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2008年度研究内容

ビタミンE固定化膜による白血球を介した血小板への刺激の低減

研究者
塚尾 浩北里大学医療衛生学部医療工学科臨床工学専政
高橋 治子北里大学医療衛生学部医療工学科臨床工学専政
小久保 謙一北里大学医療衛生学部医療工学科臨床工学専政
新保 年弘北里大学医療衛生学部医療工学科臨床工学専政
廣瀬 稔北里大学医療衛生学部医療工学科臨床工学専政
小林 弘祐北里大学医療衛生学部医療工学科臨床工学専政

キーワードビタミンE、ポリスルホン膜、血小板活性化、白血球、スーパーオキシド

要旨

ビタミンE固定化が白血球を介した血小板活性化に及ぼす影響を明らかにすることを目的とし、血小板のみ、また血小板・白血球共存下においてビタミンE固定の有無による血小板活性化の差異を比較検討した。

ブタ血液を遠心分離し、多血小板血漿(PRP) とそれに白血球を加えたPRP+白血球溶液を作製した。PRPおよびPRP十白血球溶液にキサンチンオキシダーゼを添加して発生したスーパーオキシドの量を測定するとともに、血小板の活性化率の変化をP-セレクチンの発現より評価した。APS-15MD(ポリスルホン膜)、VPS-13H(ビタミンE固定化ポリスルホン膜)に、PRPもしくは白血球を含むPRPを4時間循環させ、血小板の活性化率の変化をP-セレクチンの発現より評価した。PRPに白血球を加えるとスーパーオキシド濃度が上がり、血小板の活性化率が上昇した。このとき、キサンチンオキシダーゼを加えるとさらに血小板が活性化した。また、PRPでは、VPSの方が透析膜との接触により大きく血小板が活性化した。白血球が共存する場合、透析膜と接触した血小板は、接触していない血小板に比べ活性化率が大きく上昇し、APSとVPSでの活性化率は同程度の増加であった。このときVPSでは白血球による血小板活性化の増加が少なかった。また、PRPに比べ、白血球の共存下では、血小板が活性化されやすかった。この活性化の一因として白血球で産生されるスーパーオキシドの関与が考えられた。ビタミンEの抗酸化能によりスーパーオキシドが消去されていること白血球や血小板への刺激が抑制されていることがその一因と考えられた。ビタミンE固定化ポリスルホン膜は、非固定化ポリスルホン膜に比べて血小板を直接刺激して血小板を活性化させるが、白血球を介した血小板への刺激を低減していると考えられる。

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Suppression of platelet activation mediated by leukocytes using vitamin E-coated polysulfone dialyzer.

Researcher
Hiroshi TsukaoDepartment of Medical Engineering and Technology, Kitasato University School of Allied Health Sciences
Haruko TakahashiDepartment of Medical Engineering and Technology, Kitasato University School of Allied Health Sciences
Kenichi KokuboDDepartment of Medical Engineering and Technology, Kitasato University School of Allied Health Sciences
Toshihiro ShinboDepartment of Medical Engineering and Technology, Kitasato University School of Allied Health Sciences
Minoru HiroseDepartment of Medical Engineering and Technology, Kitasato University School of Allied Health Sciences
Hirosuke KobayashiDepartment of Medical Engineering and Technology, Kitasato University School of Allied Health Sciences

KeywordsVitamin E-coated dialyzer, platelet activation, leukocyte, superoxide

Abstract

Dialysis patients are known to be subject to increased oxidative stress, which have been reported to be attenuated by the use of vitamin E-coated dialyzer. The effects of vitamin E coating on the platelet activation, however, have not yet been examined in detail. In the present study, to clarify the mechanism involved in platelet activation and vitamin E coating, we investigated the change in platelet activation after the platelet were contacted with vitamin E-coated dialyzer with or without leukocytes.

Platelet-rich plasma (PRP) was prepared as supernatant solution separated from porcine whole blood by centrifugation of 140 x g for 20 min, and PRP with leukocytes was prepared by adding leukocyte interface layer on sedimented erythrocytes to PRP. The expression of P-selectin was measured by flowcytometry as a marker of platelet activation for the PRP or PRP with leukocytes after the platelets were stimulated by superoxide anion generated by xanthine oxidase. PRP or PRP with leukocytes was circulated for 4 hrs through the blood circuit connected to a dialyzer; VPS-13H comprised of vitamin E-coated polysulfone membrane or APS-15MD comprised of non-coated polysulfone membrane (Asahi Kasei Medical, Japan). During the experiments, samples were collected every hour, and the expression of P-selectin was also measured as a marker of platelet activation.

The ratio of activated platelets in PRP increased more in the PRP with leukocytes than that in the PRP without leukocytes. The platelets in both PRPs were further activated by the addition of superoxide anion. Ratios of activated platelet in PRP increased after the platelets were contacted with both dialyzers. The ratios of activated platelets for VPS-13H indicated higher values compared with that for APS-15MD. On the other hand, ratios of activated platelet in PRP with leukocytes were similar for both dialyzers. These results indicate that the vitamin E-coated membrane suppressed the platelet activation via leukocyte activation, which was due to the superoxide anion-scavenging activity by vitamin E-coated membrane and possibly due to the suppression of leukocyte activation by dialysis membranes in the vitamin E-coated membrane.

In conclusion, Vitamin E-coated dialyzer activated the platelet compared to non-coated dialyzer but it can suppress the platelet activation mediated by leukocytes, possibly by blocking superoxide-mediated stimulation by its antioxidative action.

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