱导淋巴管与血管生成,最终导致肿瘤转移的发生。S1PR1与获得性放射抗性的产生也有密切关联。本文就S1PR1及其在肿瘤转移和放疗抵抗中的作用进行综述。     

1-磷酸鞘氨醇对淋巴管内皮细胞生物学特性调节作用的研究

目的：研究1-磷酸鞘氨醇（S1P）对淋巴管内皮细胞（HDLEC）的生物学特性的调节作用及其调节机制。方法：应用RT-PCR方法检测HDLEC是否表达SPK及S1P受体,应用MTT法、细胞扩散盒技术检测S1P对HDLEC增殖、迁移特性的影响,并用Western印迹方法检测S1P对HDLEC的MAPK信号通路的调节。结果：HDLEC表达SPK及S1P受体,包括S1P1,S1P2,S1P3和S1P4。外源性S1P对HDLEC没有促增殖作用,但可促进其迁移,并可通过S1P1和S1P2诱导MAPK快速磷酸化。结论：初步研究结果提示,S1P可以影响淋巴管内皮细胞的生物学特性,有可能成为调控淋巴管生成及其功能的新靶点。     

1-磷酸鞘氨醇促进大鼠血管平滑肌细胞迁移的实验研究

目的探讨1-磷酸鞘氨醇（sphingosine 1-phosphate,S1P）对大鼠血管平滑肌细胞（rat vascular smooth mus-cle cells,rVSMc）迁移的作用机制,为肿瘤血管新生和心血管疾病发生机制的研究提供新的思路。方法体外分离培养、鉴定rVSMc细胞,建立低氧培养箱和氯化钴诱导的细胞低氧模型,应用RT-PCR方法对rVSMc细胞的S1P受体表达水平进行检测,运用微孔隔离室穿越方法研究S1P对rVSMc细胞迁移的作用,并用S1P受体阻断剂加以证实。结果与结论rVSMc细胞表达SPK1和S1P受体rS1P1、rS1P2和rS1P3,低氧状态下rVSMc细胞SPK1的表达和活性均高于对照,S1P主要通过与rS1P2受体结合促进rVSMc细胞的迁移。     

鞘磷脂与细胞生长,凋亡调控

鞘脂分子包括鞘磷脂和鞘糖脂两大类。它们不仅是维持细胞膜结构的重要组分，而且它们的代谢产物参与了细胞的生长、分化和凋亡等重要生理活动。１－磷酸鞘氨醇（ＳＰＰ）和神经酰胺是两类重要的代谢产物，它们通过活化或抑制蛋白激酶、磷酸酶、离子通道，传递细胞生长和凋亡的信号。在细胞内，它们的作用是拮抗的，细胞的生与死往往取决于哪一种作用占了主导地位。本文就鞘磷脂的结构与代谢、神经酰胺和ＳＰＰ的信号转导机制及其在治     

FTY720在多发性硬化治疗作用中的研究进展

多发性硬化（Muhipesclerosis，MS）是一种中枢神经系统自身免疫性疾病，激素等免疫抑制剂是其主要治疗方法。芬戈莫德（rrY720）是一种从冬虫夏草中分离出来的鞘氨醇1-磷酸盐受体抑制剂，具有促进淋巴细胞归巢、诱导淋巴细胞凋亡及免疫耐受、抑制T淋巴细胞活性等作用，而发性硬化（MS）是一种中枢神经系统免疫性疾病，FTY720作为一种新型的免疫抑制剂治疗复发-缓解型MS，在二期临床试验和三期临床试验中显示着安全有效。     

