NLRP3炎症小体激活对胰腺星状细胞增殖、迁移及细胞外基质沉积的影响  

Effects of NLRP3 inflammasome activation on the proliferation,migration and extracellular matrix deposition of pancreatic stellate cells

作　　者：顾海涛 董汉光 阎九亮 亓子豪 胡倍源 武春涛 龙江 Gu Haitao;Dong Hanguang;Yan Jiuliang;Qi Zihao;Hu Beiyuan;Wu Chuntao;Long Jiang(Department of Pancreatic Surgery,Shanghai General Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200080,China;Shanghai Key Laboratory of Pancreatic Disease,Shanghai 200080,China)

机构地区：[1]上海交通大学医学院附属第一人民医院胰腺外科,上海200080 [2]上海市胰腺疾病重点实验室,上海200080

出　　处：《中华胰腺病杂志》2023年第2期108-113,共6页Chinese Journal of Pancreatology

基　　金：国家自然基金青年项目(81400661)。

摘　　要：目的观察核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体激活对胰腺星状细胞(PSCs)增殖、迁移及细胞外基质沉积的影响。方法对大鼠PSCs进行分离培养与鉴定,根据是否给予细菌脂多糖(LPS)10μg/ml预处理24 h分为对照组和LPS组,ELISA法检测PSCs培养液中NLRP3炎症小体相关分子的表达;通过携带靶向NLRP3基因的shRNA慢病毒载体感染PSCs的方法构建NLRP3基因表达抑制的PSCs细胞株,根据有无LPS预处理和是否慢病毒干扰NLRP3表达,将PSCs分为LPS+阴性对照组和LPS+慢病毒组,采用CCK-8法和Transwell小室实验分别检测细胞增殖及迁移能力变化;采用免疫荧光染色检测PSCs细胞外基质α-SMA和collagen沉积;采用RT-qPCR检测PSCs促纤维化因子TGF-β的变化。结果培养24 h的PSCs胞质内富含高亮环形脂滴,细胞表达desmin。培养7 d后,细胞体积变大,脂滴基本消失,细胞活化并表达α-SMA。LPS组PSCs上清液中caspase-1、IL-1β、IL-18水平均显著高于对照组(1.55±0.04比0.65±0.03;2.02±0.04比1.05±0.05;1.70±0.05比0.97±0.03)。慢病毒感染PSCs后,慢病毒组NLRP3蛋白表达量(0.25±0.04)显著低于阴性对照组(0.68±0.05)。对照组、LPS组、LPS+阴性对照组、LPS+慢病毒组PSCs培养48 h后的A490值分别为0.61±0.02、1.15±0.06、0.96±0.05、0.56±0.01,迁移细胞数分别为(64.12±4.58)、(121.67±8.02)、(111.67±4.67)、(69.67±8.08)个/高倍视野,α-SMA蛋白沉积量分别为0.78±0.05、4.12±0.04、3.81±0.06、0.88±0.05,collagen蛋白沉积量分别为0.65±0.03、3.43±0.02、2.67±0.02、0.48±0.03,TGF-βmRNA表达量分别为0.22±0.03、0.89±0.01、0.86±0.03、0.43±0.02。LPS组、LPS+阴性对照组的A490值、迁移细胞数、α-SMA和collagen蛋白表达水平及TGF-βmRNA表达量均显著高于对照组及LPS+慢病毒组,差异均有统计学意义(P值均<0.05)。结论NLRP3炎症小体可通过调控PSCs生物学功能,促进其细胞增殖和迁移能力,从而加剧细胞外基质Objective To investigate the effects of NOD-like receptor protein 3(NLRP3)inflammasome activation on the proliferation,migration and extracellular matrix desposition of activated pancreatic stellate cells(PSCs).Methods The rat PSCs were isolated,cultured and identified,and were divided into control group or LPS group based on the pretreatment with LPS(10μg/ml for 24 hours)or without.The expression of NLRP3 inflammasome associated molecules in PSCs culture medium was detected by ELISA.The PSCs with NLRP3 inhibition were constructed by shRNA carrying lentivirus infection and were divided into LPS+negative control group and LPS+lentivirus group based on whether the cells were treated with LPS and infected by lentivirus or not.The alteration in cell proliferation and migration were detected by CCK-8 kit and transwell chamber method.The expression of extracellular matrixα-SMA and collagen in PSCs was detected by immunofluorescence staining and the expression of TGF-βmRNA was analyzed by RT-qPCR.Results The cytoplasm of PSCs which were cultured for 24 hours was rich in bright annular lipid droplets,and the cells expressed desmin.After 7 days of culture,the cell became larger in size,the lipid droplets basically disappeared,and the cells were activated and expressedα-SMA.The expression of caspase-1,IL-1βand IL-18 in the supernatant of PSCs culture medium in LPS group were significantly higher than those in control group(1.55±0.04 vs 0.65±0.03),(2.02±0.04 vs 1.05±0.05)and(1.70±0.05 vs 0.97±0.03),respectively.After inhibiting by lentivirus infection,the expression of NLRP3 in the lentivirus group(0.25±0.04)was significantly lower than that in negative control group(0.68±0.05).In control group,LPS group,LPS+negative control group and LPS+lentivirus group,the A490 values was 0.61±0.02,1.15±0.06,0.96±0.05,and 0.56±0.01,respectively;the migrating PSCs number was(64.12±4.58),(121.67±8.02),(111.67±4.67)and(69.67±8.08)/HF,respectively;the relative expression ofα-SMA was 0.78±0.05,4.12±0.04,3.81±0.06 and

关 键 词：NLRP3炎症小体 胰腺星形细胞 细胞外基质 纤维化 

分 类 号：R576[医药卫生—消化系统]