利用Seahorse XF-96探讨不同细胞浓度和血清对线粒体功能的影响

Effects of different cell concentrations and serum on mitochondrial function by Seahorse XF-96

目的利用Seahorse XF-96探讨不同细胞浓度和血清对HUVEC细胞和U87细胞线粒体功能的影响。方法利用细胞线粒体应激试剂盒,在不同浓度的HUVEC细胞和U87细胞中加入抑制剂(寡霉素、解偶联剂、鱼藤酮和抗霉素A),根据细胞浓度分为2000组、4000组、6000组和8000组,根据有无血清和不同的解偶联剂浓度分为血清+低解偶联剂组、血清+高解偶联剂组、无血清+低解偶联剂组和无血清+高解偶联剂组。在Seahorse XF-96中测定线粒体的耗氧率(OCR)和细胞外酸化率(ECAR),并比较HUVEC细胞和U87细胞各组线粒体的基础氧耗、质子漏、最大呼吸能力、非线粒体的氧耗、储备的呼吸能力、三磷酸腺苷(ATP)生成能力、线粒体呼吸潜力和偶联效率;比较HUVEC细胞和U87细胞之间上述指标的差异;观察血清和解偶联剂浓度对HUVEC细胞线粒体功能的影响。结果在HUVEC细胞和U87细胞中,2000组OCR显著低于4000组、6000组和8000组(P<0.01);2000组的ECAR显著低于4000组(P<0.01),4000组显著低于6000组和8000组(P<0.01)。在HUVEC细胞中,与2000组比较,6000组和8000组的基础氧耗、储备的呼吸能力、ATP生成能力和最大呼吸能力增强(P<0.05),8000组非线粒体的氧耗和质子漏增强(P<0.05);在U87细胞中,与2000组比较,6000组和8000组的基础氧耗、非线粒体的氧耗和最大呼吸能力增强(P<0.05),8000组ATP生成能力、储备的呼吸能力和质子漏增强(P<0.05);HUVEC细胞和U87细胞各组间线粒体的偶联效率和线粒体呼吸潜力比较差异均无统计学意义(P>0.05)。U87细胞8000组的质子漏高于HUVEC细胞(P<0.05);HUVEC细胞2000组、6000组和8000组的偶联效率高于U87细胞(P<0.05);HUVEC细胞6000组的ATP生成能力和储备的呼吸能力高于U87细胞(P<0.05);HUVEC细胞和U87细胞各组基础氧耗、最大呼吸能力、非线粒体的氧耗、线粒体呼吸潜力比较差异均无统计学意义(P>0.05)。血清+低解偶联剂组储备的呼吸能力和线粒体呼吸潜力高于无血清+低解偶联剂组(P<0.05),其余指标组间比较差异均无统计学意义(P>0.05);血清+高解偶联剂组和无血清+高解偶联剂组的线粒体功能各指标差异均无统计学意义(P>0.05)。结论随着细胞浓度的不断增加,HUVEC细胞和U87细胞的OCR和ECAR不断升高,细胞浓度会影响两种细胞线粒体的OCR和ECAR,HUVEC细胞和U87细胞的质子漏和偶联效率存在显著差异,血清会影响HUVEC细胞线粒体储备的呼吸能力和线粒体呼吸潜力。

Objective To investigate the effects of different cell concentrations and serum on mitochondrial function of HUVEC cells and U87 cells using Seahorse XF-96.Methods Inhibitors(oligomycin,uncoupler,rotenone,and antimycin A)were added into different concentrations of HUVEC cells and U87 cells using the cell mitochondrial stress kit.According to cell concentrations,the cells were divided into the 2000 group,4000 group,6000 group and 8000 group;according to the presence or absence of serum and different concentrations of uncoupler,the cells were divided into the serum+low uncoupler group,serum+high uncoupler group,serum free+low uncoupler group and serum free+high uncoupler group.The oxygen consumption rate(OCR)and extracellular acidification rate(ECAR)of mitochondrion were tested in the Seahorse XF-96.The basic oxygen consumption,proton leak,maximal respiration capacity,non-mitochondrial oxygen consumption,spare respiratory capacity,adenosine triphosphate(ATP)production capacity,mitochondrial respiration potential and coupling efficiency of HUVEC cells and U87 cells were compared,and the differences of the above indexes between HUVEC cells and U87 cells were compared.The effects of serum and different concentrations of uncoupler on mitochondrial function of HUVEC cells were observed.Results In HUVEC cells and U87 cells,the OCR of the 2000 group was significantly lower than that in the 4000,6000 and 8000 groups(P<0.01);ECAR of the 2000 group was significantly lower than that in the 4000 group(P<0.01),and that in the 4000 group was significantly lower than that in the 6000 and 8000 groups(P<0.01).In HUVEC cells,compared with the 2000 group,the basic oxygen consumption,spare respiratory capacity,ATP production capacity and maximal respiration capacity of the 6000 and 8000 groups were increased(P<0.05),and the non-mitochondrial oxygen consumption and proton leak of the 8000 group were increased(P<0.05).In U87 cells,compared with the 2000 group,the basic oxygen consumption,non-mitochondrial oxygen consumption and maximal respiration capacity of the 6000 and 8000 groups were increased(P<0.05),and the ATP production capacity,spare respiration capacity and proton leak of the 8000 group were increased(P<0.05).There was no statistically significant difference in the mitochondrial coupling efficiency or mitochondrial respiration potential of each group in HUVEC cells and U87 cells(P>0.05).The proton leak of the 8000 group in U87 cells was higher than that in HUVEC cells(P<0.05),the coupling efficiencies of the 2000,6000 and 8000 groups in HUVEC cells were higher than those in U87 cells(P<0.05),the ATP production capacity and spare respiration capacity of the 6000 group in HUVEC cells were higher than those in U87 cells(P<0.05).There was no statistically significant difference in the basic oxygen consumption,maximal respiration capacity,non-mitochondrial oxygen consumption or mitochondrial respiration potential of each group between HUVEC cells and U87 cells(P>0.05).The spare respiration capacity and mitochondrial respiration potential of the serum+low uncoupler group were higher than those of the serum free+low uncoupler group(P<0.05),but there was no statistically significant difference in other indexes between the groups(P>0.05);there was no statistically significant difference in the mitochondrial function indexes between the serum+high uncoupler group and serum free+high uncoupler group(P>0.05).Conclusion With the increasing of cell concentration,the OCR and ECAR of HUVEC cells and U87 cells increased continuously,cell concentrations can affect the OCR and ECAR of the two cells,there are significant differences in the proton leak and coupling efficiency between HUVEC cells and U87 cells,and serum can affect the mitochondrial spare respiration capacity and mitochondrial respiration potential of HUVEC cells.