论文部分内容阅读
目的观察吡格列酮对体外培养的HepG2细胞增殖和凋亡的影响,并探讨其是否通过PPARγ依赖途径发挥上述药理作用。方法将不同浓度的吡格列酮作用于体外培养HepG2细胞,以MTT比色法检测HepG2细胞增殖情况,以3H-TdR参入实验检测细胞DNA合成速率,采用RT-PCR和Western blot检测PPARγmRNA和蛋白的表达,以流式细胞术检测细胞凋亡和细胞周期;同时观察PPARγ特异性拮抗剂GW9662和(或)瞬时转染pSG5-PPARγ真核表达质粒对吡格列酮细胞增殖作用的影响;并将PPARγ小干扰RNA(pGCsi-PPARγ)表达质粒稳定转染HepG2细胞,观察PPARγ沉默后吡格列酮对HepG2细胞增殖作用的影响。结果吡格列酮作用于HepG2细胞后,导致HepG2细胞的增殖受到抑制、DNA合成速率减慢,并诱导细胞凋亡,呈一定的剂量依赖关系;在此过程中,G0/G1期细胞比例明显增加,S期细胞比例明显减少,但PPARγmRNA和蛋白的表达没有变化;GW9662部分拮抗吡格列酮的增殖抑制作用,但转染pSG5-PPARγ真核表达质粒可以逆转GW9662的作用;吡格列酮在高浓度(20μmol.L-1)时对pGCsi-PPARγ表达质粒稳定转染的HepG2细胞仍表现出增殖抑制作用。结论吡格列酮能够抑制HepG2细胞的增殖并诱导凋亡,具有潜在的抗瘤作用,这种作用与其诱导细胞G0/G1期的停滞有关,PPARγ依赖和非依赖途径参与上述过程。
Objective To observe the effect of pioglitazone on the proliferation and apoptosis of HepG2 cells cultured in vitro and to explore whether it exerts the above pharmacological effects through PPARγ-dependent pathway. Methods Different concentrations of pioglitazone were used to culture HepG2 cells in vitro. MTT assay was used to detect the proliferation of HepG2 cells. 3H-TdR incorporation assay was used to detect the DNA synthesis rate. RT-PCR and Western blot were used to detect the mRNA and protein expression of PPARγ. The effects of PPARγ-specific antagonist GW9662 and / or transiently transfected pSG5-PPARγ eukaryotic expression plasmids on the proliferation of pioglitazone were observed by flow cytometry. The effects of PPARγ small interfering RNA ( pGCsi-PPARγ) plasmid was stably transfected into HepG2 cells to observe the effect of pioglitazone on the proliferation of HepG2 cells after PPARγ silencing. Results After pioglitazone treatment on HepG2 cells, the proliferation of HepG2 cells was inhibited, and the DNA synthesis rate was slowed down and apoptosis was induced in a dose-dependent manner. During the process, the proportion of cells in G0 / G1 phase increased significantly, and S However, the expression of PPARγ mRNA and protein did not change. GW9662 partially antagonized the inhibitory effect of pioglitazone. However, the transfection of pSG5-PPARγ eukaryotic expression plasmid could reverse the effect of GW9662. The effect of pioglitazone at high concentration (20μmol.L-1 ) Still inhibited the proliferation of HepG2 cells stably transfected with the pGCsi-PPARγ expression plasmid. Conclusion Pioglitazone inhibits the proliferation and induces apoptosis of HepG2 cells, and has the potential antitumor effect. The effect of pioglitazone is related to the arrest of G0 / G1 phase. The PPARγ-dependent and non-dependent pathway participates in the above process.