| Budget Amount *help |
¥16,640,000 (Direct Cost: ¥15,500,000、Indirect Cost: ¥1,140,000)
Fiscal Year 2007: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000)
Fiscal Year 2006: ¥11,700,000 (Direct Cost: ¥11,700,000)
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| Research Abstract |
In order to investigate the feasibility of protection against ischemia-reperfusion injury (IRI) using a short interfering RNA (siRNA) that inhibits the activity of myeloperoxidase (MPO) of human neutrophils, the following studies were Performed.
The siRNA to diminish the MPO activity was designed and synthesized. Both effective (positive) and ineffective (negative) siRNAs were prepared. Then, the siRNA was electrically transferred into neutrophils using NucleofectorR system. The Suction of the expression of mRNA of MPO by a positive siRNA was successfully performed, which was confirmed using a real-time PCR. However, when the neutrophils were activated by opsonized zymosan, the generation of reactive oxygen species (ROS) was not diminished in the neutrophils incorporated with a positive siRNA compared to that in the neutrophils incorporated with a negative siRNA. That is, the RNA interference was not effective to inhibit the MPO activity of neutrophils.
As an alternative capture, we tried to inhibit the ROS generation in endothelial cells using human umbilical vein endothelial cells (HUVECs). First, it was revealed that some stimulants, such as lipopolysaccaride (LPS), induced the expression of interleukin-8 (IL-8) and that IL-8, in turn, induced ROS generation in HUVECs. The siRNA to diminish the expression of IL-8 was designed and synthesized. Both positive and negative siRNAs were prepared. Then, the siRNA was electrically transferred into HUVECs using NucleofectorR system. The Suction of the expression of mRNA of IL-8 by a positive siRNA was successfully performed, which was confirmed using a real-time PCR. And, when the HUVECs were stimulated by LPS, the generation of ROS was diminished in the HUVECs incorporated with a positive siRNA compared to that in the HUVECs incorporated with a negative siRNA. It was known that the ROS generated by endothelial cells hurt themselves during IRI. So, we can say that protection against WI using a siRNA was achieved.
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