藏红花素对乳腺癌血管生成作用的实验研究

目的 探讨藏红花素（Crocin）对乳腺癌血管生成作用及可能机制。方法 MTT法检测Crocin对人乳腺癌细胞MDA-MB-231及人脐静脉内皮细胞（HUVEC）的增殖抑制作用，并筛选出合适的Crocin浓度；流式细胞术检测Crocin对MDA-MB-231细胞凋亡和周期的影响；Transwell及小管形成实验检测Crocin对HUVEC细胞迁移和小管形成的抑制作用；构建乳腺癌MDA-MB-231裸鼠移植瘤模型，给予7次Crocin（5 mg/ml）治疗，免疫组化检测Crocin治疗后裸鼠移植瘤组织中CD34及Ki-67的表达。结果 MTT法检测显示，Crocin对乳腺癌细胞MDA-MB-231有显著的增殖抑制作用（P＜0.05），其48 h的半数抑制浓度（IC50）为5.0 mg／ml；而Crocin作用24 h时对HUVEC细胞有轻度增殖抑制作用，但不呈剂量依赖性，当Crocin作用48 h和72 h时，其增殖抑制作用明显增加，呈剂量依赖性，其48 h的IC50为5.97 mg／ml。流式细胞仪检测显示，Crocin可诱导MDA-MB-231细胞凋亡（P＜0.05），还可诱导细胞阻滞于G2／M期，呈剂量依赖性（P＜0.05）。Transwell及小管形成实验显示，Crocin可抑制HUVEC细胞迁移（P＜0.05）和小管形成（P＜0.05），均呈剂量依赖性。裸鼠皮下移植瘤实验显示，Crocin 5.0 mg／ml治疗后，治疗组较空白对照组移植瘤生长缓慢。空白对照组与治疗组CD34表达量分别为26.00±2.65和14.67±4.16（P＜0.05），Ki-67表达量分别为502.67±88.48和262.67±75.08，差异均有统计学意义（P＜0.05）。结论 Crocin具有一定的抗血管生成作用，可能与其可以在体内抑制肿瘤细胞增殖、降低微血管密度，在体外可抑制血管内皮细胞增殖、迁移和小管形成的作用相关，具体的分子机制还有待进一步研究。

Experimental study on antiangiogenic effect of crocin on breast cancer

Objective To observe the antiangiogenic effect of crocin on breast cancer and explore its possible mechanism. Methods In order to screen of appropriate drug concentrations, MTT assay was performed to test the inhibitory effect of crocin on breast cancer cell MDA-MB-231 and human umbilical vein endothelial cells(HUVECs). The effect of crocin on MDA-MB-231 cell ap-optosis and cell cycle was detected by flow cytometry. Transwell and Tubular formation test were used to detect the inhibitory of crocin on HUVECs migration and forming tubular. Nude mouse model of breast MDA-MB-231 cell was constracted, and the mice were given crocin( 5 mg/ ml ) for 7 times. Immunohistochemistry was applied to detect CD34 and Ki-67 expression after MDA-MB-231 subcutaneous xeno-transplanted tumors treated with crocin. Results MTT method showed that crocin had a significant inhibitory effect on the proliferation of MDA-MB-231 cells(P<0. 05) and the IC50 of 48 h was 5. 0 mg/ ml. Crocin had a mild inhibitory effect on HU-VECs without a dose-dependent manner at 24 h. But the effect was quite opposite at 48 h and 72 h, and the IC50 of 48 h was 5. 97 mg/ml. Crocin induced the apoptosis and cell cycle arrest in G2 / M phase of MDA-MB-231 cells in a dose-dependent manner(P<0. 05). Transwell and Tubule formation test showed that crocin could inhibit HUVECs migration(P<0. 05) and tube formation(P<0. 05) in a dose-dependent manner. The subcutaneous transplantation tumor growth in 5 mg/ ml crocin group was slower than that in blank control group. And the expression of CD34 and Ki-67 in blank group and 5 mg/ ml crocin group were 26. 00± 2. 65 and 14. 67± 4. 16( P<0. 05), 502. 67±88. 48 and 262. 67±75. 08(P<0. 05). Conclusion Crocin has certain effect of anti-angiogenesis, which may be re-lated to the inhibition of tumor cell proliferation, reduce the micro vascular density in vivo and can inhibit vascular endothelial cell pro-liferation, migration, as well as tubule formation in vitro. The exact molecular mechanism remains to study further.