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首页> 中文期刊> 《新乡医学院学报》 >P2X7受体阻断剂对三磷腺苷诱导的人神经母细胞瘤SH-SY5Y细胞内Ca2+水平的影响

P2X7受体阻断剂对三磷腺苷诱导的人神经母细胞瘤SH-SY5Y细胞内Ca2+水平的影响

         
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摘要

目的 观察三磷腺苷(ATP)诱导的人神经母细胞瘤SH-SY5Y细胞内Ca2+水平变化的特点,并探讨P2X7受体阻断剂对其的影响.方法 取对数生长期的SH-SY5Y细胞接种于含体积分数10％胎牛血清高糖达尔伯克改良伊格尔培养基(DMEM)中,在37℃、体积分数5％ CO2培养箱中培养24 h,随机分为4组,分别加入含0、1、3、5 mmol·L-1ATP的培养液,每孔100 μL,各组均分别作用15、30、60 min,然后用Hank's溶液洗涤细胞3次,Fluo-3/AM染细胞内Ca2+,Hoechst 33258染细胞核.荧光显微镜下观察细胞内荧光发射强度.另取对数生长期的SH-SY5Y细胞悬液接种于含体积分数10％胎牛血清高糖DMEM的96孔板中,在37℃、体积分数5％ CO2培养箱中培养24h,随机分为正常对照组、ATP组及KN-62低、中、高剂量组,正常对照组细胞在每孔100 μL培养液中常规培养,ATP组细胞在每孔100 μL含5 mmol·L-1 ATP的培养液中培养,KN-62低、中、高剂量组细胞分别每孔先用100 μL含100、500、1 000 nmol·L-1 KN-62的培养液孵育30 min,每孔再用100 μL的5mmol·L-1 ATP培养液处理1h,用Hank's溶液洗涤细胞3次,Fluro-3/AM染色细胞内Ca2,Hoechst 33258染色细胞核.荧光显微镜下观察细胞内荧光发射强度变化,图像分析软件记录结果,测每组细胞中Ca2+的平均荧光强度.每次实验每组设3个平行复孔,实验重复3次.结果 0 mmol·L-ATP组呈现荧光的细胞数量少,荧光强度弱;1、3、5 mmol·L-1 ATP组随着ATP剂量增加,呈现荧光的细胞数量逐渐增多,荧光强度逐渐增强;若ATP剂量相同,则随着ATP作用时间的延长,呈现荧光的细胞数量逐渐增多,荧光强度逐渐增强.正常对照组、ATP组、KN-62低、中、高剂量组细胞荧光强度分别为468.24±45.67、3 292.35±159.64、3 013.27 ±321.42、2 515.77 ±320.98、2 486.32 ±318.31,ATP组细胞平均荧光强度显著强于正常对照组(P＜0.05);KN-62低、中、高剂量组细胞平均荧光强度均低于ATP组(P＜0.05).结论 ATP诱导SH-SY5Y细胞内Ca2+升高具有浓度和时间依赖性,KN-62可部分阻断ATP的这一作用.%Objective To observe the changes of Ca2+ level in human neuroblastoma SH-SY5Y cells induced by adenosine triphosphate (ATP),and to investigate the effect of P2X7 receptor blocker on it.Methods The SH-SY5Y cells in logarithmic phase were inoculated in high glucose Dulbecco's modified Eagle's medium (DMEM) containing volume fraction 10％ fetal bovine serum,and cultured for 24 hours in incubator containing volume fraction 5％ CO2 at 37 ℃.Then the cells were randomly divided into four groups,and the culture medium containing 0,1,3,5 mmol · L-1 ATP was added respectively,100 μL per well.After 15,30,60 minutes,the cells were washed with Hank's solution for three times,and stained by Fluo-3/ AM and Hoechst 33258.The intracellular fluorescence intensity was observed under fluorescence microscope.The SH-SY5Y cells in logarithmic phase were inoculated in high glucose DMEM containing volume fraction 10％ fetal bovine serum,and cul-tured for 24 hours in incubator containing volume fraction 5％ CO2 at 37 ℃.Then the cells were randomly divided into normal control group,ATP group and low,middle and high dose of KN-62 group.The cells in the normal control group were cultured in 100 μL conventional culture medium.The cells in ATP group were cultured in 100 μL culture medium containing 5 mmol · L-1 ATP.The cells in the low,middle and high dose of KN-62 group were cultured for 30 minutes in 100 μL culture medium containing 100,500 and 1 000 umol · L-1 KN-62 respectively;and cultured for one hour in 5 mmol · L-1 culture medium 100 μL.Then the cells were washed with Hank's solution for three times,and stained by Fluo-3/AM and Hoechst 33258.The intracellular fluorescence intensity was observed under fluorescence microscope,and recorded by image analysis software.The average fluorescence intensity of Ca2 + in cells was measured.In each experiment,three parallel wells were set in each group,and the experiment was repeated 3 times.Results The number of fluorescent cells was few,and the fluorescence intensity was weak in 0 mmol · L-1 ATP group.With the increase of ATP dose,the number of fluorescent cells and fluorescence intensity increased gradually.Under the same dose of ATP,with the extension of ATP action time,the number of fluorescent cells and the fluorescence intensity increased gradually.The fluorescence intensity in the normal control group,ATP group and low,middle,high dose of KN-62 group was 468.24 ± 45.67,3 292.35 ± 159.64,3 013.27 ± 321.42,2 515.77 ± 320.98 and 2 486.32 ± 318.31,respectively;the fluorescence intensity in ATP group was significantly stronger than that in the normal control group (P ＜ 0.05);the fluorescence intensity in low,middle and high KN-62 group were significantly lower than those in ATP group (P ＜ 0.05).Conclusion The rise of Ca2+ concentration induced by ATP in SH-SY5Y cells has a concentration and time-dependent manner.KN-62 can partially block the action of ATP.

著录项

  • 来源
    《新乡医学院学报》 |2017年第7期|555-559|共5页
  • 作者

    李秀娟; 王国红; 张利彬; 杨坤丽; 李超堃; 王璐; 宋景贵; 赵红岗; 李东亮;

  • 作者单位

    新乡医学院生理学与神经生物学教研室,河南新乡 453003;

    新乡医学院生理学与神经生物学教研室,河南新乡 453003;

    新乡医学院生理学与神经生物学教研室,河南新乡 453003;

    新乡医学院生理学与神经生物学教研室,河南新乡 453003;

    新乡医学院生理学与神经生物学教研室,河南新乡 453003;

    新乡医学院生理学与神经生物学教研室,河南新乡 453003;

    新乡医学院第一附属医院,河南 新乡453100;

    新乡医学院生理学与神经生物学教研室,河南新乡 453003;

    新乡医学院生理学与神经生物学教研室,河南新乡 453003;

  • 原文格式 PDF
  • 正文语种 chi
  • 中图分类 神经系肿瘤;
  • 关键词

    三磷腺苷; 人神经母细胞瘤; SH-SY5Y细胞; Ca2+; P2X7受体;

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