ABRO1基因敲除小鼠骨髓来源巨噬细胞的永生化及其LPS/TLR4通路活化的检测

Immortalization of ABRO1 deficiency BMDMs and detection of LPS/TLR4 signaling pathway activation

目的永生化ABRO1基因敲除(KO)小鼠骨髓来源的巨噬细胞系(BMDM),并确定缺失ABRO1对巨噬细胞LPS/TLR4通路活化的影响。方法用p CDH-SV40LT-puro慢病毒分别感染野生型(WT)及ABRO1 KO小鼠的BMDM,连续传代培养>50代,得到永生化细胞系。利用MTS法和Ed U掺入法检测细胞增殖,AnnexinⅤ/7-AAD双染色后用流式细胞仪检测细胞凋亡。脂多糖(LPS)刺激细胞后,Western印迹法检测NF-κB及MAPK信号通路,并用流式微球阵列(CBA)检测细胞产生IL-6、TNF-α的水平。结果成功构建了ABRO1 WT和KO的永生化BMDM(i BMDM)。正常培养下,WT及KO i BMDM细胞增殖能力和细胞凋亡均无明显差异,但撤除血清后,KO i BMDM细胞凋亡明显高于WT。LPS刺激后,NF-κB、MAPK信号通路激活及IL-6、TNF-α的产生无明显差异。结论ABRO1缺失不影响LPS/TLR4信号通路。敲除ABRO1促进血清撤除诱导的i BMDM凋亡。

Objective To immortalize the ABRO1 knockout（KO） mouse bone marrow-derived macrophages（BMDMs） cell line and to determine the effect of knockout ABRO1 on the LPS/TLR4 signaling pathway activation in macrophages. Methods The p CDH-SV40 LT-puro lentivirus was infected into ABRO1 wild type（WT） and KO BMDMs.The cells were cultured for more than 50 passages without any crisis,which were immortalized BMDMs（i BMDMs）. The proliferation ability of the i BMDMs was measured by MTS assay and Ed U assay. The apoptosis detection was analyzed after Annexin Ⅴ/7-AAD staining by FACS. The NF-κB and MAPK pathways were detected by Western blot,and the IL-6 and TNF-α levels in the supernatant of cells were measured by cytometric bead array（CBA） assay in LPS-primed i BMDMs.Results The immortalized WT and KO i BMDMs were established. The unchanged proliferation ability and apoptosis of KO i BMDMs were observed under normal conditions. But the apoptosis of KO i BMDMs was significantly higher than that of WT after serum deprivation. ABRO1 deficiency had no effect on LPS-induced NF-κB and MAPK pathway activation,or on inflammasome-independent IL-6 and TNF-α secretion. Conclusion ABRO1 deficiency has no effect on LPS/TLR4 signaling pathway activation,but promotes the serum deprivation-induced apoptosis of i BMDMs.