论文部分内容阅读
目的:探讨由慢病毒载体p UL介导的p UL-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、Annexin V-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p UL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 investigate the effect of p UL-CDK2-shRNA3 (CDK2-shRNA) mediated by lentiviral vector on the malignant biological behavior of mouse melanoma B16-F1 cells after silencing CDK2 gene, and to explore its mechanism. Methods: B16-F1 cells were transfected with recombinant lentiviral vector CDK2-shRNA. Cell proliferation was measured by MTT assay. Cell apoptosis was detected by DAPI staining and Annexin V-FITC / PI double staining. Flow cytometry The changes of cell adhesion, invasion and migration were detected by cell adhesion assay, cell adhesion assay and transwell chamber assay. The expressions of RB protein (retinoblastoma protein), pRB protein, cell cycle related transcription factor E2F1 And the expression levels of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). Results: Compared with the blank control group and negative control group, the relative proliferation rate, cell adhesion rate, cell invasion and migration ability of CDK2-shRNA group were significantly decreased (all P <0.05). (P <0.05). The cells in G1 phase increased significantly while those in S phase and G2 phase decreased significantly (P <0.05). The expressions of pRB, E2F1 and RB were significantly increased (P <0.05), and the expressions of MMP-2 and MMP-9 were significantly decreased (P <0.05). CONCLUSION: The silencing of CDK2 by lentiviral vector p UL can significantly inhibit the malignant biological behavior of B16-F1 cells. The mechanism is mainly related to the changes of cell cycle and cell invasion and migration related proteins.