目的观察三磷腺苷(ATP)诱导的人神经母细胞瘤SH-SY5Y细胞内Ca~(2+)水平变化的特点,并探讨P2X_7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%CO_2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~(-1)ATP的培养液,每孔100μL,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Ca~(2+),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%CO_2培养箱中培养24 h,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100μL培养液中常规培养,ATP组细胞在每孔100μL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100μL含100、500、1 000 nmol·L~(-1)KN-62的培养液孵育30 min,每孔再用100μL的5 mmol·L~(-1)ATP培养液处理1 h,用Hank’s溶液洗涤细胞3次,Fluro-3/AM染色细胞内Ca~(2+),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Ca~(2+)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~(-1)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Ca~(2+)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 intracellular Ca2 + levels induced by adenosine triphosphate (ATP) in human neuroblastoma SH-SY5Y cells and to explore the effect of P2X7 receptor blockers. Methods SH-SY5Y cells in logarithmic growth phase were inoculated into modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and high glucose, and cultured in a volume fraction 5% CO 2 incubator at 37 ℃. 24 h, were randomly divided into 4 groups, respectively, adding 0, 1, 3, 5 mmol·L -1 ATP in culture medium, each hole 100μL, each group were treated for 15,30,60 min, The cells were washed three times with Hank’s solution and stained with Ca ~ (2 +) and Hoechst 33258 in Fluo-3 / AM cells. Fluorescence microscopy was used to observe intracellular fluorescence emission intensity. Another logarithmic growth phase SH-SY5Y cell suspension was inoculated into 96-well plates containing 10% fetal bovine serum and high glucose DMEM. The cells were cultured in 37 ℃, 5% CO 2 incubator for 24 h. The normal control group, ATP group and KN-62 low, medium and high dose group, the normal control group of cells cultured in 100μL per well culture medium, ATP group cells in each well 100μL containing 5 mmol·L -1, ATP were cultured in low, middle and high doses of KN-62. Each well was incubated with 100μL KN-62 containing 100, 500, 1000 nmol·L -1 KN-62 for 30 min. The wells were treated with 100 μL of 5 mmol·L -1 ATP for 1 h. The cells were washed three times with Hank’s solution. The nuclei of Ca 2 + and Hoechst 33258 were stained with Fluro-3 / AM. Fluorescence microscopy was used to observe the change of intracellular fluorescence emission intensity. The image analysis software recorded the average fluorescence intensity of Ca 2+ in each group of cells. Each experiment set up three parallel wells, the experiment repeated three times. Results The number of cells in 0 mmol·L -1 ATP group was less than that of the control group, and the fluorescence intensity was weak. In 1,3 and 5 mmol·L -1 ATP group, the number of fluorescent cells gradually increased with the increase of ATP dose Increased, the fluorescence intensity gradually increased; if the same dose of ATP, with the extension of ATP time, the number of cells showing fluorescence gradually increased, the fluorescence intensity gradually increased. The fluorescence intensity of the normal control group, ATP group and KN-62 low, medium and high dose groups were 468.24 ± 45.67,3 292.35 ± 159.64,3130.27 ± 321.42,2 515.77 ± 320.98,2 486.32 ± 318.31, The fluorescence intensity of KN-62 cells was significantly lower than that of the normal control group (P <0.05). The average fluorescence intensity of KN-62 cells in low, medium and high dose groups was lower than that in ATP group (P <0.05). Conclusion ATP induced a concentration-and time-dependent increase of Ca 2+ in SH-SY5Y cells. KN-62 partially blocked this effect of ATP.