野百合碱诱导胰腺癌细胞BxPC-3呈巨型多倍体化研究

Effect of monocrotaline on induction of pancreatic cancer cell BxPC-3 into polyploid giant cells

目的观察野百合碱体外诱导人胰腺癌细胞株BxPC-3形成多倍体巨细胞的作用。方法 BxPC-3细胞以1×104/mL的浓度接种于含野百合碱(0、5、10、20μg/mL)的RPMI 1640培养液内72 h,Giemsa染色分析细胞形态,流式细胞术分析DNA量,Annexin V-FITC/PI双标法流式细胞术观察野百合碱对BxPC-3细胞凋亡的诱导作用,染色体展示分析野百合碱处理后BxPC-3多倍体巨细胞的染色体组,Western blotting检测周期蛋白Cyclin B1的表达。结果 Giemsa染色显示,多倍体细胞体积大,多核。流式细胞仪检测细胞DNA量表明,野百合碱处理72 h出现4N、8N巨型BxPC-3多倍体细胞,诱导细胞凋亡,下调Cyclin B1蛋白表达,并与野百合碱的质量浓度呈依赖关系。染色体展示分析再次证明了巨型多倍体细胞的形成。结论野百合碱通过抑制周期蛋白Cyclin B1的表达,导致多倍体巨细胞增加,出现4N、8N巨型BxPC-3多倍体细胞,促使BxPC-3细胞凋亡或死亡。

Objective To observe the effect of monocrotaline （MCT） extract from Crotalaria sessiliflora on inducing human pancreatic cancer cell BxPC-3 into polyploid giant cells in vitro. Methods BxPC-3 cells （1 ×104/mL） were inoculated with MCT （0, 5, 10, and 20 μg/mL） in RPMI- 1640 for 72 h, respectively, then the cell morphology was observed by Giemsa staining, and the DNA content was measured by flow cytometry; BxPC-3 cells-induced apoptosis was checked by AnnexinV-FITC/PI double staining using flowcytometry, BxPC-3 polyploid giant cell genorne was checked by chromosome spreading assay, and Cyclin B 1 expression also was analysed by Western blotting. Results Giemsa staining and DNA content by flow cytometry showed that MCT induced BxPC-3 cell into polyploid giant cells in vitro. MCT treatment for 72 h appeared 4N, and 8N polyploid giant of BxPC-3 cells, induction of apoptosis, decreased the expression ofcyclin B1 in a dose dependent manner. Chromosome analysis demonstrated once again that polyploid giant cell was mainly in 8N. Conclusion Monocrotaline might exert its antitumor effect by blocking the cell cycle, inducing apoptosis, and down-regulating Cyclin B 1 protein.