【摘要】 目的观察谷氨酰胺(Gln)对小细胞肺癌H446细胞增殖和生存的影响,并探究其机制。方法应用CCK-8试剂盒检测Gln(+)组和Gln(-)组H446细胞在0、24、48、72、96 h的增殖情况,筛选出最佳时间,采用Annexin V-FITC/PI双染法、Cell Titer-Glo?发光法和流式细胞仪分别检测这2组细胞的存活比例、三磷酸腺苷(ATP)和活性氧(ROS)水平;以Gln(-)组为对照组,实验组中加入草酰乙酸(OAA)或α-酮戊二酸二甲酯(DM-αKG),检测各组H446细胞的ATP水平、增殖和存活情况;以Gln(-)组为对照组,实验组中加入ROS清除剂N-乙酰-L-半胱氨酸(NAC),检测2组细胞的ROS水平、增殖、克隆和存活情况;在Gln(+)条件下,用0、2、5、10μmol/L谷氨酰胺酶抑制剂BPTES处理H446细胞,通过克隆实验筛选最佳作用浓度,在此浓度下检测Gln(+)组和Gln(+)+BPTES组细胞的ATP、ROS水平和增殖水平。最后,单独应用BPTES或ROS诱导剂过氧化氢(H2O2)和二者联合应用情况下检测细胞的存活比例。结果相比Gln(+)组,Gln(-)组H446细胞的增殖水平在24、48、72、96 h均降低(P<0.05),72 h降低最明显,取72 h为最佳时间;Gln(-)组细胞的存活比例和ATP水平低于Gln(+)组(P<0.05),ROS水平高于Gln(+)组;相比Gln(-)组,Gln(-)+OAA组和Gln(-)+DM-αKG组H446细胞的ATP和增殖未升高,而存活比例升高(P<0.05);相比Gln(-)组,Gln(-)+NAC组ROS水平降低,增殖、克隆水平和存活比例均升高(均P<0.05)。克隆实验结果显示10μmol/L BPTES为最佳浓度;相比Gln(+)组,Gln(+)+BPTES组细胞的ATP和增殖降低(均P<0.05),ROS水平升高;相比单独应用,BPTES+H2O2组H446细胞存活比例明显降低。结论 Gln缺乏可通过提高ROS水平抑制H446细胞的增殖和生存;BPTES和H2O2对H446细胞有联合杀伤作用。更多还原

【Abstract】 Objective To investigate the effects of glutamine(Gln) on proliferation and survival of small cell lungcancer H446 cells,and further to explore the potential mechanism.Methods The proliferation of H446 cells was detectedat different time points(0,24,48,72 and 96 h) by CCK-8 assay in Gln(+) group and Gln(-) group,and an optimal time wasselected.Under the optimal time,Annexin V-FITC/PI staining,Cell Titer-Glo?assay kit and flow cytometer were used todetect cell survival,cellular adenosine triphosphate(ATP) and reactive oxygen species(ROS) levels.Gln(-) group was usedas the control group,under the condition of Gln deficiency,cellular ATP,cell proliferation and survival were detected afteradding oxaloacetic acid(OAA) or dimethyl-α-ketoglutarate(DM-αKG).Gln(-) group was used as the control group,cellular ROS,cell proliferation,colony and survival were detected after treated with ROS scavenger N-acetyl cysteine(NAC).With different concentrations(0,2,5,10 μmol/L) of glutaminase inhibitor BPTES,the optimal concentration wasselected through the colony assay.The cellular ATP and ROS levels and cell proliferation were detected under the optimalconcentration.H446 cells were treated with bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulfide(BPTES),ROSinducer hydrogen peroxide(H2O2) or the combination of them,and cell survival ratio was compared between two groups.Results The proliferation levels of H446 cells at 24,48,which were decreased most significantly in 72 h in Gln(-) group.When 72 h was used as the optimal time,the cell survival ratio and ATP level were decreased,and the ROS level wasincreased,in Gln(-) group compared with those of Gln(+) group(P<0.05).There was a higher survival ratio in H446 cellsin Gln(-)+OAA group and Gln(-) +DM-αKG group than that of Gln(-) group(P<0.05),but there were no significantdifferences in cell proliferation and ATP levels between Gln(-) group,Gln(-)+OAA group and Gln(-)+DM-αKG group.The ROS level was reduced,the cell proliferation,colony level and survival ratio were increased in Gln(-)+NAC groupcompared with those of Gln(-) group(P<0.05).Cloning assay showed that 10 μmol/L was the optional concentration.Underthis concentration,the proliferation and ATP level were decreased in Gln(+)+BPTES group(P<0.05),and cellular ROSlevel was up-regulated compared with Gln(+) group.The survival ratio was significantly lower in BPTES + H2O2 groupcompared with BPTES(+) group or H2O2(+) group.Conclusion Glutamine deficiency inhibits the proliferation and survivalratio of H446 cells through enhancing ROS level.BPTES and H2O2 show synergistically inhibitory effect on the survival ofH446 cells.glutamine;lung neoplasms;carcinoma,small cell;adenosine triphosphate;reactive oxygen species;cell更多还原