灵猫方抑制饥饿诱导HepG2.2.15细胞自噬研究

Lingmao Recipe Inhibits Starvation Induced Autophagy in HepG2.2.15 Cell

目的研究灵猫方对饥饿诱导HepG2.2.15细胞自噬的作用。方法选取15只SD大鼠制备灵猫方药物血清,设立厄尔平衡盐缓冲液(Earle's balanced salt solution,EBSS)组、EBSS+空白血清组、EBSS+5倍药物血清组、EBSS+10倍药物血清组及DMEM组,每组3个样本。培养HepG2.2.15细胞1、2、4及6h,构建pEGFP-N1-LC3B表达载体,转染HepG2.2.15细胞,在荧光显微镜下观察细胞浆中GFP-LC3B分布表达变化,计算点状样GFP-LC3B的细胞数与GFP-LC3B转染HepG2.2.15细胞数比值。透射电镜观察各组HepG2.2.15细胞浆内自噬体。Western Blot检测各组HepG2.2.15细胞LC3BⅡ/Ⅰ比值(microtubule-associ-ated protein 1 light chain 3 betaⅡ/Ⅰ)。结果培养2h时,与EBSS+空白血清组比较,EBSS+5倍药物血清、EBSS+10倍药物血清明显抑制GFP-LC3B由散在分布向点状分布改变(P<0.01),电子透射电镜显示EBSS+5倍药物血清组、EBSS+10倍药物血清组抑制自噬体形成,Western Blot检测显示EBSS+10倍药物血清组LC3BⅡ/Ⅰ比值较EBSS+空白血清组明显下降(P<0.01)。结论灵猫方药物血清抑制饥饿诱导HepG2.2.15的自噬。

Objective To study the effects of Lingmao Recipe（LR） on starvation-induced autophagy in HepG 2.2.15 cell.Methods Fifteen SD rats were selected to prepare LR drug serum.The Earle＇s balanced salt solution（EBSS） group,the EBSS＋vehicle serum group,the EBSS＋5 times LR serum group,the EBSS＋10 times LR serum group,and the DMEM group were set up,3 samples in each group.The HepG2.2.15 cells were cultured for 1,2,4,and 6 h respectively.The pEGFP-N1-LC3B eukaryotic expression vector was constructed and transfected with HepG2.2.15 cell.The GFP-LC3B morphological changes were observed under fluorescent microscope.The ratio of the dotty GFP-LC3B number and the GFP-LC3B transfected HepG2.2.15 number was calculated.The intracytoplasma autophagosome changes were observed using electronic transmission electron.The microtubule-associated protein 1 light chain 3 beta Ⅱ/Ⅰ was detected in HepG2.2.15 of each group using Western blot.Results Two h after culture,when compared with the EBSS＋vehicle serum group,GFP-LC3B changing from diffused distribution to dotted distribution was obviously inhibited in the EBSS＋5 times LR serum group and the EBSS＋10 times LR serum group（P0.01）.The electronic transmission electron showed that the formation of autophagosome was inhibited in the EBSS＋5 times LR serum group and the EBSS＋10 times LR serum group.Results of Western blot showed that microtubule-associated protein 1 light chain 3 beta Ⅱ/Ⅰ obviously decreased more in the EBSS＋10 times LR serum group than in the EBSS＋vehicle serum group（P0.01）.Conclusion LR could inhibit starvation-induced autophagy in HepG 2.2.15.