磁性纳米粒介导沉默PI3Kγ基因调控肿瘤相关巨噬细胞抗小鼠肺癌细胞效应的研究  

Silencing PI3Kγ Gene via Magnetic Nanoparticles Regulates the Effect of Tumor Associated Macrophages on Mouse Lung Cancer Cells

作　　者：陈全[1] 刘佳霖 贺静荣 范晓霞 吴蕊鑫 CHEN Quan;LIU Jialin;HE Jingrong;FAN Xiaoxia;WU Ruixin(Immune Research Centre,Department of Immunology,College of Basic Medicine,Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学免疫学研究中心,基础医学院免疫学教研室,重庆400016

出　　处：《中国细胞生物学学报》2020年第2期296-311,共16页Chinese Journal of Cell Biology

摘　　要：为了探讨超顺磁性Fe3O4纳米粒子(superparamagnetic iron oxide nanoparticles,SPIONs)介导的磷脂酰肌醇3激酶γ(phosphatidylinositol 3 kinaseγ,PI3Kγ)抑制表达调控的肿瘤相关巨噬细胞(tumor-associated macrophages,TAM)对小鼠Lewis肺癌细胞(Lewis lung carcinoma,LLC)增殖和凋亡的影响,该研究构建了能启动巨噬细胞(macrophage,MФ)特异性表达PI3Kγ催化亚基p100 siRNA的pSilencer-EGFP-SP-p110质粒,通过SPIONs负载成磁性纳米质粒复合物(SPIONs-DNA),在强磁作用下转染MФ,通过普鲁士蓝染色法检测SPIONs-DNA在细胞内的分布,Real-time PCR和Western blot检测细胞PI3Kγp110亚基的表达水平。建立M1、M2型MФ模型,将SPIONs-DNA在强磁作用下转染M2型MФ,通过Real-time PCR和Western blot鉴定细胞表型,明确M2型MФ转化为M1型的强度。采用Transwell系统建立SPIONs-DNA转染的M2型MФ与小鼠LLC细胞的共培养模型,通过锥虫蓝染色法检测LLC细胞的活细胞数并绘制细胞生长曲线,CCK-8法检测LLC细胞增殖情况,硝酸还原酶法检测共培养液上清中NO含量,流式细胞术检测LLC细胞凋亡情况。结果显示,制备的SPIONs-DNA在强磁作用下成功转染MФ并大量分布在细胞胞核周围,SPIONs-DNA转染组细胞PI3Kγp110 mRNA和蛋白表达水平显著低于空白细胞对照组(P<0.05)。建立的M1型MФ高表达iNOS(P<0.001),M2型MФ高表达ARG-1(P<0.001)。M2型MФ转染SPIONs-DNA后细胞iNOS mRNA和蛋白的表达显著增加(P<0.001),ARG-1 mRNA和蛋白的表达显著降低(P<0.01)。在共培养组中,SPIONs-DNA转染的M2型MФ组能大量分泌NO,LLC细胞生长和增殖能力显著降低(P<0.05),凋亡率显著增高(P<0.01)。结果表明,磁性纳米粒负载pSilencer-EGFP-SP-p110重组质粒能够特异性靶向抑制巨噬细胞PI3Kγp110的表达,诱导M2型MФ转化为M1型;其转染的M2型MФ可显著抑制LLC细胞的生长和增值,促进细胞凋亡,这与其大量分泌NO有关。该磁性纳米质粒复合物可诱导TAM发挥抗肿瘤作�The purpose of this research was to explore the effect of silencing PI3Kγ(phosphatidylinositol 3 kinaseγ)gene via SPIONs(superparamagnetic iron oxide nanoparticles)to regulate TAM(tumor-associated macrophages)on the proliferation and apoptosis of mouse LLC(Lewis lung carcinoma)cells.The pSilencer-EGFP-SPp110 plasmid which can specifically promote MФ(macrophages)to express siRNA of PIK3CG(PI3Kγ catalytic subunit p100)was constructed and loaded into SPIONs-DNA(magnetic nano-plasmid complexes)via SPIONs which was transfected into macrophages under strong magnetism.The distribution of SPIONs-DNA in the cells was detected by Prussian blue staining,and the expression of PI3Kγ p110 of transfected cells was detected by Real-time PCR and Western blot.The M1 and M2 MФ models were established,and SPIONs-DNA was transfected into M2 MФunder strong magnetism.The phenotype of the transfected cells was identified by Real-time PCR and Western blot,and the intensity of M2 MФ transformed into M1 was determined.The Transwell system was used to establish the co-culture models of SPIONs-DNA transfected M2 MФ with mouse LLC cells.The number of live cells of LLC was detected by trypan blue staining and the cell growth curve was drawn.The proliferation of LLC cells was detected by CCK-8 assay.The NO concentrations in supernatant of co-culture medium was detected by Nitrate reductase method.The apoptosis level of LLC cells was detected by flow cytometry.The results showed that the prepared SPIONs-DNA complex was successfully transfected into MФ under strong magnetism and distributed in a large amount around the cell nucleus.The mRNA and protein levels of PI3Kγ p110 in SPIONs-DNA transfected cells were significantly lower than those in the blank control group(P<0.05).The established M1 MФ highly expressed iNOS(P<0.001),and the M2 MФ highly expressed ARG-1(P<0.001).After SPIONs-DNA transfection,the expression levels of iNOS mRNA and protein in M2 cells increased significantly(P<0.01),and the expression of ARG-1 mRNA and protein

关 键 词：巨噬细胞 PI3Kγ 超顺磁性Fe3O4纳米粒子 Lewis肺癌 增殖 凋亡 

分 类 号：R735.2[医药卫生—肿瘤]