罗格列酮和全反式维甲酸对多发性骨髓瘤裸鼠移植瘤生长及血管形成的影响

Influence of rosiglitazone and all-trans-retinoic acid on angiogenesis and growth of myeloma xenograft in nude mice

目的研究罗格列酮（RGZ）和全反式维甲酸（ATRA）对多发性骨髓瘤裸鼠移植瘤生长和血管形成的影响。方法分别以不同浓度的RGZ和ATRA或RGZ＋ATRA处理U266细胞24h，采用半定量RT—PCR法检测各组U266细胞中血管内皮细胞生长因子（VEGF）的表达。将U266细胞接种于4周龄Balb／c裸鼠一侧肩胛部皮下，接种1周后分别以RGZ和RGZ＋ATRA腹腔注射干预，连续给药21d。第21天处死裸鼠，剥离皮下肿瘤并称重，将肿瘤组织分别进行HE和CD34、VEGF免疫组化染色。结果未经处理的对照组U266细胞中VEGFmRNA高表达；经RGZ处理后，VEGFmRNA的表达水平下调；经RGZ＋ATRA处理后，VEGFmRNA的表达水平较RGZ组更低。U266细胞接种后，在裸鼠皮下均可形成肿瘤。腹腔用药后，RGZ组裸鼠移植瘤的体积和重量分别为（785±262）mm3和（1748±365）mg，均明显低于对照组（均P〈0．01）；RGZ＋ATRA组移植瘤的体积和再量分别为（154±89）mm3和（626±102）mg3均明显低于RGZ组（均P〈0．05）。各组裸鼠移植瘤中均有VEGF蛋白的表达，其中对照组VEGF蛋白的平均染色强度（IS）为2．20±0．40，明显高于RGZ组（1.48±0．37，P〈0．01）；RGZ＋ATRA组的Is为0．58±0．26，明显低于RGZ组（P〈0．01）。各组裸鼠移植瘤中均有CD34的表达，其中对照组CD34表达最强，微血管密度（MVD）为（56．4±15．2）个／高倍视野，明显高于RGZ组[（44．6±11．2）个／高倍视野，P〈0．05]；RGZ＋ATRA组CD34表达最弱，MVD为（21．5±8．6）个／高倍视野，明显低于RGZ组（P〈0．01）。结论RGZ和ATRA存体内环境中也能发挥抑制骨髓瘤细胞增殖和生长的作用。通过抑制骨髓瘤细胞中VEGF的表达从而阻断血管形成，可能也足RGZ和ATRA抗肿瘤作用的重要机制之一。

Objective To observe the effect of rosiglitazone （RGZ） and all-trans-retinoic acid （ ATRA ） on the growth of myeloma xenograft in nude mice and to explore the influence of RGZ and ATRA on VEGF expression and angiogenesis in the tumor. Methods VEGF gene expression in myeloma cell line U266 ceils was analyzed by semi-quantitative RT-PCR after incubation with RGZ, ATRA, or RGZ ＋ ATRA for 24 h. Myeloma xenograft was established by subcutaneous injection of 107 U266 cells in the scapula area of 4-week old nude mice. 7 days later, the nude mice were administered with RGZ, ATRA or RGZ ＋ ATRA, respectively,by intraperitoneal injection once every day for 21 days. The control mice were given equal volume of normal saline instead of the drug. On the 21 ~＇ day of treatment, the mice were sacrificed and the tumors were taken off, and the tumor volume and weight were measured. The tumors were examined by histopathology with HE staining, and microvessel density （MVD） ,CD34 and VEGF expression in the tumors were analyzed by immunohistochemieal staining. Results VEGF mRNA was highly expressed in U266 cells and was decreased in a dose-dependent manner after incubation with RGZ. The VEGF mRNA level was further more decreased after RGZ ＋ ATRA treatment. Xenografts of U266 cells were developed in all nude mice. The volume and weight of xenografts in the RGZ group were （785±262）mm3 and （1748±365）mg, respectively, significantly lower than those of the control group （both P 〈 0.01 ）. More significant inhibition was in the RGZ ＋ ATRA group, （ 154± 89） mm3 and （626±102） mg, respectively, both were P 〈 0.05 vs. the RGZ group. RGZ inhibited the angiogenesis in U266 xenografts and immunohistochemical staining showed that the tumor MVD and VEGF expression were significantly decreased by RGZ treatment, and further more inhibited in the RGZ ＋ ATRA group. VEGF protein was expressed in all xenografts in the nude mice. Its immunohistochemical staining intensity was 2.20 ± 0.40 in the control group, significantly higher than that of 1.48 ±0.37 in the RGZ group （ P 〈 0.01 ）, and that of RGZ ＋ ATRA group was 0.58±0.26, further significantly lower than that of the RGZ group （ P 〈 0.01 ）. CD34 was expressed in all xenografts, most highly in the control group and lowest in the RGZ ＋ ATRA group. The microvessel density （MVD） was highest in the control group （56. 4 ±15. 2） , significantly lower in the RGZ group （44. 6±11. 2） （P〈0.05）, and lowest in the RGZ ＋ATRA group （21.5±8.6, P〈0.01）. Conclusions The growth of myeloma cells can also be inhibited by RGZ and ATRA in nude mice in vivo. In addition to differentiation and apoptosis induction, RGZ can inhibit the formation of myeloma xenograft probably also through the downregulation of VEGF expression and subsequent angiogenesis.