论文部分内容阅读
目的：研究AnnexinⅠ基因cDNA转染对永生化人支气管细胞系BEP2D生长的影响。方法：pT7T3D质粒经NotⅠ和XhoⅠ双酶切获得人AnnexinⅠ基因cDNA片段，亚克隆至真核表达载体pCI－neo，构建人AnnexinⅠ基因其核表达载体pCI－AXI。通过采用脂质体介导转染技术，将AnnexinⅠ的真核表达重组质粒和空载体质粒分别导入永生化人支气管细胞系BEP2D；合成AnnexinⅠ反义寡核苷酸，观察AnnexinⅠ基因对细胞生长的影响。结果：pCI－AXI转染永生化BEP2D细胞后，AnnexinⅠ的表达比永生化BEP2D细胞和空载体转染细胞提高了1．5倍。pCI－AXI转染细胞倍增时间为27h，较永生化细胞（37h）、空载体转染细胞（33h）明显缩短，流式细胞仪分析表明，AnnexinⅠ表达升高后促进G0／G1期细胞进入S期和G2／M期。永生化细胞、pCI－neo和pCI－AXI转染三种BEP2D细胞的接种效率分别为49．4％，57．87％，82．53％，pCI－AXI转染细胞形成的集落数明显高于其它两种细胞（P＜0．002），并且细胞呈复层生长趋势；pCI－AXI转染细胞在软琼脂中的克隆形成率为0．01％，永生化细胞和空载体转染细胞在软琼脂中不能形成克隆。反义寡核苷酸（50mol／L）能显著抑制BEP2D细胞的生长（P＜0．05）。结论：AnnexinⅠ具有促进BEP2D细胞增殖，提高BEP2D细胞生存
Objective: To study the effect of Annexin I cDNA transfection on the growth of immortalized human bronchial cell line BEP2D. Methods: The human Annexin I cDNA fragment was digested with NotI and XhoI by pT7T3D plasmid and subcloned into the eukaryotic expression vector pCI-neo to construct the human Annexin I gene nuclear expression vector pCI-AXI. By using liposome-mediated transfection technology, the eukaryotic expression recombinant plasmids and empty vector plasmids of Annexin I were respectively introduced into the immortalized human bronchial cell line BEP2D; Annexin I antisense oligonucleotides were synthesized and the effect of Annexin I gene on cell growth was observed. . RESULTS: After immortalized BEP2D cells were transfected with pCI-AXI, the expression of Annexin I was 1.5-fold higher than that of immortalized BEP2D cells and empty vector-transfected cells. The doubling time of pCI-AXI transfected cells was 27 h, which was significantly shorter than that of immortalized cells (37 h) and empty vector transfected cells (33 h). Flow cytometry analysis showed that the expression of Annexin I promoted G0/G1 phase cells into S Period and G2/M period. The inoculation efficiencies of the three types of BEP2D cells transfected with immortalized cells, pCI-neo and pCI-AXI were 49.4%, 57.87%, and 82.53%, respectively. The number of colonies formed by pCI-AXI transfected cells was significantly higher than that of the other cells. The other two kinds of cells (P<0.002), and the cells showed multi-layer growth tendency; the cloning rate of pCI-AXI transfected cells in soft agar was 0.01%, immortalized cells and empty vector transfected cells were Clones cannot form in soft agar. Antisense oligonucleotides (50 mol/L) significantly inhibited the growth of BEP2D cells (P<0.05). Conclusion: Annexin I can promote the proliferation of BEP2D cells and increase the survival of BEP2D cells.