CRMP1基因慢病毒干扰质粒构建及其对SH-SY5Y细胞NLRP3炎症小体蛋白表达的影响  

Construction of CRMP 1 lentiviral interference plasmid and its effect on expression of NLRP 3 inflammasome protein in SH-SY 5 Y cells

作　　者：王松豪 秦坤 韩雨 张微微 徐绍业 邵晓云[1] WANG Songhao;QIN Kun;HAN Yu;ZHANG Weiwei;XU Shaoye;SHAO Xiaoyun(Department of Human Anatomy,Guilin Medical University,Guilin 541199,China;Scientific Experiment Center,Guilin Medical University,Guilin 541199,China)

机构地区：[1]桂林医学院人体解剖学教研室,桂林541199 [2]桂林医学院科学实验中心,桂林541199

出　　处：《中国免疫学杂志》2025年第2期433-438,共6页Chinese Journal of Immunology

基　　金：广西自然科学基金(2021GXNSFAA075004,2022GXNSFAA035456,2017GXNSFAA198003);桂林医学院硕士研究生科研项目(GYYK2023009)。

摘　　要：目的:构建靶向坍塌反应调节蛋白1(CRMP1)基因的慢病毒干扰质粒,建立稳定敲减CRMP1的人神经母细胞瘤细胞(SH-SY5Y)稳转细胞系,探讨其对NLRP3炎症小体蛋白表达的影响。方法:以h-CRMP1 mRNA为靶序列设计合成shRNA双链,克隆至PLKO.1载体,构建CRMP1重组干扰质粒。将重组正确的shCRMP1质粒转染HEK-293T细胞进行慢病毒包装,获得的慢病毒上清浓缩后感染SH-SY5Y细胞,Western blot检测SH-SY5Y细胞内CRMP1的干扰效果及其对NLRP3炎症小体各组分蛋白表达的影响。结果:DNA测序结果显示,pLKO.1-shCRMP1慢病毒干扰质粒构建成功;转染HEK-293T细胞包装慢病毒后,细胞内CRMP1蛋白表达下降;慢病毒浓缩液感染SH-SY5Y细胞经嘌呤霉素筛选,Western blot发现shCRMP1慢病毒能显著下调SH-SY5Y细胞中CRMP1蛋白表达。同时稳定敲减CRMP1的SH-SY5Y细胞系中发现,MPP^(+)诱导下,抑制CRMP1表达可有效抑制NLRP3炎症小体活化。结论:成功构建pLKO.1-shCRMP1慢病毒干扰质粒,且干扰CRMP1可抑制MPP^(+)诱导的SH-SY5Y细胞NLRP3炎症小体活化,为进一步研究CRMP1在帕金森病等神经退行性疾病中的发生机制奠定基础。Objective:To construct a lentiviral interference plasmid targeting collapse response regulatory protein 1(CRMP1)gene,to establish a human neuroblastoma cell line(SH-SY5Y)with stable CRMP1 knockdown,and to investigate its impact on expression of NLRP3 inflammasome protein.Methods:Double-stranded shRNA was designed and synthesized targeting h-CRMP1 mRNA sequence,and cloned into PLKO.1 vector.Recombinant shCRMP1 plasmids were constructed correctly,which was transfected into HEK-293T cells for lentiviral packaging.Obtained lentivirus supernatant was concentrated and then infected into SH-SY5Y cells.The interference effect of shCRMP1 plasmid and protein expressions of NLRP3 inflammasome components in SH-SY5Y cells were detected by Western blot.Results:DNA sequencing results showed that insertion sequences of recombinant interference plasmids pLKO.1-shCRMP1 were consistent with designed sequences,which confirmed successful construction of shCRMP1 lentivirus interfering plasmids and transfected into HEK-293T cells for lentivirus packaging,and protein level of CRMP1 in HEK-293T cells were decreased.SH-SY5Y cells were infected with lentivirus concentrate obtained from packaging and screened with puromycin.Western blot results showed that shCRMP1 recombinant lentiviral plasmids could significantly down-regulate CRMP1 protein expression in SH-SY5Y cells.It was also found that in SH-SY5Y cell line with stable CRMP1 knockdown,inhibition of CRMP1 expression could effectively inhibit NLRP3 inflammasome activation under MPP+induction.Conclusion:pLKO.1-shCRMP1 lentiviral interfering plasmids have been successfully constructed,and interference with CRMP1 can inhibit activation of NLRP3 inflammasome in MPP+-induced SH-SY5Y cells.This study provides guidance for further research on mechanism of CRMP1 in neurodegenerative diseases such as Parkinson's disease.

关 键 词：坍塌反应调节蛋白1 慢病毒干扰 人神经母细胞瘤细胞 NLRP3炎症小体 

分 类 号：Q74[生物学—分子生物学] R34[医药卫生—基础医学] R392.1