乔礼芬, 徐永健, 刘先胜, 谢俊刚, 杜春玲, 张建, 倪望, 陈士新. 周期蛋白D1正反义表达质粒的构建鉴定及其对哮喘大鼠气道平滑肌增殖的影响J. 药学学报, 2008, 43(3): 247-252.
引用本文: 乔礼芬, 徐永健, 刘先胜, 谢俊刚, 杜春玲, 张建, 倪望, 陈士新. 周期蛋白D1正反义表达质粒的构建鉴定及其对哮喘大鼠气道平滑肌增殖的影响J. 药学学报, 2008, 43(3): 247-252.
QIAO Li-fen, XU Yong-jian, LIU Xian-sheng, XIE Jun-gang, DU Chun-ling, ZHANG Jian, NI Wang, CHEN Shi-xin. Recombination and identification of sense and antisence CyclinD1 eukaryotic expression vectors and the effects of the vectors on the proliferation of airway smooth muscle cell in asthmatic ratsJ. Acta Pharmaceutica Sinica, 2008, 43(3): 247-252.
Citation: QIAO Li-fen, XU Yong-jian, LIU Xian-sheng, XIE Jun-gang, DU Chun-ling, ZHANG Jian, NI Wang, CHEN Shi-xin. Recombination and identification of sense and antisence CyclinD1 eukaryotic expression vectors and the effects of the vectors on the proliferation of airway smooth muscle cell in asthmatic ratsJ. Acta Pharmaceutica Sinica, 2008, 43(3): 247-252.

周期蛋白D1正反义表达质粒的构建鉴定及其对哮喘大鼠气道平滑肌增殖的影响

Recombination and identification of sense and antisence CyclinD1 eukaryotic expression vectors and the effects of the vectors on the proliferation of airway smooth muscle cell in asthmatic rats

  • 摘要: 观察哮喘大鼠气道平滑肌周期蛋白D1的表达,并构建大鼠周期蛋白D1 (CyclinD1)正反义表达质粒,转染支气管哮喘大鼠气道平滑肌细胞(ASMC),探讨CyclinD1在哮喘大鼠ASMC增殖过程中的影响。大鼠雾化吸入卵清蛋白建立哮喘模型,免疫荧光检测CyclinD1的表达。以气道平滑肌条总RNA为模板,通过RT-PCR获取大鼠CyclinD1全长cDNA,插入到真核表达载体pcDNA3.1(+)上,构建CyclinD1正义表达质粒(pcDNA3.1-CyclinD1)和反义表达质粒(pcDNA3.1-asCyclinD1)。用脂质体介导的基因转染方法,将正反义重组体和空质粒(vector)分别转染哮喘大鼠和正常大鼠ASMC,用Western blotting方法鉴定CyclinD1基因的表达。采用流式细胞术、四甲基偶氮唑盐(MTT)法、增殖细胞核抗原(PCNA)染色等方法观察构建质粒对哮喘大鼠ASMC增殖的影响。结果表明:(1)与对照组相比,哮喘组CyclinD1表达显著增高;(2)酶切鉴定和测序分析证实,试验成功构建了CyclinD1正反义表达质粒。与转染pcDNA3.1-CyclinD1组和vector组相比,转染pcDNA3.1-asCyclinD1组大鼠ASMC中CyclinD1表达水平下降(P<0.01);(3)与vector组S+G2M期比例、吸收度(A)值、PCNA阳性表达率相比,转染pcDNA3.1-CyclinD1组增殖指标均明显增加(P<0.01)。转染pcDNA3.1-asCyclinD1组增殖指标均明显下降(P<0.01)。正常组大鼠转染质粒后各组间变化趋势与哮喘组一致。pcDNA3.1-CyclinD1可促进哮喘大鼠ASMC增殖,pcDNA3.1-asCyclinD1可抑制哮喘大鼠ASMC增殖,提示CyclinD1在哮喘ASMC增殖的信号转导中具有重要作用。

     

    Abstract: This study is to investigate the expression of CyclinD1 in asthmatic rats and construct expression plasmids of sense and antisense CyclinD1 gene and transfect them to asthmatic airway smooth muscle cell to study the effects of CyclinD1 on the proliferation of airway smooth muscle cells in asthmatic rats. CyclinD1 cDNA was obtained by RT-PCR of total RNA extracted from the airway smooth muscle in asthmatic rats. The sequence was inserted into eukaryotic expression vector pcDNA3.1(+) to recombinate the sense and antisense pcDNA3.1-CyclinD1 eukaryotic expression vector. The two recombinations and vector were then separately transfected into airway smooth muscle cell in asthmatic rats by using liposome. The expression level of CyclinD1 was certificated by Western blotting analysis. The proliferations of ASMCs isolated from asthmatic rats were examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. Results showed (1) Compared with control group, the content of CyclinD1 was significantly increased; (2) It was comformed by restriction endonucleasa digestion and DNA sequence analysis that the expression plasmid of sense and antisense CyclinD1 were successfully recombinated. There was significant change of CyclinD1 expression between vector and sense CyclinD1 transfected cells, and the expression level of CyclinD1 in ASMC transfected with antisense CyclinD1 was lower than that in vector transfected cells (P<0.01); (3) In the asthmatic groups, compared with the vecter group, the percentage of S+ G2M phase, absorbance A value of MTT and the expression rate of PCNA protein in ASMC transfected with pcDNA3.1-CyclinD1 vector significantly increased. The values decreased remarkably in the pcDNA3.1-asCyclinD1 group. Statistical analysis revealed that there were significant differences in these indicators of cell proliferation in three groups (P<0.01). In the normal groups, statistical analysis revealed that there were significant differences in the percentage of S+G2M phase, a value of MTT and the expression rate of PCNA protein in three groups (P<0.01). Sense CyclinD1 eukaryotic expression vectors could have a positive effect on the proliferation of ASMC, however the antisence one have a negative effect, which implicated that CyclinD1 might contribute to the process of airway smooth muscle cell proliferation.

     

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