lncRNA MRAK050699对二氧化硅粉尘诱导大鼠肺泡Ⅱ型上皮细胞上皮-间质转换的影响  

Effect of lncRNA MRAK050699 on epithelial-mesenchymal transition in rat typeⅡalveolar epithelial cells induced by SiO_(2) dust

作　　者：高芳瑜 刘煊 田娣 陈溪渊 田雪艳 焦子铭 侯朋邑 王发选 GAO Fangyu;LIU Xuan;TIAN Di;CHEN Xiyuan;TIAN Xueyan;JIAO Ziming;HOU Pengyi;WANG Faxuan(School of Public Health and Management,Ningxia Medical University,Yinchuan,Ningxia 750004,China)

机构地区：[1]宁夏医科大学公共卫生与管理学院,宁夏银川750004

出　　处：《环境与职业医学》2021年第3期231-237,共7页Journal of Environmental and Occupational Medicine

基　　金：国家自然科学基金项目(81660534);宁夏高等教育科学研究项目(NGY2020035);宁夏自然科学基金项目(2020AAC02019)。

摘　　要：[背景]矽肺发病机制未明且没有特效治疗方法,长链非编码RNA(lncRNA)在矽肺纤维化进程中或可发挥作用。[目的]探讨lncRNA MRAK050699对SiO_(2)粉尘诱导大鼠肺泡Ⅱ型上皮细胞(RLE-6TN细胞)发生上皮-间质转换(EMT)过程的影响。[方法]建立大鼠肺泡巨噬细胞(NR8383细胞)与RLE-6TN细胞共培养体外矽肺模型,分为正常组、模型组、敲减对照组、敲减组。采用CCK8法检测NR8383细胞在10、20、40、80、160、320μg·cm^(-2)SiO_(2)粉尘刺激下的活力,酶联免疫吸附试验(ELISA)法检测在0、20、40、80μg·cm^(-2)粉尘刺激下的转化生长因子-β(TGF-β)含量,实时荧光定量PCR(RT-qPCR)法检测MRAK050699敲减效率。取四个6孔板,在前两个6孔板中正常培养RLE-6TN细胞24 h,后两个6孔板分别培养转染阴性对照病毒的RLE-6TN细胞24 h,和转染敲减MRAK050699病毒的RLE-6TN细胞24 h;同时在另四个6孔板中,插入Transwell小室,正常培养NR8383细胞24 h。NR8383细胞与RLE-6TN细胞铺板比例为1∶2,之后将Transwell小室连同NR8383细胞一同转入RLE-6TN细胞的6孔板中,向后三组共培养体系的Transwell小室中加入40μg·cm^(-2)SiO_(2)粉尘悬液150μL后,四组同时继续培养24 h,取Transwell小室下室的RLE-6TN细胞置于显微镜下观察,使用Western blotting及RT-qPCR法检测E-cadherin、N-cadherin、α-SMA的mRNA和蛋白表达水平。[结果]CCK8、ELISA法结果显示SiO_(2)剂量在40μg·cm^(-2)时,细胞活力下降较为明显且可保证后续实验的有效进行,上清液中TGF-β表达水平是对照组的2.94倍,可用于后续实验;敲减组MRAK050699的表达水平相比敲减对照组明显下调,敲减效率约为50%(P<0.05)。显微镜下观察共培养后RLE-6TN细胞形态,正常组细胞呈现典型的上皮样特征,为多边形、卵圆形且紧密连接。模型组细胞呈现间质样细胞形态,表现为长梭形、纺锤形,并且细胞间隙变宽。敲减对照组与模型组类似,呈现间质细胞形态。�[Background]The pathogenesis of silicosis is unknown and there is no specific treatment for the disease.Long-chain non-coding RNA(lncRNA)may play a role in the process of silicosis fibrosis.[Objective]This experiment aims to investigate the effect of lncRNA MRAK050699 on epithelial-mesenchymal transition(EMT)in rat typeⅡalveolar epithelial cells induced by SiO_(2) dust.[Methods]The rat alveolar macrophages(NR8383 cells)and RLE-6TN cells were co-cultured in vitro and divided into a normal group,a model group,a knockdown control group,and a knockdown group.The viabilities of NR8383 cells stimulated by 10,20,40,80,160,and 320μg·cm^(-2)SiO_(2) dust were detected by CCK8 method,the levels of transforming growth factor-β(TGF-β)stimulated by 0,20,40,and 80μg·cm^(-2)SiO_(2) dust were detected by ELISA,and the knockdown efficiency was tested by real-time quantitative PCR(RT-qPCR).RLE-6TN cells in two 6-well plates,RLE-6TN cells transfected with knockdown control virus in a plate,and RLE-6TN cells transfected with knockdown virus in a plate were cultured for 24 h.At the same time,the other four 6-well plates were inserted into the Transwell chamber,and NR8383 cells were cultured normally for 24 h.The ratio of NR8383 cells to RLE-6TN cells was 1:2.Then the Transwell chamber together with NR8383 cells were transferred into the 6-well plates of RLE-6TN cells,and 40μg·cm^(-2)SiO_(2) dust suspension was added to the Transwell chamber of the latter three groups of co-culture system.After further 24 h,the RLE-6TN cells in the lower compartment of the Transwell chamber were observed under microscope.The mRNA and protein expression levels of E-cadherin,N-cadherin,andα-SMA were detected by Western blotting and RT-qPCR respectively.[Results]CCK8 and ELISA results showed that the viability of NR8383 cells decreased significantly,and the survival rate was considered eligible when SiO_(2) was 40μg·cm^(-2),as the level of TGF-βin supernatant was high enough for subsequent experiments;the knockdown efficiency of the knockdow

关 键 词：矽肺 纤维化 长链非编码RNA 肺泡Ⅱ型上皮细胞 肺泡巨噬细胞 上皮-间质转换 

分 类 号：R11[医药卫生—公共卫生与预防医学]