| Budget Amount *help |
¥14,100,000 (Direct Cost: ¥14,100,000)
Fiscal Year 2000: ¥5,400,000 (Direct Cost: ¥5,400,000)
Fiscal Year 1999: ¥8,700,000 (Direct Cost: ¥8,700,000)
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| Research Abstract |
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
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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
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