目的：探讨由慢病毒载体pUL介导的pUL-CDK2-shRNA3(CDK2-shRNA)沉默 CDK2 基因后对小鼠黑素瘤B16-F1细胞恶性生物学行为的影响，初步探索其作用机制。 方法: 利用重组慢病毒载体CDK2-shRNA转染B16-F1细胞，MTT法检测其对细胞增殖的影响，DAPI染色、AnnexinV-FITC/PI双染流式术检测细胞凋亡情况，流式术检测细胞周期变化，细胞黏附实验、transwell 小室实验分别检测细胞黏附、侵袭和迁移能力变化，Western blotting检测细胞周期信号通路上RB蛋白（成视网膜细胞瘤蛋白）、pRB蛋白、细胞周期相关转录因子E2F1及侵袭迁移相关蛋白基质金属酶-2（MMP-2）、基质金属酶-9（MMP-9）的表达水平。 结果: 与空白对照组和阴性对照组相比，CDK2-shRNA组细胞相对增殖率、细胞黏附率、细胞侵袭和迁移能力均显著下降（均P<0.05）。细胞凋亡率显著增加（均P<0.05）； G1期细胞显著增加而S期和G2期显著降低（P<0.05）。细胞周期相关蛋白pRB、E2F1明显降低而RB显著升高（P<0.05），细胞侵袭和迁移相关蛋白MMP-2、MMP-9明显降低（P<005）。 结论: 慢病毒载体pUL介导的shRNA沉默 CDK2 基因对B16-F1细胞恶性生物学行为有显著的抑制作用，其机制主要与细胞周期和细胞侵袭迁移相关蛋白表达变化有关。

Objective:To study the effect of silencing CDK2 gene induced by the recombinant lentiviral vector pUL-CDK2-shRNA 3 (CDK2-shRNA) on the malignant biological behavior of mouse melanoma B16-F1 cells, and preliminarily investigate its mechanism.Methods: Melanoma B16-F1 cells were transfected by recombinant lentivirus vector CDK2-shRNA, and effect of which on proliferation of the B16-F1 cells was examined by MTT assay. Apoptosis of the cells was detected by DAPI staining and AnnexinV-FITC/ PI staining flow cytometry assay. Cell cycles were evaluated with flow cytometry assay. Cell adhesion assay and transwell chamber experiment were used to detect the changes of adhesion, invasion and migration abilities of the cells, respectively. Expression levels of proteins RB and pRB on signaling pathway, cell cycle related transcription factor E2F1 and migration related protein MMP-2 and MMP-9 were detected with Western blotting assay. Results: Compared with the blank control and negative control groups, relative proliferation and adhesion rates, migration and invasion abilities of the cells in the CDK2-shRNA group significantly decreased (P<0.05); while the apoptosis rate significantly increased (P<0.05); Number of the cells at G1 phase significantly increased, but number of the cells at S and G2 phases significantly decreased (P<0.05); expression of pRB and E2F1 proteins significantly decreased, but expression of RB significantly increased (P<0.05), which were related with cell cycles; and expression of MMP-2 and MMP-9 proteins for invasion and migration of the cells significantly decreased (P<0.05). Conclusions: Silencing CDK2 gene mediated by recombinant lentiviral vector pUL-CDK2-shRNA could significantly inhibit malignant biological behavior of the B16-F1 cells, and its mechanism might associate with the expression changes of the proteins for cell cycles, invasion and migration of the cells.

河南省科技计划项目（No.122300410193）和河南省高等学校青年骨干教师资助计划项目（No.2011GGJS-262）