中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2015年
1期
1-6
,共6页
张林%张英可%桂连%章旭之%黄俊琪
張林%張英可%桂連%章旭之%黃俊琪
장림%장영가%계련%장욱지%황준기
THP-1细胞%let-7e%凋亡%细胞因子
THP-1細胞%let-7e%凋亡%細胞因子
THP-1세포%let-7e%조망%세포인자
THP -1 cell%let-7e%Apoptosis%Cytokine
目的:研究let-7e对单核细胞系THP-1细胞凋亡和细胞因子分泌的影响及其机制。方法免疫荧光法检测cy3标记的mimic 对照转染THP-1细胞转染率,qRT-PCR法检测let-7e mimic及对照、let-7e inhibitor及对照转染THP1-细胞后let-7e的表达;MTT法检测转染后THP-1细胞活性,流式细胞术检测细胞凋亡,Western blot法检测let-7e靶基因IFI6(interferon alpha-inducible protein 6)、EZH2(enhancer of zeste homolog 2)、caspase-3的表达;转染48h 后THP-1细胞用LPS 刺激2 h收集细胞培养上清,细胞因子芯片法检测上清中炎性因子的表达。结果 miRNA模拟物可以转染THP-1细胞,转染率约为75%,转染 let-7e mimic 12h 、24 h、48 h 后THP-1细胞的 let-7e 表达倍数分别为8.551±0.365、83.893±15.941、38.858±2.743,转染 let-7e inhibitor 12 h、24 h、48 h后THP-1细胞的let-7e表达倍数分别为0.594±0.174、2.427±1.229、3.053±0.207;let-7e mimic转染组THP-1细胞活性升高、凋亡率降低;miranda数据库分析证实let-7e与EZH2、IFI6、caspase-3结合的mirSVR score 分别为-0.1608、-0.5693、-09.423,证实IFI6、EZH2、caspase-3可能是let-7e的靶基因;let7-e mimic转染组IFI6、EZH2、caspase-3表达都有所下降;let-7e mimic 转染组 CD154、粒细胞集落刺激因子( G-CSF)、CD54、IL-13、IL-1RA和IL-23表达有所下降。结论 let-7e有抗THP-1细胞凋亡的作用,可影响LPS诱导的细胞因子的表达。 let-7e可能通过调节其靶基因caspase-3、IFI6、EZH2对THP-1细胞的生物学功能产生影响。
目的:研究let-7e對單覈細胞繫THP-1細胞凋亡和細胞因子分泌的影響及其機製。方法免疫熒光法檢測cy3標記的mimic 對照轉染THP-1細胞轉染率,qRT-PCR法檢測let-7e mimic及對照、let-7e inhibitor及對照轉染THP1-細胞後let-7e的錶達;MTT法檢測轉染後THP-1細胞活性,流式細胞術檢測細胞凋亡,Western blot法檢測let-7e靶基因IFI6(interferon alpha-inducible protein 6)、EZH2(enhancer of zeste homolog 2)、caspase-3的錶達;轉染48h 後THP-1細胞用LPS 刺激2 h收集細胞培養上清,細胞因子芯片法檢測上清中炎性因子的錶達。結果 miRNA模擬物可以轉染THP-1細胞,轉染率約為75%,轉染 let-7e mimic 12h 、24 h、48 h 後THP-1細胞的 let-7e 錶達倍數分彆為8.551±0.365、83.893±15.941、38.858±2.743,轉染 let-7e inhibitor 12 h、24 h、48 h後THP-1細胞的let-7e錶達倍數分彆為0.594±0.174、2.427±1.229、3.053±0.207;let-7e mimic轉染組THP-1細胞活性升高、凋亡率降低;miranda數據庫分析證實let-7e與EZH2、IFI6、caspase-3結閤的mirSVR score 分彆為-0.1608、-0.5693、-09.423,證實IFI6、EZH2、caspase-3可能是let-7e的靶基因;let7-e mimic轉染組IFI6、EZH2、caspase-3錶達都有所下降;let-7e mimic 轉染組 CD154、粒細胞集落刺激因子( G-CSF)、CD54、IL-13、IL-1RA和IL-23錶達有所下降。結論 let-7e有抗THP-1細胞凋亡的作用,可影響LPS誘導的細胞因子的錶達。 let-7e可能通過調節其靶基因caspase-3、IFI6、EZH2對THP-1細胞的生物學功能產生影響。
목적:연구let-7e대단핵세포계THP-1세포조망화세포인자분비적영향급기궤제。방법면역형광법검측cy3표기적mimic 대조전염THP-1세포전염솔,qRT-PCR법검측let-7e mimic급대조、let-7e inhibitor급대조전염THP1-세포후let-7e적표체;MTT법검측전염후THP-1세포활성,류식세포술검측세포조망,Western blot법검측let-7e파기인IFI6(interferon alpha-inducible protein 6)、EZH2(enhancer of zeste homolog 2)、caspase-3적표체;전염48h 후THP-1세포용LPS 자격2 h수집세포배양상청,세포인자심편법검측상청중염성인자적표체。결과 miRNA모의물가이전염THP-1세포,전염솔약위75%,전염 let-7e mimic 12h 、24 h、48 h 후THP-1세포적 let-7e 표체배수분별위8.551±0.365、83.893±15.941、38.858±2.743,전염 let-7e inhibitor 12 h、24 h、48 h후THP-1세포적let-7e표체배수분별위0.594±0.174、2.427±1.229、3.053±0.207;let-7e mimic전염조THP-1세포활성승고、조망솔강저;miranda수거고분석증실let-7e여EZH2、IFI6、caspase-3결합적mirSVR score 분별위-0.1608、-0.5693、-09.423,증실IFI6、EZH2、caspase-3가능시let-7e적파기인;let7-e mimic전염조IFI6、EZH2、caspase-3표체도유소하강;let-7e mimic 전염조 CD154、립세포집락자격인자( G-CSF)、CD54、IL-13、IL-1RA화IL-23표체유소하강。결론 let-7e유항THP-1세포조망적작용,가영향LPS유도적세포인자적표체。 let-7e가능통과조절기파기인caspase-3、IFI6、EZH2대THP-1세포적생물학공능산생영향。
