稳定敲除Mcart⁃1下调RAW264.7巨噬细胞增殖能力和氧化磷酸化水平

Stable knockout of Mcart-1 down-regulates the proliferation and oxidative phosphorylation of RAW264.7 macrophages

目的基于CRISPR/Cas9原理构建Mcart-1稳定敲除的小鼠单核巨噬细胞白血病细胞株RAW264.7,检测其细胞增殖能力和能量代谢水平。方法用Cas9慢病毒和sgRNA慢病毒分两步感染RAW264.7巨噬细胞,并挑选单克隆细胞:qPCR和Western blot检测Cas9表达水平和Mcart-1敲除效果;单细胞测序分析突变位点;细胞能量代谢分析仪Seahorse检测细胞耗氧速率(OCR)和能量代谢表型;试剂盒测定细胞内总ATP含量;细胞计数仪计数和羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)染色检测细胞增殖。结果成功构建Mcart-1稳定敲除的RAW264.7细胞株(记为Mcart-1^(-/-)-RAW264.7);该细胞株中Mcart-1发生移码突变;与野生型RAW264.7细胞系(记为RAW264.7)相比,Mcart-1^(-/-)-RAW264.7细胞氧气消耗速率(OCR)下降,胞外酸化速率(ECAR)升高;与RAW264.7细胞相比,Mcart-1^(-/-)-RAW264.7细胞内总ATP含量下降(P<0.05),线粒体ATP产生速率下降(P<0.01),而糖酵解ATP产生速率升高(P<0.01);与RAW264.7细胞相比,Mcart-1^(-/-)-RAW264.7细胞增殖能力下降(P<0.001)。结论稳定敲除Mcart-1后RAW264.7细胞增殖能力和氧化磷酸化水平均下降。该细胞株可作为探索肿瘤微环境细胞功能的重要工具。

Objective To construct a RAW264.7 macrophage cell strain with stable knockout of Mcart-1 by using CRISPR/Cas9 gene editing technique,and to detect its biological function.Methods Cas9 and sgRNA lentivirus were used to infect RAW264.7 macrophages in two steps.Positive cells were screened with puromycin and hygromycin,and single cells were plated by flow cytometry to obtain monoclonal cells;Expression of Cas9 and Mcart-1 was detected by qPCR and Western blot,and the mutationsite was confirmed by sequence analysis.Cells number counting and carboxyfluorescein diacetate succinimdyl ester(CFSE)staining were used to detect cell proliferation;ATP detection kit was used to measure total ATP content in cells;Seahorse bioenergy analyzer was used to detect cell oxygen consumption rate(OCR)and glycolysis rate.Results Successfully constructed RAW264.7 cell strain with Mcart-1 stably knocked out,denoted as Mcart-1^(-/-)-RAW264.7;A frameshift mutation occurred in Mcart-1 gene in this cell strain;Compared with the wild-type RAW264.7 cell line(RAW264.7),the OCR increased and the extracellular acidification rate(ECAR)of Mcart-1^(-/-)-RAW264.7 cells decreased;The total ATP content in Mcart-1 knocked out cells decreased(P<0.05),mitochondrial ATP production rate decreased(P<0.01),while glycolytic ATP production rate increased(P<0.01);The proliferation activity of RAW264.7 cells decreased after Mcart-1 was knocked out(P<0.001).Conclusions The proliferation activity and oxidative phosphorylation level of RAW264.7 cells were both down-regulated after the Mcart-1 was stably knocked out.This cell strain is an important tool for exploring the function of cells in the tumor microenvironment.