細胞を安定化し低温保存できる抗氷核物質(Antifreeze product, AFP)を用いた肺の低温(氷温)保存が臓器細胞保存と移植後再還流障害の抑制に及ぼす効果についてラットを用いた動物実験を行った。【対象と方法】保存液を糖分を含まないEuro-Collins(EC)液にした.EC液にブドウ糖(3.5g/100ml)を加えたものをECG液,EC液に保存効果の高いトレハロース(6.84g/100ml)を加えたものをECT液,ECG液にBacillus thuringiensis由来のAFP(200μg/ml)を加えたものをAFP液とした.250-300gのWister系ラットで心停止後、心臓と肺を取り出し,次の8群に分類した保存法で1時間および24時間保存した.各群でtrypan blue排泄・ATP, LDHによる細胞活性・病理所見による肺損傷など保存肺の組織活性を検討した.(各群n=4)1群:-1℃EC液,2群:4℃EC液,3群:-1℃ECG液,4群:4℃ECG液,5群:-1℃ECT液,6群:4℃ECT液,7群:-1℃AFP液,8群:4℃AFP液.また,250-300gのWister系ラットを用いて,3群,5群,7群で左肺同種移植を行った.移植後2時間で移植肺を摘出し,病理学的評価と乾湿重量比を測定した.(各群n=2)結果の数値は平均値で表し、Mann-Whitney U testで比較検定した。p<0.05以下を有意差ありとした。【結果】保存の評価:保存1時間に対する保存24時間の細胞ATP, LDH比率の結果は上記群の順に,肺ATP(%、Fig.3):69.5,34.5,84.1,73.5,91.4,76.7,86.1,76.5,肺LDH(%、Fig.4):112.0,151.0,104.0,93.7,92.5,92.4,83.8,98.0.肺ATPは各保存液ともに,4℃群は-1℃群に対し有意に低下した.ATPの温存は,4℃ではECT≧AFP≧ECG>EC,-1度ではECT>AFP≧ECG>EC.肺LDHの逸脱は4℃ではEC>AFP≧ECG≧ECT,-1度ではEC>ECG>ECT>AFP.同様にtrypan blue排泄能の低下・細胞浮腫などの細胞障害は-1℃群で4℃群に比べ強かった.EC群は他の3群に比べ細胞障害が強かったが,他の3群間で有意差はなかった.左肺同種移植後,摘出肺の評価:病理学的には肺水腫の所見であったが3群,5群,7群で差は確認できなかった.乾湿重量比は3群,5群,7群でそれぞれ0.45,0.41,0.43と差を認めなかった.【結論】AFP添加による肺保存効果はトレハロースに比べ少ないが,特に低温での保存に有用である可能性がある.
[Purpose] Several factors affect preservation of the lung : one is the temperature to which the organ is exposed. Preserving it at a low temperature reduces the metabolic rate of the organ to be transplanted, thus reducing energy demand and suppressing the development of ischemic disturbances. If the preservation temperature approaches 0℃, however, the interior of the organ freezes, cellular structures and eventually the entire organ may be destroyed, an effect contrary to the purpose of preservation.It has been noted recently that there are various cryoprotective substances in the body of organisms that are capable of surviving subfreezing temperatures. Furthermore the result of a preclinical study indicated that a lung can be preserved for an extended period at a low temperature if a cryoprotective agent (such as ethanol) has been added to the preservative fluid. Thus a hypothesis is proposed that the antifreeze product added to a preserving fluid keeps the organ to be transplanted f rom freezing and prolongs the preservation period by keeping the organ at a temperature even lower than what has been applied (freezing temperature). In the current study, anti-freeze polysaccharides that stabilize cells and preserve them at a low temperature were added to the preservation fluid. An animal experiment was conducted using rats and the effects of preserving a lung at a low temperature (freezing temperature) using such additives were observed on the preservation of the cells and prevention of post-transplantation reperfusion disorders.[Subjects and method] Euro-Collins (EC) solution containing no sugar was used as a preservative fluid. The following were prepared from this EC solution : ECG solution with the addition of glucose (3.5 g/100 ml) ; ECT solution, an EC solution to which trehalose (6,84 g/100 ml), a highly effective preservative, was added ; and AFP solution, an ECG solution to which an AFP derived from Bacillus thuringiensis (200 μg/ml) was added. Cardiac arrest was induced in Wistar rats weighing 250 to 300 g and their hearts and lungs were removed and preserved for 1 and 24 hours according to one of the following 8 methods of preservation (n=4 for each group) : group 1, -1℃ EC solution ; group 2, 4℃ EC solution ; group 3, -1℃ ECG solution ; group 4, 4℃ ECG solution ; group 5, -1℃ ECT solution ; group 6, 4℃ ECT solution ; group 7, -1℃ AFP solution ; and group 8, 4℃ AFP solution.Tissue activities of the preserved lungs-such as trypan blue excretion, cellular activities expressed by ATP and LDH, and pulmonary destruction recognized in pathological examinations-were evaluated (n=4 in each group).The left lungs collected from groups 3,5, and 7 were used for allotransplantation to Wister rats weighing 250 to 300g. Within 2 hours of transplantation, the transplanted lungs were removed for pathological evaluation and determination of the dry and wet weight ratio (n=2 for each group).[Results] Evaluation of organ preservation : cellular ATP and LDH ratios in the lungs preserved for one hour vs.24 hours were listed for groups 1 to 8 : (ATP %), 69.5, 34.5, 84.1, 73.5, 91.4, 76.7, 86.1, and 76.5 ; (LDH %), 112.0, 151.0, 104, 0, 93.7, 92.5, 92.4, 83.8, and 98.0. The pulmonary ATP activity was significantly reduced in the 4℃ groups in comparison with the -1℃ groups. Successful preservation of ATP was noted in the following order : ECT【greater than or equal】AFP【greater than or equal】ECG>EC at 4℃ and ECT>AFP【greater than or equal】ECG>EC at -1℃. The extent of the escape of pulmonary LDH was eminent in the following sequence, EC>AFP【greater than or equal】ECG【greater than or equal】ECT at 4℃ and EC>ECG>ECT>AFP at -1℃. Similarly, cytological disruptions, expressed by trypan blue excretion and cellular edema, were more pronounced in the -1℃ group in comparison with the 4℃ group. Cytological deviations were more exaggerated in the EC group than in the other 3 groups, whereas there was no significant differences among these 3 groups.Evaluation of the excised left lungs after allotransplantation : a pathological examination detected pulmonary edema in groups 3, 5 and 7 but no outstanding difference was found among these groups. The ratio of wet and dry organ weights were 0.45, 0.41 and 0.43 for groups 3, 5 and 7, respectively, with few intergroup differences.[Conclusion] The lung preserving effect of AFP is inferior to that of trehalose but the former may be effective for organ preservation, in particular at low temperatures.
研究課題/領域番号:16591387, 研究期間(年度):2004-2005
出典：「抗氷核活性物質、不凍蛋白質を用いた移植肺の低(氷)温保存に関する基礎的研究」研究成果報告書 課題番号16591387 (KAKEN：科学研究費助成事業データベース（国立情報学研究所）) 本文データは著者版報告書より作成