不同浓度沙利度胺对K562细胞凋亡及血管内皮生长因子分泌的影响

【】　目的　研究沙利度胺对人慢性粒细胞白血病急变株K562细胞凋亡及血管内皮生长因子（vascularendotheliagrowthfactor，VEGF）分泌的影响。　方法　采用不同浓度的沙立度胺（0.5、1.0、2.0mmol/L）作用于K562细胞24、48、72、96h，瑞-姬（Wright-Giemsa）染色法观察细胞形态；四甲基偶氮唑盐（methylthiazolyltetrozolium，MTT）法检测细胞增殖；流式细胞仪膜联蛋白V-异硫氰酸荧光素/碘化丙啶双染法检测凋亡率；琼脂糖凝胶电泳法检测脱氧核糖核酸梯状条带；酶联免疫吸附法检测VEGF浓度。　结果　培养24、48h后，沙立度胺对K562细胞生长无抑制作用；作用72h后，1.0、2.0mmol/L浓度组开始出现对K562细胞生长的抑制（P<0.001）；作用96h后，0.5mmol/L浓度组也产生对K562细胞生长的抑制（P<0.001），呈一定的浓度和时间依赖性。沙立度胺处理72h后，K562细胞出现形态学改变，其体积缩小，出现空泡化，边缘出现突起，染色质浓缩、边集，核固缩、出现凋亡小体。经沙立度胺处理后，流式分析结果显示K562细胞凋亡率增加（P<0.001）。沙立度胺作用72h后，琼脂糖凝胶电泳可见典型的DNA梯状条带。K562细胞培养48h后，沙立度胺抑制VEGF的分泌（P<0.001），并且VEGF浓度与凋亡率呈负相关（r=-0.789）。　结论　沙利度胺抑制K562细胞的增殖，呈一定的浓度和时间依赖性；沙利度胺对K562细胞凋亡有明显诱导作用；沙利度胺抑制K562细胞VEGF的分泌。【Abstract】　Objective　Toinvestigatetheeffectofthalidomideonapoptosisofk562cellsanditsvascularendothelialgrowthfactorsecretion.　Methods　K562cellswereculturedinvitrowith0.5,1.0,and2.0mmol/Lthalidomidefor24,48,72,and96hours.MorphologyoftheK562cellswasobservedbytheWright-Giemsastainingmethod.Methylthiazolyltetrozolium（MTT）assaywasusedtodeterminethecellgrowth.Therateofapoptosiswasanalyzedbyflowcytometry(FCM)withannexinV-fluoresceinisothiocyanate/propidiumiodide（AnnexinV-FITC/PI）double-stainingmethod.AgarosegelelectrophoresiswasusedtodetectedDeoxyribonucleicacidLadder（DNALadder）.TheconcentrationofVEGFwasquantifiedbytheenzyme-linkedimmunosorbentassay(ELISA).　Results　Culturedfor24or48hours,thalidomidehadnoeffectontheproliferationoftheK562cells.Butafterculturedfor72hours,thalidomidebegantoinhibitthegrowthoftheK562cellsattheconcentrationof1.0and2.0mmol/L（P<0.001）.Afterculturedfor96hours,theproliferationoftheK562cellswasinhibitedtooattheconcentrationof0.5mmol/Lthalidomide（P<0.001）.Thus,thalidomideinhibitedthegrowthoftheK562cellswithadose-andtime-dependentmannertosomeextent.Afterexposuretothalidomidefor72hours,K562cellsunderwenttypicalmorphologicalchangesofapoptosissuchasvaculization,thebuddingofcytoplasm,chromatincondensation,margination,shrunkennucleusandapoptoticbody.TheresultsofflowcytometryshowedthatthalidomidecouldobviouslyincreasetheratesoftheapoptosisofK562cells（P<0.001）.Aftertreatedwiththalidomidefor72hours,DNAwasextractedforAgarosegelelectrophoresisandtypicalDNAladderstripswereobserved.ThesecretionofVEGFwasinhibitedwhenexposuretothalidomidefor48hours（P<0.001）,andtherewasnegativecorrelationbetweenVEGFconcentrationsandapoptoticrates（r=-0.789）.　Conclusions　ThalidomidecouldinhibitethegrowthoftheK562cellswithadose-andtime-dependentmannertosomeextent.ThalidomidecouldobviouslyinducetheapoptosisoftheK562cellsandinhibititssecretionofVEGF.