CAJ | 학술논문

目的：构建针对NLRP3基因的短发夹RNA（shRNA）慢病毒表达载体，在HepG2细胞中鉴定该载体对NLRP3的抑制效果。方法：设计针对NLRP3基因的shRNA序列，将其插入慢病毒表达载体pWPT-U6-shRNA-CMV-GFP中，将载体与包装质粒psPAX2、pMD2.G共转染293T细胞，进行病毒包装；得到的病毒原液浓缩后系列稀释为9个浓度递减的病毒液，分别转染293T细胞后进行滴度测定；用获得的慢病毒液感染人肝癌细胞系HepG2，获得稳定沉默NLRP3的细胞株；利用实时定量PCR和Western印迹检测稳定细胞株中NLRP3的沉默效应。结果：构建了具有沉默效应的慢病毒干扰载体；稀释法测定干扰病毒滴度为2×108 TU/mL；实时定量PCR和Western印迹实验均证实慢病毒转染后，细胞株中NLRP3表达水平明显降低。结论：构建了人NLRP3基因特异性慢病毒干扰载体,获得NLRP3基因稳定干扰的HepG2细胞株。
목적：구건침대NLRP3기인적단발협RNA（shRNA）만병독표체재체，재HepG2세포중감정해재체대NLRP3적억제효과。방법：설계침대NLRP3기인적shRNA서렬，장기삽입만병독표체재체pWPT-U6-shRNA-CMV-GFP중，장재체여포장질립psPAX2、pMD2.G공전염293T세포，진행병독포장；득도적병독원액농축후계렬희석위9개농도체감적병독액，분별전염293T세포후진행적도측정；용획득적만병독액감염인간암세포계HepG2，획득은정침묵NLRP3적세포주；이용실시정량PCR화Western인적검측은정세포주중NLRP3적침묵효응。결과：구건료구유침묵효응적만병독간우재체；희석법측정간우병독적도위2×108 TU/mL；실시정량PCR화Western인적실험균증실만병독전염후，세포주중NLRP3표체수평명현강저。결론：구건료인NLRP3기인특이성만병독간우재체,획득NLRP3기인은정간우적HepG2세포주。
Objective: To construct a recombinant lentiviral vector of short hairpin RNA(shRNA) targeting NLRP3 gene, and to identify its inhibitory effect in HepG2 cells. Methods: The NLRP3 shRNA sequence was designed and inserted into the lentiviral vector pWPT-U6-shRNA-CMV-GFP. The lentiviral vector was further packaged with accessory plasmids into lentivirus in 293T cells. The virus solution was concentrated and serially diluted into 9 levels, and transfected into 293T cells respectively for the titer determination. The resulted lentivirus was infect?ed HepG2 cell to stably suppress NLRP3 expression, and the inhibition effect was detected by quantitative real-time PCR(qRT-PCR) and Western blotting. Results: A lentiviral vector carrying NLRP3 shRNA was constructed. The virus titer was 2×108 TU/mL detected by dilution method. The NLRP3 expression in HepG2 cells was down-regulated significantly, confirming by the qRT-PCR and Western blotting. Conclusion: The constructed lentiviral vector targeting human NLRP3 gene can stably suppress NLRP3 expression in HepG2 cell lines.