Experimental study of lung low temperature preservation using antifreeze product

【目的】影响肺保存的因素有几个：一个是器官所处的温度。低温保存可降低待移植器官的代谢率，从而减少能量需求，抑制缺血性紊乱的发生。然而，如果保存温度接近0℃，器官内部就会冻结，细胞结构甚至整个器官都可能被破坏，这与保存的目的是相反的。最近人们注意到，生物体体内有各种各样的低温保护物质，它们能够在冰点以下的温度下生存。此外，一项临床前研究的结果表明，如果在保存液中加入冷冻保护剂（如乙醇），肺可以在低温下保存较长时间。因此，提出了一种假设，即添加到保存液中的防冻产品可以使待移植器官免于冻结，并通过将器官保持在比所施加的温度（冷冻温度）更低的温度来延长保存期限。在本研究中，在保存液中加入了稳定细胞并在低温下保存细胞的抗冻多糖。采用大鼠进行动物实验，观察低温（冷冻温度）下使用该添加剂保存肺对细胞的保存和预防移植后再灌注紊乱的作用。【对象和方法】采用不含糖的Euro-Collins （EC）溶液作为保鲜液。从该EC溶液中制备如下：添加葡萄糖的ECG溶液（3.5 g/100 ml）；ECT溶液，在EC溶液中加入海藻糖（6,84 g/100 ml），这是一种高效的防腐剂；甲胎蛋白溶液，加入苏云金芽孢杆菌甲胎蛋白（200 μg/ml）的ECG溶液。取体重为250 ~ 300 g的Wistar大鼠进行心脏骤停，取其心脏和肺，按以下8种保存方法中的一种保存1和24小时（每组n=4）： 1组，-1℃EC溶液；第2组，4℃EC溶液；3组，-1℃心电液；4组，4℃心电液；第5组，-1℃ECT溶液；6组，4℃ECT溶液；第7组，-1℃AFP溶液；8组为4℃AFP溶液。评估保存肺的组织活性，如台盼蓝排泄、ATP和LDH表达的细胞活性以及病理检查中发现的肺破坏（每组n=4）。取第3、5、7组左肺移植给体重250 ~ 300g的Wister大鼠。移植后2小时内取出移植肺进行病理评价，测定干、湿重比（每组n=2）。[结果]器官保存评价：1 ~ 8组肺组织保存1小时与24小时的细胞ATP和LDH比值分别为：（ATP %）、69.5、34.5、84.1、73.5、91.4、76.7、86.1、76.5；(LDH %), 112.0, 151.0, 104, 0, 93.7, 92.5, 92.4, 83.8和98.0。与-1℃组相比，4℃组肺ATP活性明显降低。成功保存ATP的顺序如下：4℃时ECT（大于等于）AFP（大于等于）ECG>EC， -1℃时ECT>AFP（大于等于）ECG>EC。4℃时EC>AFP【大于等于】ECG【大于等于】ECT， -1℃时EC>ECG>ECT>AFP，肺LDH的逃逸程度显著。同样，与4℃组相比，-1℃组的细胞学破坏（表现为台盼蓝排泄和细胞水肿）更为明显。EC组细胞学偏差明显高于其他3组，但3组间差异无统计学意义。同种异体移植术后切除左肺的评价：病理检查发现3、5、7组肺水肿，但各组间无明显差异。3、5、7组干、湿器官重比分别为0.45、0.41、0.43，组间差异不大。[结论]甲胎蛋白的保肺效果不如海藻糖，但海藻糖可能对器官保存有效，特别是在低温下。少

[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 … More 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. Less