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目的 研究反义基因在细胞中持续、稳定表达对平滑肌细胞生长增殖的影响。方法 将分别载有c(myc第1、2和3外显子反义片段的三种重组逆转录病毒表达载体aM1、aM2和aM3导入大鼠主动脉平滑肌细胞(SMC),并于持续筛选后1个月进行稳定表达检测。结果 aM1和aM2能持续抑制靶细胞Myc蛋白表达(分别为对照表达量的59.7%和81.3%)、增殖细胞核抗原(PCNA)表达(分别为对照表达量的52.4%和51.9%),抑制靶细胞的生长增殖活性并以aM2作用最强(抑制率达到30%),同时,aM2还有一定的延长细胞周期和减少DNA合成的作用,而aM3作用相反。另外,与瞬时表达相比,外源基因在靶细胞中的作用并非一成不变,而基因转染操作对细胞的影响会随着细胞的代偿逐渐减少并在短期内消失。结论 对c(myc中不同区域进行反义封闭会产生不同的效应,对其转录活性区进行封闭可取得最强的细胞生长抑制。而靶细胞对对外源基因导入可能具有“纠正作用”。
Objective To study the effect of continuous and stable expression of antisense gene on smooth muscle cell proliferation. Methods Three recombinant retroviral vectors aM1, aM2 and aM3 carrying antisense c, myc 1, 2, and 3 exons, respectively, were introduced into rat aortic smooth muscle cells (SMCs). After continuous screening 1 month respectively.Results aM1 and aM2 could inhibit Myc protein expression in target cells (59.7% and 81.3%, respectively) and PCNA expression in control cells (52.4% And 51.9%, respectively) inhibited the growth and proliferation of target cells and the effect of aM2 was the strongest (30% inhibition rate). Meanwhile, aM2 could prolong cell cycle and decrease DNA synthesis, while aM3 had the opposite effect.In addition, Compared with the transient expression, the effect of exogenous gene in target cells is not constant, and the influence of gene transfection operation on cells gradually decreases with the compensation of cells and disappears in a short time.Conclusion The effect of c (myc in different regions Antisense blockade will produce different effects, the transcription of the active region can be closed to obtain the strongest inhibition of cell growth, and target cells on the introduction of foreign genes may have a “corrective effect.”