CAJ | 학술논문

目的探讨小鼠黑素瘤细胞B16F10与小鼠单核巨噬细胞RAW264.7共培养中Notch-RBP-J信号通路对巨噬细胞表型分化的影响.方法设计针对CBF1/RBP-Jκ基因的siRNA编码序列,转染至小鼠单核巨噬细胞RAW264.7细胞.实验分4组:沉默前共培养组,B16F10细胞与RAW264.7细胞共培养;沉默后共培养组,B16F10细胞与RBP-Jκ基因沉默后的RAW264.7细胞共培养;空白对照组,单独培养的RAW264.7细胞;阳性对照组,白细胞介素4刺激诱导的RAW264.7细胞.Western印迹法、ELISA法及流式细胞仪检测各组巨噬细胞的表型;RT-PCR法检测各组notch1、notch2、DLL1、DLL4及Hes1的表达.采用SPSS17.0软件进行重复测量方差分析及单因素方差分析、线性趋势检验、Bonferroni两两比较.结果 Western印迹结果显示,RAW264.7与B16F10共培养不同时间后,RAW264.7细胞的CD163在空白对照组、共培养24 h、48 h、72 h组、阳性对照组的相对表达量分别为1.016±0.018、1.274±0.034、2.065±0.094、3.615±0.144、3.099±0.071,经单因素方差分析(n=4),F=527.42,P＜ 0.01,共培养后RAW264.7细胞CD163表达量较空白对照组增加;ELISA检测结果显示,共培养24、48、72 h,RAW264.7细胞培养上清液白细胞介素10的水平分别为(167.610±3.527)、(433.433±5.558)、(679.673±8.101) ng/L.沉默后共培养24、48、72 h,RAW264.7细胞培养上清液白细胞介素10表达量分别为(63.403±0.856)、(103.427±2.072)、(202.297±3.610) ng/L,较未沉默时降低(F=8.01,P＜ 0.05).Western印迹法及流式细胞仪分别检测沉默后共培养RAW264.7细胞CD163、CD206的表达量,均较未沉默时减少(P＜0.05).RT-PCR示,沉默后共培养组较沉默前共培养组及对照组的Notch信号通路相关基因mRNA表达均降低.结论沉默Notch-RBP-J信号通路后共培养组中RAW264.7细胞向M2型极化较未沉默共培养组减少,总体表型仍为M2型;该通路的活化促进RAW264.7细胞向M2型极化.
목적 탐토소서흑소류세포B16F10여소서단핵거서세포RAW264.7공배양중Notch-RBP-J신호통로대거서세포표형분화적영향.방법 설계침대CBF1/RBP-Jκ기인적siRNA편마서렬,전염지소서단핵거서세포RAW264.7세포.실험분4조:침묵전공배양조,B16F10세포여RAW264.7세포공배양;침묵후공배양조,B16F10세포여RBP-Jκ기인침묵후적RAW264.7세포공배양;공백대조조,단독배양적RAW264.7세포;양성대조조,백세포개소4자격유도적RAW264.7세포.Western인적법、ELISA법급류식세포의검측각조거서세포적표형;RT-PCR법검측각조notch1、notch2、DLL1、DLL4급Hes1적표체.채용SPSS17.0연건진행중복측량방차분석급단인소방차분석、선성추세검험、Bonferroni량량비교.결과 Western인적결과현시,RAW264.7여B16F10공배양불동시간후,RAW264.7세포적CD163재공백대조조、공배양24 h、48 h、72 h조、양성대조조적상대표체량분별위1.016±0.018、1.274±0.034、2.065±0.094、3.615±0.144、3.099±0.071,경단인소방차분석(n=4),F=527.42,P＜ 0.01,공배양후RAW264.7세포CD163표체량교공백대조조증가;ELISA검측결과현시,공배양24、48、72 h,RAW264.7세포배양상청액백세포개소10적수평분별위(167.610±3.527)、(433.433±5.558)、(679.673±8.101) ng/L.침묵후공배양24、48、72 h,RAW264.7세포배양상청액백세포개소10표체량분별위(63.403±0.856)、(103.427±2.072)、(202.297±3.610) ng/L,교미침묵시강저(F=8.01,P＜ 0.05).Western인적법급류식세포의분별검측침묵후공배양RAW264.7세포CD163、CD206적표체량,균교미침묵시감소(P＜0.05).RT-PCR시,침묵후공배양조교침묵전공배양조급대조조적Notch신호통로상관기인mRNA표체균강저.결론 침묵Notch-RBP-J신호통로후공배양조중RAW264.7세포향M2형겁화교미침묵공배양조감소,총체표형잉위M2형;해통로적활화촉진RAW264.7세포향M2형겁화.
Objective To investigate the effect of the Notch-RBP-J signaling pathway on the phenotypic differentiation of RAW264.7 murine macrophage-like cells cocultured with B16F10 murine melanoma cells.Methods A small interference RNA (siRNA) targeting the CBF1/RBP-Jκ gene was designed.RAW264.7 murine macrophage-like cells were divided into four groups:unsilenced co-culture group cocultured with B16F10 cells,silenced co-culture group transfected with the designed siRNA and cocultured with B16F10 cells,blank control group cultured alone,positive control group induced by interleukin-4 (IL-4).After additional culture for different durations,Western blot,enzyme-linked immunosorbent assay (ELISA) and flow cytometry were conducted to determine the phenotype of macrophages,and reverse transcription (RT)-PCR was performed to detect the expressions of notch1,notch2,DLL1,DLL4 and Hes1 genes in macrophages.Statistical analysis was carried out by one-way analysis of variance (ANOVA),repeated measures ANOVA,linear trend test and the Bonferroni method with the SPSS17.0 software.Results Western blot showed that the relative expression level of CD163 in RAW264.7 cells was 1.016 ± 0.018 in the blank control group,1.274 ± 0.034,2.065 ± 0.094 and 3.615 ± 0.144 in the unsilenced co-culture group at 24,48 and 72 hours respectively,and 3.099 ± 0.071 in the positive control group,with significant differences between these groups (n =4,F =527.42,P ＜ 0.01).There was a significant increase in CD163 expression in RAW264.7 cells in the unsilenced co-culture group compared with the blank control group.As ELISA revealed,the levels of IL-10 in the culture supernatant of RAW264.7 cells were (167.61 ± 3.527),(433.433 ± 5.558) and (679.673 ± 8.101) ng/L in the unsilenced co-culture group at 24,48,and 72 hours respectively,significantly higher than those in the silenced co-culture group ((63.403 ± 0.856),(103.427 ± 2.072),(202.297 ± 3.61) ng/L,respectively,F =8.01,P ＜ 0.05).Western blot and flow cytometry both demonstrated a statistical reduction in the expressions of CD163 and CD206 in RAW264.7 cells in the silenced co-culture group compared with the unsilenced co-culture group (both P ＜ 0.05).The mRNA expressions of notch signaling pathwayrelated genes in RAW264.7 cells were also attenuated in the silenced co-culture group in comparison with the unsilenced co-culture group.Conclusions Although most of the RAW264.7 cells in the silenced co-culture group exhibited the M2 phenotype,their polarization toward M2 phenotype was weakened compared with those in the unsilenced co-culture group,implying that the activation of the Notch-RBP-J signaling pathway promotes the M2-polarization of RAW264.7 cells.