HepG2细胞胰岛素抵抗模型建立影响因素研究

摘要

目的 建立体外肝癌(HepG2)细胞胰岛素抵抗(IR)模型,研究血清添加、诱导剂浓度和培养时间对模型建立的影响.方法 采用不同浓度胰岛素对肝癌HepG2细胞进行不同时间的诱导,建立肝细胞IR模型;以葡萄糖氧化酶法研究不同诱导浓度、培养时间及血清添加对胰岛素抵抗模型细胞葡萄糖消耗量(ΔGC)的影响.采用噻唑蓝(MTT)法评价细胞活性,并计算葡萄糖比消耗率(ΔGC/R),得到最佳建模条件.结果适当提高胰岛素诱导浓度可缩短诱导时间,或可通过延长诱导时间来降低诱导浓度.胰岛素能促进HepG2细胞增殖,且在含血清培养基中增殖较迅速.诱导后的细胞在含血清培养基中培养24 h即能得到理想的胰岛素抵抗模型.结论 可通过胰岛素诱导培养法建立稳定的IR模型,在含5×10-8 mol/L胰岛素的培养基中诱导48 h,再换含血清培养基继续培养24 h,易形成明显的胰岛素抵抗模型.此HepG2细胞模型在较长时间内(72 h)均可维持胰岛素抵抗特性.%Objective To establish a hepatoma carcinoma cell (HepG2 cell) model of insulin resistance (IR) in vitro and to explore the effect of serum addition, inducer concentration and culture time on the model. Methods The HepG2 cells were induced by different concentrations of insulin for different time to establish IR model. The effect of inducer concentration, culture time and serum addition on the glucose consumption (△GC) of IR cell model was determined by glucose oxidase method. The cell activity was estimated by MTT method, and the rate of glucose consumption (△GC/R) was calculted to obtain the optimal condition for establishment of IR model. Results The induction time could be shortened by proper raise of the insulin concentration, or the insulin concentration could be reduced by prolongation of the induction time. Insulin may promote the proliferation of HepG2 cells especially in medium containing serum. The induced cell cultured 24 h in the medium containing serum could establish ideal IR cell model. Conclusion A stable IR cell model can be established by insulin induction culture method. The results suggest that after culture in the medium containing 5×10-8 mol/L insulin for 48 h,and sequential culture in the medium containing serum for 24 h, an obvious IR model was successfully established. This HepG2 cell model can maintain insulin resistance for a long time (72 h).