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

我们检测了Bax基因是否能诱导培养的大鼠血管平滑肌细胞或巨噬细胞发生凋亡。Northern印迹分析显示Bax基因的表达，Western印迹分析表明，与未转染组相比，转染组培养的SMCs和巨噬细胞中Bax蛋白的表达明显增加。TUNEL阳性细胞百分率、Taq-聚合酶原位连接阳性细胞百分率、细胞凋亡率也分别高于未转染组。DNA梯状条带在转染组呈阳性。电子显微镜下可见凋亡的SMCs和巨噬细胞出现皱缩、核染色质凝集和/或凋亡小体，表明这些细胞在形态上是凋亡性的。另一方面，在si…中观察到pfu阳性细胞，表明有坏死。结论：Bax基因转染组可诱导培养的大鼠颈动脉内膜细胞和巨噬细胞发生凋亡。2.用腺病毒载体通过特定的导管观察了Bax基因在大鼠颈动脉球囊损伤后是否诱导了SMC的凋亡和对内膜增生的保护作用。分别于球囊损伤后1、5、14、30d用PTCA导管处死大鼠。Northern印迹分析显示，转染组的颈动脉内膜有Bax基因的过度表达，而Western印迹分析则显示出Bax蛋白的过度表达。原位杂交和免疫组织化学染色显示，在内膜增生的血管内膜细胞中，Bax mRNA和Bax蛋白均有过表达。TUNEL阳性和Taq聚合酶原位结扎阳性SMC的百分率在转染组明显高于未转染组。超微结构分析显示SMC出现典型的细胞凋亡。在转染组和未转染组中，具有阳性增殖细胞核抗原的SMC的百分比相似。但转染组与未转染组的颈动脉内膜增生程度无明显差异，提示转导Bax基因可诱导球囊损伤后颈动脉内膜增生的SMC发生凋亡。然而，没有证据表明对内膜增生有保护作用。其原因可能是本研究中Bax基因的转染量太小，不能降低内膜的增生程度。因此，新型强势载体的开发具有重要意义。较少

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