Zhang Li, Liu Hou-jia, Li Tie-jun, Yang Yang, Guo Xian-ling, Wu Meng-chao, Rui Yao-cheng, Wei Li-xin
Tumor Immunology and Gene Therapy Center, Second Military Medical University, Shanghai 200438, China.
Acta Pharmacol Sin. 2008 Jul;29(7):847-52. doi: 10.1111/j.1745-7254.2008.00823.x.
To investigate the expression of scavenger receptor that binds phosphatidylserine and oxidized lipoprotein (SR-PSOX)/CXC chemokine ligand 16 (CXCL16) in the human monocyte-derived cell line THP-1, and the effect of lentiviral vectors for the stable delivery of SR-PSOX/CXCL16 short hairpin RNA on foam cell formation.
A lentiviral expression vector containing enhanced green fluorescence protein (GFP) and SR-PSOX small interfering RNA (siRNA) (Lenti-SR-PSOXsi), or the control siRNA (Lenti-NC) gene was constructed. A human monocyte-derived cell line THP-1 was transfected with a different multiplicity of infection (MOI) of Lenti-SR-PSOXsi or Lenti-NC, and cultured to obtain stably-transfected THP- 1KD and THP-1NC cells. After incubation with oxidatively-modified, low-density lipoprotein (Ox-LDL), the expression of SR-PSOX/CXCL16 mRNA was determined by real-time PCR. The expression of the SR-PSOX/CXCL16 protein was detected by flow cytometry analysis. The effect of Lenti-SR-PSOXsi on foam cell formation was assessed by Oil red O-stain analysis.
Ox-LDL increased the expression of SR-PSOX/CXCL16 mRNA in a time- and dose-dependent manner in THP-1 cells. Four days after transfection with Lenti-SR-PSOXsi (MOI: 100), the percentage of GFP expression cells was over 89.3%. The expression of the SR-PSOX/ CXCL16 mRNA and protein in THP-1KD cells significantly decreased compared with the parent cells, even the THP-1KD cells stimulated with 40 mg/L Ox-LDL. Ox-LDL uptake experiments in THP-1- and THP-1KD-derived macrophages indicated that SR-PSOX/CXCL16 deficiency decreased the development of macrophage- derived foam cell formation.
The above data showed that SRPSOX siRNA delivered by using lentiviral vectors in THP-1 cells was a powerful tool for studying the effect of SR-PSOX, and decreased the expression of the SRPSOX gene by inhibiting macrophage-derived foam cell formation.
研究磷脂酰丝氨酸和氧化脂蛋白结合清道夫受体（SR-PSOX）/CXC趋化因子配体16（CXCL16）在人单核细胞衍生细胞系THP-1中的表达，以及慢病毒载体稳定递送SR-PSOX/CXCL16短发夹RNA对泡沫细胞形成的影响。
构建含增强型绿色荧光蛋白（GFP）和SR-PSOX小干扰RNA（siRNA）的慢病毒表达载体（Lenti-SR-PSOXsi）或对照siRNA（Lenti-NC）基因。用不同感染复数（MOI）的Lenti-SR-PSOXsi或Lenti-NC转染人单核细胞衍生细胞系THP-1，培养获得稳定转染的THP-1KD和THP-1NC细胞。用氧化修饰的低密度脂蛋白（Ox-LDL）孵育后，通过实时PCR测定SR-PSOX/CXCL16 mRNA的表达。通过流式细胞术分析检测SR-PSOX/CXCL16蛋白的表达。通过油红O染色分析评估Lenti-SR-PSOXsi对泡沫细胞形成的影响。
Ox-LDL以时间和剂量依赖性方式增加THP-1细胞中SR-PSOX/CXCL16 mRNA的表达。用Lenti-SR-PSOXsi（MOI：100）转染4天后，GFP表达细胞的百分比超过89.3%。与亲本细胞相比，THP-1KD细胞中SR-PSOX/CXCL16 mRNA和蛋白的表达显著降低，即使是用40 mg/L Ox-LDL刺激的THP-1KD细胞。THP-1和THP-1KD来源的巨噬细胞的Ox-LDL摄取实验表明，SR-PSOX/CXCL16缺乏会减少巨噬细胞衍生的泡沫细胞形成的发展。
上述数据表明，在THP-1细胞中使用慢病毒载体递送SRPSOX siRNA是研究SR-PSOX作用的有力工具，并通过抑制巨噬细胞衍生的泡沫细胞形成降低了SRPSOX基因的表达。
