CAJ | 학술논문

目的：：构建针对S100P基因的shRNA慢病毒载体,并在胃癌MGC-803细胞上鉴定沉默效率,观察其对细胞生物学行为的影响。方法：筛选的S100P基因特异性siRNA靶点,合成短发卡结构shRNA,并与GV115-GFP慢病毒载体重组形成shRNA表达载体,与pHelper 1.0和pHelper 2.0质粒共转染293T细胞,产生慢病毒。用病毒感染MGC-803细胞,RT-PCR和Western blot检测靶基因的沉默效率、克隆形成情况、细胞周期变化和凋亡实验观察靶基因沉默对MGC-803的影响。结果：构建的重组shRNA慢病毒载体,经293T细胞包装获得病毒颗粒,和阴性对照相比,慢病毒感染组S100 P mRNA表达下降了83.4%,蛋白表达也明显受到抑制。 S100 P沉默后MGC-803的克隆形成能力明显减弱,细胞周期发生S期阻滞,细胞凋亡明显增加。结论：成功地构建了S100P基因shRNA慢病毒表达载体,该载体能够在MGC-803细胞中沉默S100P基因的表达,导致胃癌细胞克隆形成能力降低,细胞周期阻滞,诱导细胞凋亡,表明S100 P基因有可能具有影响胃癌发生发展的作用。
목적：：구건침대S100P기인적shRNA만병독재체,병재위암MGC-803세포상감정침묵효솔,관찰기대세포생물학행위적영향。방법：사선적S100P기인특이성siRNA파점,합성단발잡결구shRNA,병여GV115-GFP만병독재체중조형성shRNA표체재체,여pHelper 1.0화pHelper 2.0질립공전염293T세포,산생만병독。용병독감염MGC-803세포,RT-PCR화Western blot검측파기인적침묵효솔、극륭형성정황、세포주기변화화조망실험관찰파기인침묵대MGC-803적영향。결과：구건적중조shRNA만병독재체,경293T세포포장획득병독과립,화음성대조상비,만병독감염조S100 P mRNA표체하강료83.4%,단백표체야명현수도억제。 S100 P침묵후MGC-803적극륭형성능력명현감약,세포주기발생S기조체,세포조망명현증가。결론：성공지구건료S100P기인shRNA만병독표체재체,해재체능구재MGC-803세포중침묵S100P기인적표체,도치위암세포극륭형성능력강저,세포주기조체,유도세포조망,표명S100 P기인유가능구유영향위암발생발전적작용。
Objective: To construct a shRNA lentiviral vector targeting S100P gene, and detect the efficiency of gene silence and its biological role on MGC-803 cells. Methods:Specific targets sequence of S100P were designed and synthesized short hairpin RNA, then were recombined into shRNA expression vector GV115-GFP. 293T cells were cotransfected with reconbined lentiviral vector(sh-S100P), pHelper 1. 0 and pHelper 2. 0 to produce shRNA lentiviral particles. MGC-803 cells were tansfected with lentivirus, the silencing efficiency of targeting sequence was detected by RT-PCR and Western blot, the biological function of S100P gene silence on MGC-803 cells was evaluated by colony formation assay, cell cycle and apoptosis analysis. Results: We successfully constructed the shRNA lentiviral expression vector and obtained the virus by packaging the 293T cell. The expression level of S100P mRNA in MGC-803 cells transfected with identificated shRNA lentiviral particles was decreased by 83. 4%. The expression of protein was suppressed notably. Downregulating S100P expression suppressed the cell colony formation, blocked cell cycle and promoted the apoptosis in MGC-803 gastric cancer cell. Conclusion: The successfully constructed recombinant vector can silence S100P expression in MGC-803 cells and suppress colony formation, induce cell cycle arrest and apoptosis, which indicates S100P may be involved in the progress of gastric cancer cells.