慢病毒介导SIRT1基因表达降低的ATDC5细胞模型建立

Establishment of an ATDC5 cell model with lentivirus-mediated SIRT1 gene knockdown

背景:沉默信息调节因子1(silent information regulator 1,SIRT1)基因是一个与衰老关系密切的基因,可以通过调节PI3K/AKT、NF-κB等信号通路影响到组织及机体的衰老过程,并参与骨科相关疾病的发生发展。建立慢病毒介导的SIRT1基因表达降低的ATDC5细胞模型,可以为研究SIRT1基因相关的骨科系统疾病如骨关节炎、骨质疏松症等提供实验基础。目的:构建携带小鼠SIRT1基因的RNAi慢病毒载体,将其转染ATDC5细胞系,建立SIRT1基因表达降低的ATDC5细胞模型。方法:根据RNAi序列设计原则设计并合成SIRT1基因的RNAi靶点序列,连接GV248(框架结构:h U6-MCS-Ubiquitin-EGFP-IRES-puromycin)载体,转化后经阳性克隆测序及PCR鉴定,质粒抽提后导入293T细胞,包装慢病毒,取上清检测滴度。将含有小鼠SIRT1基因RNAi的病毒颗粒转染ATDC5细胞系,并于荧光显微镜下进行观察,行RT-PCR检测ATDC5细胞中SIRT1 m RNA的表达情况。结果:经测序、PCR检测可知,SIRT1基因的RNAi靶点序列与GV248载体连接成功,并包装成高滴度的慢病毒。携带小鼠SIRT1基因的RNAi慢病毒感染ATDC5细胞后,荧光显微镜下观察显示感染率较高,RT-PCR检测发现,携带SIRT1基因的慢病毒感染组SIRT1基因m RNA的相对表达量为0.386±0.117,与阴性对照病毒感染组相比有统计学差异(P<0.05),敲减效率为61.4%。结论:成功建立SIRT1基因表达降低的ATDC5细胞模型,为研究SIRT1基因相关骨科系统疾病提供实验基础。

Background：Silent information regulator 1 （SIRT1） gene has a close relationship with aging. It can affect the aging process of tissue and organism through regulating PI3K/AKT, NF-κB signaling pathways and participate in the development of orthopedic diseases. An ATDC5 cell model with lentivirus-mediated SIRT1 gene knockdown can provide experi-mental basis for the study of SIRT1 gene related orthopedic diseases such as osteoarthritis and osteoporosis. Objective：To construct a lentiviral vector for RNA interference （RNAi） of mouse SIRT1 gene which was transfected into ATDC5 cell line to establish an ATDC5 cell model with SIRT1 gene knockdown. Methods：According to the design principle of the RNAi target sequence, RNAi target sequence of SIRT1 gene was designed and synthesized. And then the sequence was connected with GV248 （hU6-MCS-Ubiquitin-EGFP-IRES-puromycin） vector. After PCR identification and clone sequencing, the plas-mid was extracted and transfected into 293T cells. The lentiviruses were packed and the supernatant was collected to detect titer. The lentiviruses containing mouse SIRT1 gene RNAi sequence were transfected into ATDC5 cell line which was observed under fluorescence microscope. RT-PCR was used to detect the expression of SIRT1 mRNA in ATDC5 cells. Results：SIRT1 gene RNAi sequence was successfully connected with GV248 vector through PCR and sequence analysis and pack-aged into high titer lentivirus. After the lentivirus containing mouse SIRT1 gene RNAi sequence were transfected into AT-DC5 cells, the high infection rate was found under fluorescence microscope. RT-PCR showed that the expression of SIRT1 mRNA was 0.386±0.117, which was significantly different from the negative control group （P〈0.05）. The knockdown effi-ciency was 61.4%. Conclusions：An ATDC5 cell model with lentivirus-mediated SIRT1 gene knockdown has been successfully established. It can provide experimental basis for the study of SIRT1 gene related orthopedics diseases.