摘要
目的观察牛黄天龙胶囊(含药血清)对人子宫内膜癌HEC-B细胞能否诱导凋亡并对其抗癌机制进行探讨。方法实验组分低、中、高3个剂量组并设空白组和阳性对照组进行含药血清的制备;MTT法观察细胞毒作用;细胞排染法观察瘤细胞直接损害实验;吖啶橙/溴化乙锭(AO/EB)双荧光染色法及电镜(TEM)下观察细胞凋亡的形态学变化;流式细胞术(FCM)测定细胞凋亡率并进行细胞周期分析,测定凋亡抑制蛋白survivin和凋亡效应蛋白Caspase-3的表达。结果MTT法:人子宫内膜癌HEC-B细胞经含药血清作用后细胞生长受到明显抑制且以低剂量组抑制率最高。细胞排染法:同一时间段内以低剂量组效果最好,不同时间段内(24、48h)细胞抑制率显著提高(P<0.01)。AO/EB和TEM:实验组均可见到典型细胞凋亡的形态学变化。FCM:实验组各组细胞凋亡率与空白组相比差异有显著性(P<0.01)且以低剂量组最高,细胞周期分析显示实验组G0/G1期细胞明显增多而S期细胞明显减少与空白组相比差异有显著性(P<0.01),Survivin蛋白表达在空白组最高,在实验组表达降低与空白组相比差异有显著性(P<0.01),Caspase-3表达则相反,在实验组表达最高与空白组相比差异有显著性(P<0.01)。结论复方中药“牛黄天龙胶囊”(含药血清)能够使人子宫内膜癌HEC-B细胞生长阻滞在G0/G1期并可诱导人子宫内膜癌HEC-B细胞凋亡;并可降低凋亡抑制蛋白survivin表达同时可提高凋亡效应蛋白Caspase-3表达,这可能是其诱导人子宫内膜癌HEC-B细胞凋亡的机制之一;对人子宫内膜癌HEC-B细胞的抗肿瘤作用并非浓度越大效果越好而有一最适剂量。
Objective To explore the effect and mechanism of "Niuhuangtianlong capsule" whether can induce apoptosis on human endometrial cancer cell line HEC-B. Methods The test groups were respectively divided into three dose groups" low-dose, middle-dose and high-dose group including control group and positive group for medical serum. The cell proliferation inhibition was measured by MTT as well as by the cell refusing stain test. Additionally, HEC-B cell morphology changes of apoptosis were observed by AO/EB double fluorescent dye staining and by TEM. Finally, the cell apoptosis rates, cell cycle as well as the expression of survivin, caspase-3 were also measured by FCM. Results MTT: Endometrial cancer line was significantly inhibited by medical serum and show the low-dose group was the best effect. The assay of cell refusing stain: The results in the same or different period were the same as MTT. AO/EB double fluorescent dye staining: the early apoptosis cells stained in yellow-green and the later apoptosis cells stained in orange in the medical serum groups. TEM: the cell apoptotic morphology changes of HEC-B cells could be observed by TEM: marked condensation, Margination and acute angle project of nuclear chromatins as well as formation of vacuoles in cell cytoplasm. FCM: the apoptotic rate in medical serum groups was significantly different compared with control group ( P〈0. 01 ) . The cell apoptotic rate of low-dose group was also the highest as compared with the middle-dose, high-dose group ( P〈 0. 01 ) . The result of cell cycle show: in the test group cells in S phase were reduced and G1 /G0 phase were enhanced obviously compared with control group ( P〈0. 01 ) . The expression of surviving was decreased evidently, on the contrary caspase-3 was increased obviously compared with control group ( P〈0. 01 ) . Conclusion (1) Human endometrial cancer cell HEC-B could be induced apoptosis and inhibited at G0/G1 period by herbal complex prescription "Niuhuangtianlong capsule". (2)" Niuhuangtianlong capsule" could reduce the expression of surviving and enhance the expression of caspase-3, which may be one of the HEC-B cell inducing apoptotic mechanism. (3) "Niuhuangtianlong capsule" inducing HEC-B cell apoptosis do not display the better result with increasing dose, the low dose has the best effect in this experiment.
出处
《肿瘤防治研究》
CAS
CSCD
北大核心
2006年第6期417-421,475,共6页
Cancer Research on Prevention and Treatment
关键词
血清药理学
细胞凋亡
AO/EB双荧光染色法
TEM
FCM
Pharmacology
Cell apoptosis
AO/EB double fluorescent dye staining
Transmission electron microscope
Flow cytometry