shRNA沉默CDK2基因对B16-F1细胞小鼠皮下移植瘤的影响

Effect of CDK2 gene silencing on the subcutaneous transplantation tumor of B16-F1 cells in mice

目的早期恶性黑色素瘤可以通过手术切除的方式得到缓解,发生转移的黑色素瘤由于其耐药性,预后较差。为探索治疗黑色素瘤的新方法,本研究探讨由慢病毒载体pUL介导的shRNA沉默CDK2基因对黑色素瘤细胞B16-F1皮下移植瘤生长的影响。方法于C57BL/6小鼠右侧腹股沟皮下注射B16-F1细胞建立移植瘤模型,采用癌灶多点注射的方法分为空白对照组、阴性对照组和实验组(n=5)。阴性对照组,采用癌灶多点注射的方法将携带NC-shRNA的重组慢病毒(滴度为2×108 TU/mL)导入肿瘤灶,每只小鼠200μL,隔日1次,共6次,观察18d;空白对照组,用与阴性对照组相同的方式方法向瘤体注射PBS,观察18d;实验组,采用癌灶多点注射的方法将携带CDK2-shRNA的重组慢病毒(滴度为2×108 TU/mL)导入肿瘤灶,每只小鼠200μL,隔日1次,共6次,观察18d。观察肿瘤生长情况并绘制肿瘤生长曲线,18d后杀鼠取瘤称瘤质量,计算肿瘤生长抑制率,TUNEL检测肿瘤组织细胞凋亡情况,蛋白质印迹法检测肿瘤组织细胞中CDK2蛋白表达情况。结果 C57BL/6小鼠接种瘤细胞3d后均有肿瘤形成。治疗的第6天空白对照组、阴性对照组和实验组的肿瘤体积分别为(48.7±8.4)、(50.0±8.9)和(31.4±8.7)mm3,实验组与空白对照组和阴性对照组相比,肿瘤生长速度明显降低,差异有统计学意义,F=8.879,P=0.004。空白对照组、阴性对照组和实验组的肿瘤重量分别为(0.56±0.10)、(0.55±0.11)和(0.28±0.07)g,实验组与空白对照组和阴性对照组相比,肿瘤体质量明显降低,差异有统计学意义(F=13.659,P=0.001),实验组与空白对照组相比肿瘤生长抑制率为50.2%;空白对照组、阴性对照组和实验组的细胞凋亡指数分别为(7.73±1.08)%、(7.87±1.42)%和(32.27±3.62)%,实验组与空白对照组和阴性对照组相比,瘤细胞凋亡指数显著升高,差异有统计学意义,F=189.748,P<0.001。实验组与空白对照组和阴性对照组相比,组织细胞中CDK2蛋白的表达显著降低,差异有统计学意义(F=293.346,P<0.001),实验组相对于空白对照组CDK2蛋白表达的抑制率为50.4%。结论重组慢病毒CDK2-shRNA沉默CDK2基因的表达能有效抑制B16-F1细胞移植瘤的生长,CDK2基因可能成为黑色素瘤基因治疗的有效靶点。

OBJECTIVE To explore the new method of treating melanoma and investigate the effect of CDK2 gene silence mediated by lentivirus on the growth of melanoma cell line B16-F1 in the C57BL/6 mice. METHODS B16-F1 cells were injected into the right groin of C57BL/6 mice to establish the transplanted tumor model, which were randomly divided into three groups according to the material injected into the cancer foci., blank control group, negative control group and experimental group,5 mice in each group, each group was treated for 6 times, the growth status of the tumor was observed, tumor volume was measured, tumor growth curve was drawn, tumor weight was weighed after 18 days treatment and the tumor growth inhibition rate was calculated. Apoptosis of tumor ceils was inspected by TUNEL. Expression of CDK2 protein in tumor tissues was detected by Western blot. RESULTS Three days after inoculation of tumor cells, the tumor formation in C57BL/6 mice was observed. The mean volume of the blank control group, negative control group and experimental group were （48.7±8.4） mm^3, （50.04-8.9） mm^3 and （31.4±8.7） mm^3 at the sixth day of treatment respectively. Compared with the blank control group and the negative control group, the growth rate of tumor in the experimental group was significantly decreased from the sixth day of treatment（F=8. 879 ,P〈0. 004）. The tumor weight of the three groups at the end of experiment was （0. 56±0.10） g,（0.55±0.11） g and （0.28±0.07） g respectively. Compared with the two control groups, the tumor weight of the experimental group was significantly decreased （F= 13. 659, P = 0. 001）, the tumor growth inhibition rate was 50.2 % compared with that of the blank control group. The apoptosis index of tumor cells in the above three groups was （7.73±1.08）%, （7. 87±1.42） % and （32.27 ± 3.62）% respectively. Compared with the two control groups, the apoptosis index of tumor cells in experimental group was significantly increased （F=189. 748,P〈0. 001）. The expression of CDK2 protein in experimental group was significantly decreased compared with the two control groups （F= 293. 346, P〈0. 001）. The inhibition rate of CDK2 protein expression in the experimental group was 50.4% compared with the blank control group. CONCLUSIONS The growth of B16-F1 cell transplanted tumor can be effectively inhibited by CDK2 gene silencing. It suggests that CDK2 gene may be an effec- tive target for gene therapy of melanoma in future.