Objective To investigate the effects of a microRNA family member , let-7e, on mono-cytic cell line THP-1 with regard to cell apoptosis and cytokine secretion and to analyze the possible mecha -nism.Methods THP-1 cells were transfected with mimic negative control (cy3) and observed with immu-nofluorescence microscopy for the evaluation of transfection rate .The expression of let-7e in THP-1 cells re-spectively transfected with let-7e mimic, mimic negative control, let-7e inhibitor and inhibitor negative con-trol were detected by qRT-PCR.MTT assay and flow cytometry analysis were used to detect the activities and apoptosis of transfected THP-1 cells.Western blot assay was performed to measure the expression of the genes encoding interferon alpha-inducible protein 6( IFI6 ) , enhancer of zeste homolog 2 (EZH 2 ) and caspase -3 that were target genes of let-7e predicted by bioinformatics analysis .THP-1 cells were transfected with let-7e mimic and mimic negative control for 48 h and then stimulated with LPS for 2 h for further detec-tion.The supernatants of cell culture were collected for the detection of secreted cytokines by Human Cyto -kine Array.Results The monocytic THP-1 cells were transfected with mimic negative control with a trans-fection efficiency of about 75%.There were 8.551±0.365, 83.893±15.941, 38.858±2.743 and 0.594± 0.174, 2.427±1.229, 3.053±0.207 fold increases in let-7e expression after the transient transfection of THP-1 cells with let-7e mimic and let-7e inhibitor for 12 h, 24 h and 48 h, respectively.The transfection of let-7e mimic into THP-1 cells enhanced the cell activities and inhibited the apoptosis of the transfected cells . Bioinformatics analysis showed that let-7e bound to the genes encoding EZH 2, IFI6 and caspase-3 with the mirSVR scores of -0.1608,-0.5693 and-0.9423, suggesting them as the predicted target genes of let-7e. The expressions of IFI6, EZH2 and caspase-3 in let-7e mimic transfected THP-1 cells were decreased as in-dicated by Western blot assay .The results of Human Cytokine Array showed that the expression of LPS-in-duced cytokines including CD154, G-CSF, CD54, IL-13, IL-1RA and IL-23 were inhibited in let-7e mimic transfected THP-1 cells. Con clusion Let-7e had an anti-apoptosis effect on monocytic THP-1 cells and in-fluenced the secretion of LPS-induced cytokines in THP-1 cells.Let-7e might regulate the biological function of THP-1 cells through inhibiting the expression of target genes encoding caspase -3, IFI6 and EZH2.