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本文旨在探讨周期蛋白D1(cyclin D1)在香烟烟雾提取物(cigarette smoke extract,CSE)所致人肺动脉平滑肌细胞(human pulmonary artery smooth muscle cells,HPASMCs)增殖和迁移中的作用。构建反义cyclin D1基因真核表达载体(pIRES2-EGFP-ascyclin D1),采用脂质体介导基因转染法将空载体(pIRES2-EGFP)和pIRES2-EGFP-ascyclin D1导入正常HPASMCs后,分别进行CSE干预。细胞随机分为6组:对照组、空载体组、反义cyclin D1组、5%CSE组、空载体+5%CSE组、反义cyclin D1+5%CSE组。用实时荧光RT-PCR和Western blot法分别检测cyclin D1mRNA和蛋白的表达,采用流式细胞术、四甲基偶氮唑盐比色法(MTT)、增殖细胞核抗原(PCNA)染色法测定细胞增殖能力,Transwell小室法检测细胞迁移能力。结果显示,反义cyclin D1基因真核表达载体pIRES2-EGFP-ascyclin D1成功构建,并成功转染入HPASMCs,转染后HPASMCs中cyclin D1的mRNA和蛋白表达水平较对照组均显著下降(P<0.05)。与对照组比较,5%CSE组cyclinD1的mRNA和蛋白表达水平均明显升高(P<0.05),细胞增殖和迁移能力显著增强(P<0.05)。与5%CSE组比较,反义cyclinD1+5%CSE组cyclin D1mRNA和蛋白表达水平均明显下降(P<0.05),细胞增殖和迁移能力显著降低(P<0.05)。上述结果提示,CSE可通过上调cyclin D1表达促进HPASMCs增殖和迁移,反义cyclin D1基因真核表达载体可抑制CSE介导的HPASMCs增殖和迁移,提示cyclin D1在CSE所致HPASMCs增殖和迁移中发挥重要调控作用。
This study aimed to investigate the role of cyclin D1 in the proliferation and migration of human pulmonary artery smooth muscle cells (HPASMCs) induced by cigarette smoke extract (CSE). The antisense cyclin D1 gene eukaryotic expression vector (pIRES2-EGFP-ascyclin D1) was constructed. The pIRES2-EGFP-ascyclin D1 vector was transfected into normal HPASMCs by lipofectamine CSE intervention. The cells were randomly divided into 6 groups: control group, empty vector group, antisense cyclin D1 group, 5% CSE group, empty vector + 5% CSE group, antisense cyclin D1 + 5% CSE group. The expression of cyclin D1 mRNA and protein were detected by real-time RT-PCR and Western blot respectively. The cell proliferation was measured by flow cytometry, MTT and PCNA staining Capability, Transwell chamber assay for cell migration. The results showed that the antisense cyclin D1 gene eukaryotic expression vector pIRES2-EGFP-ascyclin D1 was successfully constructed and successfully transfected into HPASMCs. The mRNA and protein expression of cyclin D1 in HPASMCs were significantly decreased after transfection compared with the control group (P < 0.05). Compared with the control group, the mRNA and protein expressions of cyclinD1 in 5% CSE group were significantly increased (P <0.05), and the cell proliferation and migration were significantly increased (P <0.05). Compared with 5% CSE group, cyclin D1 mRNA and protein expression in antisense cyclin D1 + 5% CSE group were significantly decreased (P <0.05), and cell proliferation and migration were significantly decreased (P 0. 05). These results suggest that CSE can promote the proliferation and migration of HPASMCs by up-regulating the expression of cyclin D1. The antisense cyclin D1 gene eukaryotic expression vector can inhibit the proliferation and migration of HPASMCs induced by CSE, suggesting cyclin D1 play a role in the proliferation and migration of HPASMCs induced by CSE Important regulatory role.