罗格列酮与全反式维甲酸对骨髓瘤细胞分化的影响及其可能机制

Differentiating effect of PPARγ/ligand rosiglitazone and all trans-retinoic acid on myeloma cells and its possible mechanism

目的探讨过氧化物酶体增殖物活化受体γ（PPARγ）的配体罗格列酮（RGZ）与全反式维甲酸（ATRA）对骨髓瘤细胞分化的影响及其可能机制。方法以RGZ和ATRA处理人骨髓瘤细胞U266和RPMI-8226，用^3H-掺入法检测细胞增殖变化；碘化丙啶（PI）染色后，以流式细胞仪检测细胞周期变化；瑞氏-姬母萨染色观察细胞形态学变化；流式细胞仪分析细胞表面CD49e的表达变化；Western blot方法检测p27^Kip1和p21^Waf1的表达变化。结果RGZ能显著抑制U266和RPMI-8226细胞的增殖，并且这种抑制作用呈剂量依赖性。经5μmol／LRGZ处理后，U266和RPMI-8226细胞G0／G1期细胞的比例分别为（45．2±6．7）％和（40．3±7．3）％，较对照组明显增加（均P〈0．05）；G2／M期和S期细胞的比例分别为（52．2±7．4）％和（57．4±9．5）％，较对照组明显减少（均P〈0．05）；CIM9e的表达率分别为（8．7±1．5）％和（7．9±1．6）％，较对照组明显上调（均P〈0．05）。以10μmol／LRGZ处理后，U266和RPMI-8226细胞的G0／G1期、G2／M期和S期细胞比例、CD49e的表达率均较5μmol／LRGZ组的变化更加显著。同时，经RGZ处理后，U266和RPMI-8226细胞的形态学变化也符合骨髓瘤细胞分化成熟的规律，p27^Kip1和p21^Waf1蛋白的表达量也较对照组明显增加。RGZ和ATRA联合对U266和RPMI-8226细胞影响较RGZ单独作用更加明显。结论RGZ可能通过上调p27^Kip1和p21^Waf1蛋白的表达，介导骨髓瘤细胞的周期阻滞，并诱导骨髓瘤细胞分化，抑制骨髓瘤细胞增殖。ATRA能增强RGZ对骨髓瘤细胞的上述作用，两者联合具有协同效应。

Objective To investigate the effects of PPARγ/ligand （rosiglitazone, RGZ） as well as combined with all trans-retinoic acid （ATRA） on human myeloma cells and try to explore the possible mechanism. Methods Human myeloma cell lines U266 and RPMI-8226 cells were treated with RGZ in the presence or absence of ATRA. Cell proliferation was evaluated by [^ 3H ] thymidine incorporation, cell cycle distribution and CD49e expression were analyzed by flow cytometry, morphology changes were evaluated by Wright-Giemsa staining, and p27^Kip1 and p21^Waf1 expression was detected by Western blotting. Results The exposure to RGZ induced proliferation inhibition in both cell lines in a dose-dependent manner. After cultured with 5 μmol/L RGZ, the proportion of U266 and RPMI-8226 cells in phase G0/G1 was （45.2 ± 6. 7）% and （40.3 ±7. 3）% ,respectively （P 〈0. 05）. The proportion of the cells in phase G2/M and S was （52.2 ± 7.4 ） % and （ 57.4 ± 9.5 ） % , respectively （ P 〈 0.05 ）. These changes were more evident when the RGZ concentration was increased to 10 μmol/L. A combination of RGZ with ATRA enhanced the growth inhibition and cell cycle arrest effects of RGZ. The RGZ-treated myeloma cells displayed morphological characteristics of cell differentiation, and more evident signs of differentiation were observed when RGZ was combined with ATRA. These changes were confirmed by the detection of CD49e expression. The expression of p27^Kip1 and p21^Waf1 in myeloma cells was up-regulated by RGZ and this change was more apparent when RGZ was used in combination with ATRA. Conclusion RGZ can induce cell cycle arrest and cell differentiation in myeloma cells which maybe caused by up-regulation of p27^Kip1 and p21^Waf1 expression. ATRA can enhance these effects of RGZ on multiple myeloma cells and combined use of these two drugs may show a synergistic effect on myeloma cells.