论文部分内容阅读
目的比较腺病毒介导的小鼠过氧化物酶体增殖剂活化受体γ(PPARγ)基因过度表达与配体活化对小鼠Raw264.7巨噬细胞小窝蛋白-1(CAV-1)基因和蛋白表达的影响,探讨PPARγ基因对巨噬细胞CAV-1的调控作用及机制。方法首先构建表达小鼠PPARγ1基因的复制缺陷型腺病毒表达载体;将Raw264.7细胞随机分成对照组、PPARγ基因过度表达组、PPARγ活化剂曲格列酮干预组以及PPARγ基因过度表达和曲格列酮共刺激组进行干预;观察各组Raw264.7细胞PPARγ和CAV-1基因和蛋白的表达变化。结果RT-PCR检测对照组Raw264.7细胞有CAV-1基因的表达,免疫印迹法未发现CAV-1蛋白表达,但免疫细胞化学证实胞膜和核膜上均有少量CAV-1表达;经RT-PCR、免疫细胞化学和免疫印迹检测发现PPARγ基因过度表达组、曲格列酮干预组和二者共刺激组Raw264.7细胞CAV-1基因和蛋白表达明显增加(且共刺激组>PPARγ基因过度表达组>曲格列酮干预组,P<0.05)。对照组Raw264.7胞浆内PPARγ表达量较低,而PPARγ基因过度表达组和共刺激组PPARγ表达均明显增加(P<0.05),而曲格列酮干预组无明显变化。结论PPARγ基因活化或过度表达均能上调Raw264.7细胞CAV-1基因和蛋白表达。曲格列酮活化PPARγ基因,增加CAV-1基因和蛋白表达,但不增加PPARγ基因和蛋白表达水平。与单一作用比较,同时活化和过度表达PPARγ基因对CAV-1基因和蛋白的表达有累积效应,说明PPARγ的这一作用在配体活化下增强。推测曲格列酮上调CAV-1表达依赖于PPARγ,非本身药理特性所致。
Objective To compare the effect of adenovirus-mediated mouse peroxisome proliferator-activated receptor gamma (PPARγ) gene overexpression and ligand activation on mouse caveolass-1 (CAV-1) gene of Raw264.7 macrophages And protein expression, to investigate the regulatory effect of PPARγ gene on macrophage CAV-1 and its mechanism. Methods The recombinant adenovirus expressing mouse PPARγ1 gene was constructed. Raw264.7 cells were randomly divided into control group, PPARγ gene over-expression group, PPARγ-activated troglitazone group and PPARγ gene overexpression and trilogy The experiment was conducted to observe the changes of PPARγ and CAV-1 gene and protein expression in Raw264.7 cells. Results The expression of CAV-1 gene in Raw264.7 cells was detected by RT-PCR. The expression of CAV-1 protein was not detected by immunoblotting. However, a small amount of CAV-1 was expressed on both cell membrane and nuclear membrane by immunocytochemistry. The results of RT-PCR, immunocytochemistry and Western blotting showed that the expression of CAV-1 gene and protein in RAW264.7 cells were significantly increased (P <0.05) and the co-stimulated group> PPARγ Gene overexpression group> troglitazone intervention group, P <0.05). The expression of PPARγ in Raw264.7 cytoplasm in control group was lower than that in PPARγ gene-overexpression group and co-stimulated group (P <0.05), while no significant change was observed in troglitazone-treated group. Conclusion PPARγ gene activation or overexpression upregulates CAV-1 gene and protein expression in Raw264.7 cells. Troglitazone activated PPARγ gene, increased CAV-1 gene and protein expression, but did not increase PPARγ gene and protein expression level. Compared with the single effect, the simultaneous activation and overexpression of PPARγ gene has a cumulative effect on the expression of CAV-1 gene and protein, indicating that this effect of PPARγ is enhanced by ligand activation. Speculated that troglitazone up-regulated CAV-1 expression depends on PPARγ, non-own pharmacological properties.