CAJ | 학술논문

目的：探讨亲磷脂酸磷脂酶 A1（PA-PLA1）基因沉默对小鼠分泌胰岛素细胞 MIN6分泌功能的影响。方法：根据 GenBank 中小鼠 PA-PLA1基因 mRNA 序列构建 siRNA 表达载体（pGPU6-PA-PLA1）并转染MIN6，实时荧光定量 PCR 和 Western blot 筛选有效干扰载体。将获得的有效干扰载体转染 MIN6细胞48 h后进行葡萄糖刺激实验，检测其胰岛素分泌的变化。结果：酶切及测序证实成功构建了4个靶向 PA-PLA1的 siRNA 表达载体。实时荧光定量 PCR 和 Western blot 分析显示 siRNA 表达载体 pGPU6-PA-PLA1-1885具有较高的干扰效率，其转染的 MIN6细胞 PA-PLA1 mRNA 水平降至对照组的46．3％，PA-PLA1蛋白水平降至对照组的33．9％，同时胰岛素分泌水平降至对照组的65．0％（P ＜0．05）。结论：PA-PLA1基因沉默可降低MIN6的胰岛素分泌水平。
목적：탐토친린지산린지매 A1（PA-PLA1）기인침묵대소서분비이도소세포 MIN6분비공능적영향。방법：근거 GenBank 중소서 PA-PLA1기인 mRNA 서렬구건 siRNA 표체재체（pGPU6-PA-PLA1）병전염MIN6，실시형광정량 PCR 화 Western blot 사선유효간우재체。장획득적유효간우재체전염 MIN6세포48 h후진행포도당자격실험，검측기이도소분비적변화。결과：매절급측서증실성공구건료4개파향 PA-PLA1적 siRNA 표체재체。실시형광정량 PCR 화 Western blot 분석현시 siRNA 표체재체 pGPU6-PA-PLA1-1885구유교고적간우효솔，기전염적 MIN6세포 PA-PLA1 mRNA 수평강지대조조적46．3％，PA-PLA1단백수평강지대조조적33．9％，동시이도소분비수평강지대조조적65．0％（P ＜0．05）。결론：PA-PLA1기인침묵가강저MIN6적이도소분비수평。
Objective To explore the effect of the gene silencing of phosphatidic acid-preferring phospholipase A1 (PA-PLA1) on insulin secretion in mouse insulin-secreting cell line MIN6. Methods The siRNA expression vector of mouse PA-PLA1 gene targeting was constructed using mouse PA-PLA1 mRNA sequence available in GenBank, and MIN6 cells were transfected with the vector. Fluorescence quantitative PCR and Western-blotwere applied to screen efficient RNAi-vector. After transfection with obtained efficient RNAi-vectors for 48 hours, glucose-stimulated insulin secretion experiments were conducted, and the changes of insulin secretion were examined. Results Four siRNA expression vectors of mouse PA-PLA1 gene targeting were confirmed to be successfully constructed by the analyses of enzyme cleavage and sequencing. The results of fluorescence quantitative PCR and Western blot analyses indicated that the siRNA expression vectorpGPU6-PA-PLA1-1885was the most effective RNAi-vector in the four vectors. The expression levels of the PA-PLA1 mRNA and protein of the MIN6 cells transfectedwith pGPU6-PA-PLA1-1885 decreased to 46.3% and 33.9% of that of the control, respectively, and meanwhile the insulin secretion levels of the cells decreased to 65.0% of that of the control (P < 0.05). Conclusion The gene silencing of phosphatidic acid-preferring phospholipase A1 might decrease insulin secretion in MIN6 cells.