CAJ | 학술논문

目的：应用RNA干扰技术抑制小鼠巨噬细胞RAW264．7细胞P2X7受体（ P2X7 R）基因的表达，建立稳定干扰细胞株，并观察其对细胞增殖和凋亡的影响。方法：用脂质体法将P2X7 R shRNA重组质粒转染至RAW264．7细胞，经 G418筛选后获得稳定干扰细胞株。细胞分为野生型（ WT ）组、阴性对照（ NC ）组和干扰（shP2X7R）组。 Real－time PCR法检测细胞中P2X7R mRNA的表达，Western blot检测细胞中P2X7R蛋白的表达；CCK－8方法检测细胞生长活性，5－乙炔基－2’－脱氧尿苷（5－ethynyl－2’－deoxyuridine，EdU）掺入实验检测细胞增殖活性；流式细胞术分析细胞周期的分布和吞噬情况。结果：P2X7 R shRNA能明显抑制RAW264．7细胞的P2X7 R mR－NA和蛋白的表达，抑制率在80％以上。48 h后，shP2X7R组细胞的生长速度明显高于NC组和WT组（P＜0．05），增殖期细胞比例明显升高（P＜0．05），说明下调P2X7R基因能明显促进细胞增殖。 shP2X7R组的细胞周期出现改变，S期和G2／M期的比例明显上升，增殖指数增高（P＜0．05）。 shP2X7R组的细胞吞噬活性明显高于NC组（P＜0．05）。结论：本研究成功构建了稳定干扰P2X7 R基因表达的小鼠巨噬细胞株RAW264．7，shP2X7 R能够明显促进RAW264．7细胞的增殖，改变了细胞的吞噬活性。
목적：응용RNA간우기술억제소서거서세포RAW264．7세포P2X7수체（ P2X7 R）기인적표체，건립은정간우세포주，병관찰기대세포증식화조망적영향。방법：용지질체법장P2X7 R shRNA중조질립전염지RAW264．7세포，경 G418사선후획득은정간우세포주。세포분위야생형（ WT ）조、음성대조（ NC ）조화간우（shP2X7R）조。 Real－time PCR법검측세포중P2X7R mRNA적표체，Western blot검측세포중P2X7R단백적표체；CCK－8방법검측세포생장활성，5－을결기－2’－탈양뇨감（5－ethynyl－2’－deoxyuridine，EdU）참입실험검측세포증식활성；류식세포술분석세포주기적분포화탄서정황。결과：P2X7 R shRNA능명현억제RAW264．7세포적P2X7 R mR－NA화단백적표체，억제솔재80％이상。48 h후，shP2X7R조세포적생장속도명현고우NC조화WT조（P＜0．05），증식기세포비례명현승고（P＜0．05），설명하조P2X7R기인능명현촉진세포증식。 shP2X7R조적세포주기출현개변，S기화G2／M기적비례명현상승，증식지수증고（P＜0．05）。 shP2X7R조적세포탄서활성명현고우NC조（P＜0．05）。결론：본연구성공구건료은정간우P2X7 R기인표체적소서거서세포주RAW264．7，shP2X7 R능구명현촉진RAW264．7세포적증식，개변료세포적탄서활성。
AIM: To establish a cell line of stable silencing of P2X7 receptor (P2X7R) expression through short hairpin RNA ( shRNA)-mediated interference in murine RAW264.7 macrophages, and to investigate the proliferation and apoptosis in the cell line.METHODS:Stable silencing of P2X7 R gene in the RAW264.7 cells was achieved by re-combinant shRNA plasmid targeting murine P2X7 R gene via liposome mediated transfection, followed by G418 selection. The efficacy of plasmid transfection and P2X7 R silencing in G418 resistant cells was verified by immunofluorescent micros-copy and real-time PCR, respectively.The proliferative activity was analyzed by CCK-8 assay and EdU cell proliferation as-say.The cell cycle distribution and apoptosis were evaluated by flow cytometry.RESULTS:The expression of P2X7 R at mRNA and protein levels was down-regulated by 80% in shP2X7 R group compared with negative control ( NC) plasmid transfection.In addition, P2X7 R-silencing cells exhibited higher proliferative activity compared with NC and wild-type RAW264.7 cells (P<0.05).Compared with NC cells, P2X7R silencing resulted in an increase in the phagocytosis of the cells ( P<0.05) .CONCLUSION:A cell line RAW264.7 of stable silencing of P2X7 R expression was successfully es-tablished.P2X7 R gene silencing stimulates the proliferation, and changes phagocytic function in murine RAW264.7 macro-phages.