稳定敲降多腺苷二磷酸核糖聚合酶1的HaCaT细胞的建立

Establishment of HaCaT cells with stable knockdown of poly(ADP-ribose)polymerase-1

目的构建多腺苷二磷酸核糖聚合酶1(PARP-1)短发夹RNA(shRNA)的慢病毒载体质粒,获取稳定敲降PARP-1蛋白的人永生化表皮角化形成细胞HaCaT(PARP-1 KDHaCaT细胞)。方法针对PARP-1 mRNA设计干扰序列shRNA,用限制性内切酶技术将干扰序列shRNA插入慢病毒载体质粒pLVX-shRNA2-puro,构建含PARP-1 shRNA的慢病毒载体质粒(pLVX-shRNA2-puro-PARP-1),酶切和核酸测序鉴定该质粒是否构建成功。采用脂质体转染法将该质粒载体转染至HEK293T细胞包装慢病毒。将带有PARP-1 shRNA的慢病毒颗粒〔感染复数为100〕感染HaCaT细胞,经嘌呤霉素3 mg·L^(-1)筛选15 d,用Western印迹法和实时定量PCR(RT-qPCR)检测PARP-1蛋白和mRNA表达水平,验证PARP-1 KD HaCaT细胞PAPR-1敲降效果。用相同方式构建阴性对照细胞(NCHaCaT细胞)。随后分别将NCHaCaT和PARP-1 KDHaCaT细胞分为细胞对照组、DNA损伤剂硫芥(SM)100和1000μmol·L^(-1)组,孵育6和24 h后制备细胞裂解液,用Luminex法测定磷酸化组蛋白H2AX(γ-H2AX)表达水平。结果DNA测序和酶切结果表明,所构建的慢病毒载体质粒中含有PARP-1 shRNA序列,表明含PARP-1 shRNA的慢病毒载体质粒构建成功。RT-qPCR结果显示,PARP-1 KDHaCaT细胞中PARP-1 mRNA水平降低为NCHaCaT细胞的约14%;Western印迹法结果显示,PARP-1 KDHaCaT细胞中PARP-1蛋白表达水平降低为NCHaCaT细胞的约10%。DNA损伤剂SM 1000μmol·L^(-1)作用24 h后,与NCHaCaT细胞相比,PARP-1 KDHaCaT细胞γ-H2AX表达水平明显增加(P<0.01)。结论成功构建含PARP-1 shRNA的慢病毒载体质粒,并获得稳定敲降PARP-1的HaCaT细胞。

OBJECTIVE To construct a lentiviral vector with short hairpin RNA(shRNA)targeting poly(ADP-ribose)polymerase-1(PARP-1)gene and to obtain human immortalized keratinocytes HaCaT cells with stable PARP-1 knockdown(PARP-1 KD HaCaT cells).METHODS Interfering sequences were designed according to the mRNA sequences of PARP-1.The shRNA was inserted into the lentiviral vector plasmid pLVX-shRNA2-puro by restriction endonuclease technology.Enzyme digestion and nucleic acid sequencing technology were used to identify if the plasmid was successfully constructed.The aquired plasmid was transfected into HEK293T cells by liposome transfection to produce functional virus.Lentiviral particles with PARP-1 shRNA were transfected into HaCaT cells with 100 MOI(multiplicity of infection).Mono-clone PARP-1 KD HaCaT cells were picked out 15 d after puromycin 3 mg·L^(-1) screening.The PARP-1 protein and mRNA expressions were identified with Western blotting and real time-quanti⁃tative PCR(RT-qPCR)to find out if PARP-1 KD HaCaT cells were successfully constructed.Negative control(NC)HaCaT cells were constructed in the same way.Then,NC HaCaT cells and PARP-1 KD HaCaT cells were divided into cell control,DNA damage agent sulfur mustard(SM)100 and 1000μmol·L^(-1) groups,respectively.The cell lysate was prepared after incubation of SM for 6 and 24 h,and the expression level of phosphorylated histone H2AX(γ-H2AX)was determined using the Luminex method.RESULTS The results of sequencing and enzyme digestion showed that the constructed lentiviral vector plasmid contained PARP-1 shRNA sequence,indicating that the lentivirus vector plasmid containing PARP-1 shRNA was successfully constructed.RT-qPCR showed that the level of PARP-1 mRNA in PARP-1 KD HaCaT cells decreased to about 14%of that in NC HaCaT cells.Western blotting showed that the expression level of PARP-1 protein in PARP-1 KD HaCaT cells decreased to about 10%of that in NC HaCaT cells.After exposure to SM 1000μmol·L^(-1) for 24 h,compared with NC HaCaT cells,the expression level ofγ-H2AX in PARP-1 KD HaCaT cells significantly increased(P<0.01).CONCLU⁃SION The lentivirus plasmid vector containing PARP-1 shRNA is successfully constructed,and stable PARP-1 KD HaCaT cells are obtained.