CDK2基因沉默联合达卡巴嗪对B16-F1黑素瘤的生长抑制作用

Inhibitory effect of CDK2 gene silencing combined with dacarbazine on the growth of B16- F1 melanoma

目的 探讨CDK2基因沉默后对达卡巴嗪（DTIC）治疗B16、F1黑素瘤抑瘤效应的影响。方法 实验设对照组、CDK2、shRNA组、DTIC组、CDK2、shRNA ＋ DTIC组，MTT法检测各组细胞生长抑制情况，计算药物相互作用指数（CDI值），AnnexinV、FITC/ PI双染法检测细胞凋亡情况。建立C57 BL/6小鼠B16、F1细胞移植瘤模型，分组实验，持续观察18 d，绘制肿瘤生长曲线，计算肿瘤生长抑制率，TUNEL检测肿瘤组织细胞凋亡情况。结果 与对照组相比，CDK2、shRNA组、DTIC组、CDK2、shRNA ＋ DTIC组在72h的细胞相对存活率分别为（40.6 ± 2.8）%、（45.2 ± 3.7）%、（28.7 ± 2.1）%，细胞凋亡率分别为（25.1 ± 3.3）%、（15.6 ± 2.2）%和（45.6 ± 3.5）%，细胞相对存活率显著降低（F = 458.04，P 〈 0.05）而细胞凋亡率显著升高（F = 115.46，P 〈 0.05），其中CDK2、shRNA ＋ DTIC组比DTIC组的细胞相对存活率显著降低（P 〈 0.01），而细胞凋亡率显著升高（P 〈 0.01）；两药相互作用指数（CDI值） 〈 0.7。治疗第6天，对照组、CDK2、shRNA组、DTIC组及CDK2、shRNA ＋ DTIC组的肿瘤体积分别为（185.44 ± 68.97） mm^3、（83.91 ± 14.33） mm^3、（123.70 ± 20.85） mm^3、（34.54 ± 10.72） mm^3。从治疗的第6天开始，与对照组相比，CDK2、shRNA组、DTIC组及CDK2、shRNA ＋ DTIC组的肿瘤生长速度明显降低（F = 11.819，P 〈 0.05），而CDK2、shRNA ＋ DTIC组的肿瘤生长速度明显低于DTIC组（P = 0.04）；与对照组相比，CDK2、shRNA组、DTIC组、CDK2、shRNA ＋ DTIC组的肿瘤生长抑制率分别为52.2%、41.2%、86.4%，组织细胞凋亡指数分别为（32.93 ± 3.72）%、（21.62 ± 3.54）%、（63.29 ± 4.74）%，组织细胞凋亡指数显著升高（F = 222.25，P 〈 0.05），其中CDK2、shRNA ＋ DTIC组的组织细胞凋亡指数显著高于DTIC组（P 〈 0.01）。结论 沉默CDK2基因的表达可以增加DTIC对黑素瘤的生长抑制作用，二者体外有协同效应，通过增加肿瘤细胞的凋亡是其机制之一。

Objective To evaluate the antitumor effect of dacarbazine（DTIC）on B16-F1 melanoma after CDK2 gene silencing. Methods Cultured B16-F1 melanoma cells were divided into 4 groups： control group receiving no treatment, CDK2-shRNA group infected with a recombinant lentivirus pUL-CDK2-shRNA, DTIC group cultured in 96-well plates followed 12 hours later by the treatment with 250 μmol/L DTIC, CDK2-shRNA ＋ DTIC group infected with pUL-CDK2-shRNA followed 12 hours later by the treatment with 250 μmol/L DTIC. MTT assay was performed to evaluate the growth inhibition of B16-F1 melanoma cells, and coefficient of drug interaction（CDI） was calculated. AnnexinV-FITC/PI double staining was conducted to detect cell apoptosis. C57BL/6 mice were subcutaneously injected with B16-F1 cells at exponential growth phase into the right groin to establish melanoma-bearing mouse models. Twenty mouse models were randomly and equally divided into 4 groups： control mouse group injected with phosphate-buffered solution（PBS） into tumors, CDK2-shRNA mouse group injected with pUL-CDK2-shRNA into tumors, DTIC mouse group injected with DTIC into the abdominal cavity, and CDK2-shRNA ＋ DTIC mouse group treated with pUL-CDK2-shRNA and DTIC. The animal experiment lasted 18 days, and the tumor growth curve was drawn. After 18-day treatment, all the mice were sacrificed, and tumors were isolated and weighed. The tumor growth inhibition rate was calculated, and the tumor cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling（TUNEL）. Results After 72-hour culture, compared with the control group, the CDK2-shRNA group, DTIC group, and CDK2-shRNA ＋ DTIC group showed significantly decreased relative cell survival rates （40.6% ± 2.8%, 45.2% ± 3.7%, 28.7% ± 2.1%, respectively; F = 458.04, P 〈 0.05）, but significantly increased cell apoptosis rates （25.1% ± 3.3%, 15.6% ± 2.2%, 45.6% ± 3.5%, respectively; F = 115.46, P 〈 0.05）. Additionally, CDK2-shRNA ＋ DTIC group showed significantly lower relative cell survival rates （P 〈 0.01）, but higher cell apoptosis rates （P 〈 0.01） compared with the DTIC group. The CDI value was less than 0.7. On the sixth day after the in vivo treatment, the tumor volumes in the control mouse group, CDK2-shRNA mouse group, DTIC mouse group, and CDK2-shRNA ＋ DTIC mouse group were（185.44 ± 68.97） mm^3,（83.91 ± 14.33） mm^3, （123.70 ± 20.85） mm^3, and（34.54 ± 10.72） mm^3 respectively. From then on, the CDK2-shRNA mouse group, DTIC mouse group, and CDK2-shRNA ＋ DTIC mouse group showed significantly decreased tumor growth rates compared with the control mouse group （F = 11.819, P 〈 0.05）, and the tumor growth rate was significantly lower in the CDK2-shRNA ＋ DTIC mouse group than in the DTIC mouse group （P = 0.04）. The calculated tumor growth inhibition rates in the CDK2-shRNA mouse group, DTIC mouse group and CDK2-shRNA ＋ DTIC mouse group were 52.2%, 41.2% and 86.4% respectively. Compared with the control mouse group, the CDK2-shRNA mouse group, DTIC mouse group, and CDK2-shRNA ＋ DTIC mouse group showed significantly increased tumor cell apoptosis indice （32.93% ± 3.72%, 21.62% ± 3.54%, 63.29% ± 4.74% respectively; F = 222.25, P 〈 0.05）. Moreover, the tumor cell apoptosis index was significantly higher in the CDK2-shRNA ＋ DTIC mouse group than in the DTIC mouse group （P 〈 0.01）. Conclusion CDK2 gene silencing can enhance the inhibitory effect of DTIC on the growth of melanoma, and show a synergistic effect with DTIC, likely by increasing the apoptosis of tumor cells.