Gene therapy against restenosis after balloom injury using apoptosis inducer, bax

我们检测了凋亡诱导基因bax基因转染培养的大鼠平滑肌细胞（SMC）或巨噬细胞是否诱导培养细胞凋亡。免疫印迹分析显示，转染后培养的SMCs和巨噬细胞中Bax mRNA表达较未转染组明显增加，Western印迹分析显示Bax蛋白表达较未转染组明显增加。TUNEL阳性细胞的百分比、基于Taq聚合酶的原位连接阳性细胞、凋亡细胞的指示物在每个转染的SMC和巨噬细胞中也分别高于未转染的细胞组。DNA梯状条带在转染细胞中呈阳性。电子显微镜分析显示存在凋亡的SMC和巨噬细胞，具有皱缩、特异性核染色质凝聚和/或凋亡小体，表明这些细胞在形态学上是凋亡的。另一方面，在S1细胞中观察到指示坏死的Pfu阳性细胞。 ...更多信息 结论：bax基因转染可诱导大鼠颈动脉平滑肌细胞和巨噬细胞凋亡。II.应用腺病毒载体通过特异性导管转染大鼠颈动脉，观察bax基因转染对大鼠颈动脉平滑肌细胞凋亡的诱导作用及对球囊损伤后内膜增生的保护作用。分别于球囊损伤后1、5、14、30天处死大鼠。免疫印迹分析显示，转染组大鼠颈动脉内膜增生区bax蛋白表达明显高于对照组，而noclonal blot分析显示bax mRNA表达明显高于对照组。原位杂交和免疫组化结果显示，在内膜增生的平滑肌细胞中，bax mRNA和Bax蛋白表达明显增加。转染组内膜增生中TUNEL阳性和Taq-polymerase原位连接阳性SMC的百分比显著高于未转染组。电镜下可见平滑肌细胞典型的凋亡。转染组和未转染组中PCNA阳性的SMC百分比相似，PCNA阳性是极化的指标。结论：bax基因转染大鼠颈动脉后，可诱导球囊损伤后内膜增生的平滑肌细胞凋亡。然而，没有证据表明对内膜增生有保护作用。这可能是由于bax基因转染程度太小，不能减轻内膜增生程度所致。因此，开发新的、强有力的载体非常重要。少

I.We examined whether transfection of bax gene, apoptosis-inducing gene, into rat cultured smooth muscle cells (SMC) or macrophages induces apoptosis in the cultured cells. Nothern blot analysis revealed the expression of bax mRNA and western blot analysis also showed the definite increase of Bax protein in the transfected cultured SMCs and macrophages, compared with non-transfected cell groups. The percentage of TUNEL-positive cells, Taq-polymerase based in situ ligation-positive cells, indicators of apoptotic cells, were also higher In each of the transfected SMCs and macrophages than non-transfected cell groups, respectively. DNA ladder became posotive in the transfected cell groups. Electron microscopic analysis revealed the presence of apoptotic SMCs and macrophages with schrinkage, specific nuclear chromatin condensation, and/or apoptotic bodies, indicating these cells are morphologically apoptotic. On the other hand, Pfu-posotive cells indicating necrosis were observed in the si … More milar percentage between the transfected and non-transfected SMCs or macrophages, respectively.Thus, we conclude that the transfection of bax gene can induce apoptosis of cultured SMCs and macrophages in rats.II.It was examined whether the transfection of bax gene in rat carotid arteries induces apoptosis of SMCs and is protective against intimal hyperplasia after the balloon injury using adeno-viral vectors through the specific catheter. The rats were sacrificed 1, 5, 14 and 30 days after the balloon injury using PTCA catheter. The overexpression of bax mRNA by nothern blot analysis and Bax protein by western blot analysis ware observed in the carotid areteries with intimal hyperplasia in the transfected groups. The in situ hybridization and the immunohistochemistry ahowed the overexpressions of bax mRNA and Bax protein in the SMCs in the intimal hyperplasia. The percentages of TUNEL-positive and Taq-polymerase based in situ ligation-positive SMCs were significantly higher in the transfected intimal hyperplasia than the non-transfected intimal hyperplasia. Electron microscopic analysis showed typical apoptosis in SMCs. The SMCs with posotive PCNA, an indicator of poloriferation, was similar in the percentage between the transfected and non-transfected groups. However, the degree of intimal hyperplasia showed no siginificant difference between the transfected and non-transfected carotid arteries.Thus, we conclude that the transfection of bax gene in the rat carotid arteries can induce apoptosis of SMCs in the intimal hyperplasia after balloom injury. However, there was no evidence of protective effect against intimal hyperplasia. The descrepancy may be explained by that the degree of transfection of bax gene in the present study was too small to reduce the degree of intimal hyperplasia. Therefore, the development of new and strong vector is important. Less