摘要
目的研究谷氨酰胺酶抑制剂CB-839(简称CB-839)介导T细胞效应抑制肺癌细胞α-酮戊二酸(αKGA)、谷氨酰胺(Gln)转化的作用机制。方法10只SPF级健康SD裸鼠均建立肺癌动物模型,分为模型组及抑制剂组,模型组裸鼠常规饲养;抑制剂组裸鼠皮下注射CD-839,测量两种肿瘤体积。人肺癌细胞A549购自上海匹拓公司,将肺癌细胞分为Ⅰ组(肺癌细胞常规培养)、Ⅱ组(肺癌细胞+0.25μmol/L浓度CB-839)、Ⅲ组(肺癌细胞+0.50μmol/L浓度CB-839)、Ⅳ组(肺癌细胞+1.00μmol/L浓度CB-839)。MTT法测A549细胞增殖,流式细胞仪测A549细胞凋亡及CD80、CD86、主要组织相容性复合体Ⅱ(MHCⅡ)水平,RT-qPCR测谷氨酸脱氢酶(GLUD1)mRNA、谷氨酰胺酶(GLS)mRNA水平,αKGA试剂盒测αKGA水平。结果培养24、48及72 h时Ⅰ组肺癌A549细胞吸光度(A值)高于Ⅱ组,差异有统计学意义(P<0.05);培养24、48及72 h时Ⅳ组肺癌A549细胞A值与Ⅲ组比较,明显降低,差异有统计学意义(P<0.05)。Ⅰ、Ⅱ、Ⅲ及Ⅳ组肺癌A549细胞凋亡率分别为(3.02±0.39)%、(4.72±0.52)%、(7.95±0.84)%及(13.22±1.28)%。与Ⅲ组相比,Ⅳ组肺癌A549细胞中CD80、CD86、MHCⅡ水平升高,差异均有统计学意义(P<0.05)。Ⅳ组肺癌A549细胞中αKGA水平与Ⅲ组比较,明显降低,差异均有统计学意义(P<0.05)。GLUD1 mRNA与GLS mRNA呈正相关(r=0.671,P<0.001),GLUD1 mRNA与αKGA呈正相关(r=0.708,P<0.001)。GLS mRNA与αKGA呈正相关(r=0.671,P<0.001)。结论CB-839可能通过介导T淋巴效应提高CD80、CD86、MHCⅡ水平,抑制αKGA及Gln相关基因转化,从而降低肺癌细胞活性,促进其凋亡,为今后肺癌的临床治疗及新药研发提供参考依据。
Objective To study the mechanism of T cell effect mediated by glutaminase inhibitor CB-839(CB-839 for short)to inhibit the transformation ofα-ketoglutaric acid(αKGA)and glutamine(Gln)in lung cancer cells.Methods Lung cancer animal models were established in 10 SPF healthy SD nude mice,including model group and inhibitor group.In the inhibitor group,nude mice were injected with CD-839 subcutaneously to measure the volume of two tumors.Human lung cancer cells A549 were purchased from Shanghai pituo company.The lung cancer cells were divided into groupⅠ(conventional culture of lung cancer cells),groupⅡ(lung cancer cells+CB-839 at a concentration of 0.25μmol/L),and groupⅢ(lung cancer cells+0.50μmol/L concentration of CB-839),Ⅳgroup(lung cancer cells+1.00μmol/L concentration of CB-839).A549 cell proliferation was measured by MTT assay,flow cytometry measures A549 cell apoptosis and CD80,CD86,major histocompatibility complexⅡ(MHCⅡ)levels,RT-qPCR measures glutamate dehydrogenase 1(GLUD1)mRNA,glutaminase(GLS)mRNA levels,andαKGA kit measuresαKGA content.Results The absorbance(A value)of A549 cells in groupⅠwas higher than that in groupⅡat 24,48 and 72 h,and the difference was statistically significant(P<0.05).The A value of lung cancer A549 cells in groupⅣwas significantly lower than that in groupⅢat 24,48 and 72 h,and the difference was statistically significant(P<0.05).The apoptosis rates of A549 cells in groupsⅠ,Ⅱ,ⅢandⅣwere(3.02±0.39)%,(4.72±0.52)%,(7.95±0.84)%and(13.22±1.28)%,respectively.Compared with groupⅢ,the levels of CD80,CD86 and MHCⅡin lung cancer A549 cells in groupⅣwere increased,with statistical significance(P<0.05).TheαKGA level in A549 cells in groupⅣwas significantly lower than that in groupⅢ,with statistical significance(P<0.05).GLUD1 mRNA was positively correlated with GLS mRNA(r=0.671,P<0.001),and GLUD1 mRNA was positively correlated withαKGA(r=0.708,P<0.001).GLS mRNA was positively correlated withαKGA(r=0.671,P<0.001).Conclusion CB-839 may increase
作者
张启新
周游
黄飞
ZHANG Qixin;ZHOU You;HUANG Fei(Department of Thoracic Surgery,Haimen District People′s Hospital,Nantong,Jiangsu 226100,China;Department of Pathology,Haimen District People′s Hospital,Nantong,Jiangsu 226100,China)
出处
《国际检验医学杂志》
CAS
2023年第5期582-587,共6页
International Journal of Laboratory Medicine
基金
南通市健康委员会科研项目(QB2019019)。
