CAJ | 학술논문

目的：探讨高糖刺激上调人肾小管上皮细胞株( HK-2)骨桥蛋白(OPN)表达的分子机制。方法利用高糖(25 mmol·L-1)刺激 HK-2细胞,并应用特异性抑制剂、siRNA抑制PI3K和(或)mTOR活性,应用Real-time PCR检测OPN mRNA表达；Western blot 检测 OPN、p-AKT、p-S6、Raptor 和Rictor蛋白表达。结果高糖刺激呈时间依赖性上调HK-2细胞OPN 表达,其中, OPN mRNA 在刺激48h 后达高峰；OPN蛋白表达在72h达到最高。此外,高糖刺激激活PI3K/AKT/mTORC1信号通路。利用 PI3K 特异性抑制剂LY294002、mTORC1特异性抑制剂 rapamycin 抑制 PI3K/AKT/mTOR通路后,HK-2细胞的OPN表达明显降低。进一步,siRNA敲低Raptor降低HK-2细胞中OPN蛋白的表达,而敲低Rictor对OPN蛋白表达无影响。结论高糖通过激活PI3K/AKT/mTORC1通路上调HK-2细胞OPN的表达。
목적：탐토고당자격상조인신소관상피세포주( HK-2)골교단백(OPN)표체적분자궤제。방법이용고당(25 mmol·L-1)자격 HK-2세포,병응용특이성억제제、siRNA억제PI3K화(혹)mTOR활성,응용Real-time PCR검측OPN mRNA표체；Western blot 검측 OPN、p-AKT、p-S6、Raptor 화Rictor단백표체。결과고당자격정시간의뢰성상조HK-2세포OPN 표체,기중, OPN mRNA 재자격48h 후체고봉；OPN단백표체재72h체도최고。차외,고당자격격활PI3K/AKT/mTORC1신호통로。이용 PI3K 특이성억제제LY294002、mTORC1특이성억제제 rapamycin 억제 PI3K/AKT/mTOR통로후,HK-2세포적OPN표체명현강저。진일보,siRNA고저Raptor강저HK-2세포중OPN단백적표체,이고저Rictor대OPN단백표체무영향。결론고당통과격활PI3K/AKT/mTORC1통로상조HK-2세포OPN적표체。
Aim Toexplorethemechanismofupregu-lation of osteopontin ( OPN ) expression induced by high glucose in human renal tubular epithelial cells (HK-2cells).Methods Afterstimulationwithhigh-glucose (25 mmol·L-1 ) culture medium, HK-2 cells were then treated with the specific inhibitors or siRNA to inhibit the activity of PI3K and/or mTOR. Subse-quently, Real-time PCR was used to investigate the mRNA level of OPN, and Western blot was performed to detect the protein expression of OPN, p-AKT, p-S6,RaptorandRictor.Results Theexpressionlevel of OPN was increased in a time-dependent manner in HK-2 cells followed by high-glucose stimulation. The mRNA level of OPN peaked at 48 h; while the protein expression of OPN reached the highest level at 72h. Meanwhile, high glucose activated the PI3K/AKT/mTOR signaling pathway. Moreover, inhibition of the PI3 K/AKT/mTOR pathway by LY294002 and/or rapa-mycin led to significant down-regulation of OPN. Addi-tionally, the treatment with Raptor siRNA, but not Rictor siRNA resulted in reduction of OPN expression. Conclusion Highglucoseincreasestheexpressionof OPN through the activation of PI3 K/AKT/mTORC1 signaling pathway in HK-2 cells.