CAJ | 학술논문

目的观察大鼠骨髓间充质干细胞条件培养基(BMSCs-CM)对人HepG2细胞胰岛素抵抗(IR)的影响,并探讨其分子机制.方法根据葡萄糖利用情况筛选出最佳造模剂使用浓度及时间;以正常HepG2细胞为空白对照,给予棕榈酸处理的细胞为IR模型组,加入BMSCs-CM为条件培养基(CM)干预组,而加入同样经半透膜浓缩后的低糖培养基作为阴性对照组,分别采用细胞培养、糖原染色、葡萄糖及糖原检测试剂盒等方法来分析各组葡萄糖利用情况;免疫印迹法观察BMSCs-CM对IR HepG2细胞磷酸化的胰岛素受体底物(p-IRS)、磷脂酰肌醇-3-激酶(PI3K)与磷酸化的丝氨酸/苏氨酸蛋白激酶(p-AKT)蛋白表达的影响;分别加入PI3K抑制剂LY294002及激动剂740Y-P后继续观察上述胰岛素信号蛋白的变化情况.结果 (1)以0.25 mmol/L棕榈酸作用HepG2细胞24 h的方法成功建立IR模型;IR模型组培养液中葡萄糖含量明显高于空白对照组,其细胞内糖原含量也显著减少(P＜0.05).(2)与IR模型组相比,CM干预组HepG2/IR葡萄糖的摄取与利用明显改善(P＜0.05),而在阴性对照组则差异没有统计学意义.(3)CM干预组p-IRS、PI3K与p-AKT的蛋白表达水平显著高于IR模型组(P＜0.05);而在加入LY294002阻断PI3K后,给予BMSCs-CM后PI3K及p-AKT蛋白含量则未增加;进一步加入740Y-P过度激活PI3K后,则这些蛋白的表达水平较IR模型组显著升高.结论大鼠BMSCs-CM的胰岛素增敏作用与p-IRS、PI3K与p-AKT蛋白的表达增强有关.
목적 관찰대서골수간충질간세포조건배양기(BMSCs-CM)대인HepG2세포이도소저항(IR)적영향,병탐토기분자궤제.방법 근거포도당이용정황사선출최가조모제사용농도급시간;이정상HepG2세포위공백대조,급여종려산처리적세포위IR모형조,가입BMSCs-CM위조건배양기(CM)간예조,이가입동양경반투막농축후적저당배양기작위음성대조조,분별채용세포배양、당원염색、포도당급당원검측시제합등방법래분석각조포도당이용정황;면역인적법관찰BMSCs-CM대IR HepG2세포린산화적이도소수체저물(p-IRS)、린지선기순-3-격매(PI3K)여린산화적사안산/소안산단백격매(p-AKT)단백표체적영향;분별가입PI3K억제제LY294002급격동제740Y-P후계속관찰상술이도소신호단백적변화정황.결과 (1)이0.25 mmol/L종려산작용HepG2세포24 h적방법성공건립IR모형;IR모형조배양액중포도당함량명현고우공백대조조,기세포내당원함량야현저감소(P＜0.05).(2)여IR모형조상비,CM간예조HepG2/IR포도당적섭취여이용명현개선(P＜0.05),이재음성대조조칙차이몰유통계학의의.(3)CM간예조p-IRS、PI3K여p-AKT적단백표체수평현저고우IR모형조(P＜0.05);이재가입LY294002조단PI3K후,급여BMSCs-CM후PI3K급p-AKT단백함량칙미증가;진일보가입740Y-P과도격활PI3K후,칙저사단백적표체수평교IR모형조현저승고.결론 대서BMSCs-CM적이도소증민작용여p-IRS、PI3K여p-AKT단백적표체증강유관.
Objective To study the effect of conditioned media for rat bone marrow mesenchymal stem cells (BMSCs-CM) on palmitic acid (PA)-induced insulin resistance (IR) in HepG2 cells and its underlying molecular mechanisms.Methods HepG2 cells were treated with or without BMSCs-CM and L-DMEM in the presence or absence of PA.Glucose utilization in HepG2 cells were detected with PAS,glucose and glycogen measurements.Western blotting was used to assess the expression of phospho-insulin receptor substrate (p-IRS),phosphatidylinositol 3-kinase (PI3 K) and p-AKT.Results (1) Incubation of HepG2 cells with 0.25 mmol/L PA for 24 hours significantly increased the glucose concentration and decreased the glycogen content (P ＜ 0.05) in the media.(2) Treatment with BMSCs-CM significantly ameliorated the glucose and glycogen alteration in cells pretreated with PA (P ＜ 0.05),however,no obvious effect of BMSCs-CM on the cell glucose and glycogen production.(3) BMSCs-CM treatment also increased protein expression of p-IRS,PI3K and p-AKT in PA incubated HapG2 cells (P＜ 0.05).The effect of BMSCs-CM on PI3K and p-AKT expression could be mimicked upon addition of 740Y-P,a PI3K agonist,but abolished by LY294002,a PI3K specific inhibitor.Conclusions BMSCs-CM could improve the insulin sensitivity in HepG2 cells pretreated with PA through upregulation of insulin signaling component expression.