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目的检测细胞周期蛋白依赖性蛋白激酶抑制剂 flavopiridol 对卵巢上皮性癌(卵巢癌)细胞及移植瘤的干预作用。方法应用流式细胞仪和脱氧核苷酸末端转移酶介导的脱氧尿苷三磷酸标记法(TUNEL)检测 flavopiridol 作用后卵巢癌细胞系 AO 的细胞凋亡率和细胞周期,应用实时荧光定量 PCR 技术检测 flavopiridol 作用前、后 AO 细胞中细胞周期蛋白(cyclin)D 和活性半胱氨酸天冬氨酸蛋白酶(caspase)3的表达情况。建立卵巢癌裸鼠皮下及腹腔移植瘤模型,观测 flavopiridol 干预后裸鼠的生存情况及肿瘤体积的变化,TUNEL 和免疫组化方法分别检测肿瘤组织中的细胞凋亡情况和微血管密度(MVD)。结果 AO 细胞在150、300、500 nmol/L 浓度的 flavopiridol 作用下,凋亡率分别为4.1%、10.7%和7.6%;G_1期细胞比例显著增加,S 期比例显著降低(P<0.05)。flavopiridol作用后 AO 细胞 cyclin D 的表达量(0.25)显著下降,活性 caspase-3的表达量(2.55)轻度升高,高于flavopiridol 作用前的0.69(P<0.05)、2.49(P>0.05)。flavopiridol 干预后裸鼠的累积生存率显著提高(P<0.05);裸鼠的平均生存时间为(141±14)d,显著高于磷酸盐缓冲液(PBS)作用后的(106±11)d,两者比较,差异有统计学意义(P<0.05);在 flavopiridol 干预后53 d 时抑瘤率为40%。flavopiridol 干预后裸鼠的肿瘤组织中有细胞凋亡发生,MVD 为(12±5)个,显著高于 PBS 作用后的(35±10)个,两者比较,差异有统计学意义(P<0.05)。结论 flavopiridol 能显著抑制卵巢癌细胞及移植瘤的生长,延长荷瘤裸鼠的生存期。
Objective To investigate the intervention of cyclin - dependent kinase inhibitor flavopiridol on epithelial ovarian cancer (ovarian cancer) cells and xenografts. Methods Flow cytometry and deoxynucleotidyl transferase-mediated deoxyuridine triphosphate labeling (TUNEL) were used to detect apoptosis rate and cell cycle of ovarian cancer cell line AO after flavopiridol treatment. Real-time fluorescence quantitative PCR To detect the expression of cyclin D and caspase 3 in AO cells before and after flavopiridol treatment. The subcutaneous and abdominal xenograft model of ovarian cancer in nude mice was established. The survival and tumor volume of nude mice after the intervention of flavopiridol were observed. The apoptosis and microvessel density (MVD) were detected by TUNEL and immunohistochemistry. Results The apoptotic rates of AO cells treated with 150,300 and 500 nmol / L flavopiridol were 4.1%, 10.7% and 7.6%, respectively. The proportion of cells in G 1 phase increased significantly and the proportion of S phase decreased significantly (P <0.05). After treated with flavopiridol, the expression of cyclin D decreased significantly (0.25) and the activity of caspase-3 (2.55) increased slightly, which was significantly higher than that before flavopiridol treatment (0.69, 2.49, P <0.05) . The cumulative survival rate of nude mice increased significantly after intervention with flavopiridol (P <0.05). The mean survival time of nude mice was (141 ± 14) d, which was significantly higher than that of PBS (106 ± 11) d , The difference was statistically significant (P <0.05). The antitumor rate was 40% at 53 d after flavopiridol intervention. The apoptosis of nude mice in the presence of flavopiridol was (12 ± 5) MVD, which was significantly higher than that of PBS (35 ± 10), the difference was statistically significant (P < 0.05). Conclusion flavopiridol can significantly inhibit the growth of ovarian cancer cells and xenografts and prolong the survival of tumor-bearing nude mice.