CAJ | 학술논문

目的：建立稳定胰岛素抵抗肝癌（HepG2/IR）细胞模型，探讨模型细胞内质网应激伴侣蛋白葡萄糖调节蛋白78（GRP78）表达及其对顺铂的敏感性的关系。方法采用0．5、1．0μmol/L胰岛素诱导 HepG2细胞发生胰岛素抵抗，日立7600全自动生化仪检测培养上清液中的葡萄糖水平并计算葡萄糖消耗量；MTT 法测定顺铂对细胞的增殖抑制作用，Annexin Ⅴ/PI 双标记检测细胞的凋亡率；实时荧光定量PCR、流式细胞术和 Western-Blot法分别测定细胞胰岛素受体（InsR）及GRP78的表达水平。结果 HepG2/IR细胞葡萄糖消耗量降低、InsR 表达显著受抑，对顺铂的敏感性降低，48 h 和72 h 的 IC50为未作诱导处理HepG2细胞的165．9％和158．8％，差异有统计学意义（P＜0．05），凋亡率降低50．29％。HepG2/IR 细胞GRP78 mRNA 和蛋白的表达量分别为 HepG2细胞的2．12倍和2．27倍。结论肝癌 HepG2细胞经胰岛素诱导发生胰岛素抵抗后对顺铂的耐药性增强，其机制与内质网应激伴侣蛋白GRP78的表达增高有关。
목적：건립은정이도소저항간암（HepG2/IR）세포모형，탐토모형세포내질망응격반려단백포도당조절단백78（GRP78）표체급기대순박적민감성적관계。방법채용0．5、1．0μmol/L이도소유도 HepG2세포발생이도소저항，일립7600전자동생화의검측배양상청액중적포도당수평병계산포도당소모량；MTT 법측정순박대세포적증식억제작용，Annexin Ⅴ/PI 쌍표기검측세포적조망솔；실시형광정량PCR、류식세포술화 Western-Blot법분별측정세포이도소수체（InsR）급GRP78적표체수평。결과 HepG2/IR세포포도당소모량강저、InsR 표체현저수억，대순박적민감성강저，48 h 화72 h 적 IC50위미작유도처리HepG2세포적165．9％화158．8％，차이유통계학의의（P＜0．05），조망솔강저50．29％。HepG2/IR 세포GRP78 mRNA 화단백적표체량분별위 HepG2세포적2．12배화2．27배。결론간암 HepG2세포경이도소유도발생이도소저항후대순박적내약성증강，기궤제여내질망응격반려단백GRP78적표체증고유관。
Objective To establish the insulin resistant HepG2(HepG2/IR)cells model,and investigate the relationship of insu-lin resistance and reduced susceptibility to chemotherapy in hepatoma cells and its mechanism.Methods HepG2 cells were cultured in medium containing 0.5μmol/L insulin for different hours to induce insulin resistance.Glucose consumption of HepG2/IR cells were measured by Hitachi 7600 automatic biochemical analyzer.The cis-dichlorodiamineplatinum(DDP)sensitivity of the HepG2 and HepG2/IR cells were determined by MTT assay,the Annexin Ⅴ/PI assay was adopted to measure the apoptosis rate.In addi-tion,real-time PCR,flow cytometry (FCM)and Western-Blot were employed to detect the mRNA and protein levels of insulin re-ceptor(InsR)and endoplasmic reticulum chaperonin 78(GRP78).Results The glucose consumption decreased and expression of In-sR was down-regulated in HepG2/IR cells.The HepG2/IR cells had reduced sensitivity to DDP (P<0.05 ).The IC50 s of the HepG2/IR cells treated by DDP for 48 h and 72 h were 158.8% and 165.9% of HepG2 cells respectively,while the apoptosis rate was 50.29% lower.The mRNA and protein level of GRP78 in HepG2/IR cells were 2.12 and 2.27 times of that in HepG2 cells. Conclusion The stable HepG2/IR cells showed stronger resistance to DDP were established from HepG2 cell induced with insulin, and its mechanism may be related to the increased expression of GRP78.