鞘氨醇激酶1对氧自由基诱导心肌细胞损伤的保护作用研究

目的:研究鞘氨醇激酶1(SPK1)在氧自由基诱导心肌细胞损伤过程中的作用.方法:分离培养Wistar乳大鼠心肌细胞,用不同浓度过氧化氢(H2O2,0, 50, 100, 200, 400 μmol/L)处理细胞,检测乳酸脱氢酶(LDH)的释放和细胞SPK1酶活性变化;Northern 印迹方法分析SPK1催化产物1-磷酸鞘氨醇(S1P)受体的表达.用S1P预处理心肌细胞,然后加入100 μmol/L的H2O2继续作用48 h,检测细胞培养上清中LDH的活性;用携带人SPK1基因的复制缺陷型重组腺病毒载体(Ad-SPK1)感染Wistar乳大鼠心肌细胞,然后加入100 μmol/L的H2O2继续作用48 h,检测细胞培养上清中LDH的活性.分析外源性S1P或SPK1高表达对H2O2诱导心肌细胞损伤的保护作用.结果:H2O2作用于心肌细胞后,导致LDH释放增多,SPK1酶活性抑制,其作用呈剂量依赖性;H2O2刺激使S1P受体Edg-1表达水平升高,Edg-3表达水平降低.外源性S1P预处理使H2O2诱导的LDH释放减少,其作用呈剂量依赖性.与对照组相比,Ad-SPK1感染使心肌细胞SPK1酶活性明显升高,细胞培养上清中S1P含量增多,并明显抑制H2O2诱导心肌细胞LDH的释放.结论:SPK1高表达能保护H2O2导致的心肌细胞死亡,这种保护作用主要通过S1P的释放来实现.Edg-1和Edg-3可能参与SPK1对心肌细胞的保护作用.     

高表达鞘氨醇激酶对胃癌细胞生物学特性的影响

目的研究高表达鞘氨醇激酶（SPK）对人胃癌细胞增殖、迁移和凋亡等细胞生物学行为的影响。方法以重组腺病毒为载体,将人野生型（rAd—SPK^WT）及突变体（rAd—SPK^DV）SPK基因导入人胃癌细胞BCC-823。以Western blot检测外源SPK基因的表达,以[γ^82P]ATP掺入法测定SPK酶活性,用迁移扩散盒技术测定细胞的迁移能力。结果重组腺病毒可有效介导SPK在人胃癌细胞BGC-823中的表达。酶活性分析表明野生型SPK基因可增强SPK活性,而突变体SPK基因抑制SPK活性;高表达野生型SPK可以促进胃癌细胞的迁移,抑制5-FU对胃癌细胞的细胞毒作用,而高表达突变体SPK可以抑制胃癌细胞的迁移,增强5FU对胃癌细胞的细胞毒作用。结论SPK可以抑制5-FU诱导的胃癌细胞凋亡,促进胃癌细胞迁移,有可能成为胃癌新的治疗靶点。     

携带人鞘氨醇激酶及突变体基因重组腺病毒载体的制备及表达

目的制备携带人野生型鞘氨醇激酶基因(SPK^wt)及其突变体(SPK^DN)的腺病毒载体,为研究SPK的生物学功能提供高效基因转移载体。方法将野生型和突变体SPK基因亚克隆至穿梭载体pshuttle-cmv,重组的穿梭质粒经Pme线性化后,电转化E.coli BJ-AD-1感受态,获得带有目的基因的腺病毒重组子质粒;重组子经Pac I线性化后,以脂质体介导转染293包装细胞,获得重组腺病毒载体;分别行PCR鉴定重组腺病毒是否携带目的基因,Western blot鉴定目的基因是否表达,酶活性检测判断野生型基因和突变体基因表达活性。结果重组腺病毒带有目的基因,可以感染内皮细胞并正确表达,酶活性分析表明野生型SPK基因增强SPK活性,突变体SPK基因抑制SPK活性。结论成功构建了携带SPK野生型及其突受体的重组腺病毒,为研究SPK的功能提供了物质基础。     

肝刺激物促肝细胞增殖信号转导途径的初步研究

目的：探讨新生牛肝肝刺激物(hepatic stimulator substance，HSS)促进SMMC7721肝癌细胞增殖的信号转导途径。方法：对新生牛肝进行多步分离，采用^3H-Td RDNA掺入法检测分离物的促增殖活性，从而获得具增殖刺激活性的HSS；Western印迹方法检测其对肝癌细胞MAPK Thr202／Tyr204的磷酸化作用并检测其对SPK的激活作用。结果：经多步分离纯化，获得了活性较高的HSS粗提物，Western印迹法结果显示HSS粗提物能明显提高MAPK的磷酸化水平，并能激活细胞内的SPK活性。结论：HSS对细胞的增殖刺激可能是通过SPK和MAPK途径发挥作用。     

