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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。
Purpose Early malignant melanoma can be surgically removed and the metastatic melanoma has a poor prognosis due to its resistance. In order to explore a new method for the treatment of melanoma, we investigated the effect of silencing CDK2 gene by lentiviral vector pUL on the growth of subcutaneously transplanted B16-F1 melanoma cells. Methods B16-F1 cells were injected subcutaneously into the inguinal groves of C57BL / 6 mice to establish a transplanted tumor model. The tumor models were divided into blank control group, negative control group and experimental group (n = 5). Negative control group, the multi-point injection of cancer-carrying NC-shRNA carrying recombinant lentivirus (titer of 2 × 108 TU / mL) into the tumor focus, each mouse 200μL, every other day, a total of 6 times, The rats in the blank control group were injected with PBS for 18 days in the same way as the negative control group. The experimental group received recombinant adenovirus carrying CDK2-shRNA (titer of 2 × 108 TU / mL) into the tumor focus, each mouse 200μL, every other day, a total of 6 times, observation 18d. The growth of tumor was observed and the tumor growth curve was drawn. After 18 days, the mice were sacrificed to take tumor mass and the tumor growth inhibition rate was calculated. The apoptosis of tumor cells was detected by TUNEL. The expression of CDK2 protein in tumor cells was detected by Western blotting. Results C57BL / 6 mice inoculated tumor cells after 3 days all have tumor formation. On the sixth day after treatment, the tumor volume of the blank control group, the negative control group and the experimental group were (48.7 ± 8.4), (50.0 ± 8.9) and (31.4 ± 8.7) mm3, respectively. The experimental groups were significantly different from the blank control group and the negative control group The tumor growth rate was significantly lower than that of the control group (F = 8.879, P = 0.004). The tumor weights of the blank control group, the negative control group and the experimental group were (0.56 ± 0.10), (0.55 ± 0.11) and (0.28 ± 0.07) g, respectively. Compared with the blank control group and the negative control group, (F = 13.659, P = 0.001). Compared with the blank control group, the tumor growth inhibition rate was 50.2% in the experimental group and the apoptosis index in the blank control group, the negative control group and the experimental group (7.73 ± 1.08)%, (7.87 ± 1.42)% and (32.27 ± 3.62)%, respectively. Compared with the blank control group and the negative control group, the apoptosis index of the experimental group was significantly increased, the difference was statistically significant, F = 189.748, P <0.001. Compared with the blank control group and the negative control group, the expression of CDK2 protein in the experimental group was significantly decreased (F = 293.346, P <0.001), and the inhibition of CDK2 protein expression in the experimental group compared with the blank control group The rate was 50.4%. Conclusion The silencing of CDK2 gene expression by recombinant lentivirus CDK2-shRNA can effectively inhibit the growth of transplanted tumor in B16-F1 cells. The CDK2 gene may be an effective target for gene therapy of melanoma.