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目的:成功构建稳定沉默表达IGF-1R基因质粒,并转染至L02细胞(人正常肝细胞),观察IGF-1R基因沉默对L02细胞过氧化损伤时经内质网途径特异性凋亡蛋白Caspase12表达的影响。方法:构建针对L02细胞IGF-1R基因的短发夹状RNA(shRNA),将4种不同序列的Sh-H-IGF1R-1108、Sh-H-IGF1R-2797、Sh-H-IGF1R-3215、Sh-H-IGF1R-3787质粒(设为实验组)以及含与IGF-1R无关序列的Sh-H-IGF1R-V1质粒(设为阳性对照组)和空白质粒Sh-H-IGF1R-V2(设为阴性对照组)分别转染L02细胞,24 h后用RTPCR方法检测IGF-1R表达水平,筛选出稳定沉默IGF-1R基因的质粒。随后进行下一步实验分组:空白对照组(A组)、过氧化损伤组(B组)、阴性质粒转染组(C组)、目的基因转染组(D组)。12 h后收集细胞,采样待测,用Western Blot法检测各组Caspase12表达。结果:各实验组与Sh-H-IGF1R-V1组及Sh-H-IGF1R-V2组比较扩增倍数均降低(P<0.05),其中Sh-H-IGF1R-3215组(0.02±0.01)与Sh-H-IGF1R-3787(0.05±0.05)沉默效果最稳定,但后组重复性更好。B组(2.41±0.03)、C组(2.45±0.02)、D组(3.60±0.06)Caspase12表达均较A组(0.34±0.02)升高(P<0.01),C组较B组略升高(P>0.05),D组较B、C组显著升高(P<0.01)。结论:4种针对IGF-1R基因沉默质粒均构建成功,并成功转染至人L02细胞(人正常肝细胞),沉默效果最理想的质粒为Sh-H-IGF1R-3787。IGF-1R靶向shRNA质粒可通过沉默L02细胞IGF-1R表达加重细胞凋亡,间接验证了IGF-1/IGF-1R信号通路对L02细胞过氧化损伤时经内质网途经的细胞凋亡程序活化有一定的调控作用。
OBJECTIVE: To construct a stable and silencing IGF-1R gene plasmid and transfect it into L02 cells (normal human hepatocytes) to observe the effects of IGF-1R gene silencing on endoplasmic reticulum pathway-specific apoptosis protein Caspase12 The impact of expression. METHODS: Short hairpin RNA (shRNA) against IGF-1R gene of L02 cells was constructed and four different sequences of Sh-H-IGF1R-1108, Sh-H- IGF1R- 2797, Sh-H-IGF1R- The Sh-H-IGF1R-3787 plasmid (experimental group) and the Sh-H-IGF1R-V1 plasmid containing the unrelated IGF-IR sequence (set as positive control group) and the blank plasmid Sh-H-IGF1R- Negative control group) were transfected L02 cells, 24 h after RTPCR method to detect the expression of IGF-1R, screened plasmid stably silence IGF-1R gene. Then the next experiment group was divided into: blank control group (A group), peroxide injury group (B group), negative plasmid transfection group (C group) and target gene transfection group (D group). After 12 h, cells were harvested, samples were taken for determination, and Caspase 12 expression in each group was detected by Western Blot. Results: Compared with Sh-H-IGF1R-V1 group and Sh-H-IGF1R-V2 group, the amplification times of all the experimental groups were significantly decreased (P <0.05) Sh-H-IGF1R-3787 (0.05 ± 0.05) showed the most stable silencing effect, but the repeatability of the latter group was better. The expressions of Caspase 12 in group B (2.41 ± 0.03), group C (2.45 ± 0.02) and group D (3.60 ± 0.06) were significantly higher than those in group A (0.34 ± 0.02) (P <0.01) (P> 0.05). The D group was significantly higher than the B and C groups (P <0.01). CONCLUSION: Four plasmids targeting IGF-1R gene silencing were successfully constructed and successfully transfected into human L02 cells (normal human hepatocytes). Sh-H-IGF1R-3787 was the most ideal plasmid for silencing. IGF-1R-targeted shRNA plasmids can exacerbate apoptosis by silencing the expression of IGF-1R in L02 cells, and indirectly verify that the IGF-1 / IGF-1R signaling pathway endoplasmic reticulum apoptosis process during L02 cell peroxidative injury Activation has a certain regulatory role.