Induction of apoptosis in two mammalian cell lines results in increased levels of fructose-1,6-Bisphosphate and CDP-choline as determined by 31P MRS

Programmed cell death or apoptosis was induced in human promyelocytic leukemia (HL-60) and Chinese hamster ovary (CHO-K1) cells using several cytotoxic drugs that have different modes of action, including camptothecin, ceramide, chelerythrine, etoposide, farnesol, geranyl geraniol, and hexadecylphosphocholine. The consequent changes in cellular metabolism were monitored using ³¹P MRS measurements on intact cells and cell extracts. Cells undergoing programmed cell death exhibited characteristic changes in the levels of glycolytic and phospholipid metabolites. The most significant changes were increases in the concentration of the glycolytic intermediate, fructose-l,6-bisphosphate and in the concentration of CDP-choline, which is an intermediate in phosphatidylcholine biosynthesis. In HL-60 cells, the increase in fructose-l,6-bisphosphate levels could be explained by depletion of cellular NAD(H) levels. All of the agents used to induce apoptosis caused the accumulation of CDP-choline. Since the resonances of this compound occur in a relatively well resolved region of tissue spectra, it could provide a marker for apoptosis that would allow the noninvasive detection of the process in vivo using ³¹P MRS measurements.     

31P NMR spectroscopic studies of the effects of cyclophosphamide on perfused RIF-1 tumor cells

To determine whether direct cellular effects of chemotherapy are responsible for ³¹P NMR spectral changes observed in treated tumors in vivo, RIF-1 fibrosarcoma cells were examined in vitro before, during, and after treatment with 4-hy-droperoxycyclophosphamide (4-HC), an activated form of cyclophosphamide. When RIF-1 cells were treated with 4-HC in a metabolically stable but nonproliferating state, the ³¹P NMR spectra were identical with those of untreated cells for up to 70 h. When actively proliferating RIF-1 cells were treated with 4-W, the intensities of the nucleotide triphosphate resonances, which increased linearly during control cell growth, remained constant for 50 h or longer. These studies demonstrate that the bioenergetic improvement observed following treatment of RIF-1 tumors in vivo [S.-J. Li, J.P. Wehrle, S.S. Rajan, R.G. Steen, J.D. Glickson, and J. Hilton, Cancer Res. 48, 4736 (1988)] does not result from direct effects of cyclophosphamide metabolites on RIF-1 cell metabolism, but rather from indirect effects of treatment on tumor or host physiology. Key words: ³¹P NMR spectroscopy; RIF-1 tumor cells; cyclophosphamide; cancer.     

Endurance training selectively increases high‐density lipoprotein‐bound sphingosine‐1‐phosphate in the plasma

Sphingosine‐1‐phosphate (S1P) is a bioactive lysosphingolipid that is found in relatively high concentration in human plasma. Erythrocytes, endothelial cells, and activated platelets are the main sources of circulating S1P. The majority of plasma S1P is transported bound to high‐density lipoprotein (HDL ) and albumin. In recent years, HDL ‐bound S1P attracted much attention due to its cardioprotective and anti‐atherogenic properties. We have previously found that endurance‐trained athletes are characterized by higher plasma S1P concentration compared to untrained individuals. This finding prompted us to examine the effect of endurance training on S1P metabolism in blood. Thirteen healthy, untrained, male subjects completed an 8‐week training program on a rowing ergometer. Three days before the first, and 3 days after the last training session, blood samples were drawn from an antecubital vein. We found that total plasma S1P concentration was increased after the training. Further analysis of different plasma fractions showed that the training selectively elevated HDL ‐bound S1P. This effect was associated with activation of sphingosine kinase in erythrocytes and platelets and enhanced S1P release from red blood cells. We postulate that increase in HDL ‐bound S1P level is one of the mechanisms underlying beneficial effects of regular physical activity on cardiovascular diseases.