高压干燥保存对小鼠供心冷缺血再灌注损伤的影响

Efficacy of high-pressure mixed gas preservation of mouse hearts for transplantation subjected to cold ischemia and reperfusion injury

目的观察氧气（oxygen,O2）及一氧化碳（carbon monoxide,CO）混合高压气体干燥保存方法对小鼠离体供心冷缺血再灌注损伤的影响。方法采用C57BL/6雄性小鼠,建立同系小鼠心脏颈部异位移植模型。4~6周龄供体鼠48只,数字表法随机分为4组（n=12）：空白对照组（A组）、即刻移植组（B组）、组氨酸-色氨酸-酮戊二酸盐（histidine-tryptophan-ketoglutarate solution,HTK）液保存组（C组）及高压干燥保存组（D组）。6~8周龄小鼠36只,数字表法随机分为3组,即刻移植受体组、HTK受体组及高压干燥受体组（n=12）。供心处理如下：A组仅取供体心,不做移植;B组供心不经保存取下后即刻移植;C组供心浸泡于HTK液中;D组供心悬挂并置于高压气体罐中（PO2：3.2×101.3 k Pa＋PCO：0.8×101.3 k Pa=4×101.3 k Pa）。C、D两组供心均于4℃冰箱中保存24 h后,分别移植于受体组小鼠颈部。移植后2 h,对比观察B、C、D三组复跳情况及供心功能。移植后24 h,检测受体鼠血清心功酶变化;取各组供心组织,分别采用苏木素-伊红（HE）染色、Tunnel染色及行蛋白免疫印迹（Western-blot,WB）检测,对比观察供心病理学改变、心肌细胞凋亡及B细胞淋巴瘤/白血病-2（B cell lymphoma/leukemia-2,bcl-2）蛋白表达情况。结果 B组10只供心复跳,C组2只供心复跳,D组9只供心复跳,3组间复跳率比较,差异有统计学意义[83.3%（10/12）vs.16.7%（2/12）vs.75.0%（9/12）,P=0.0015];其中D组复跳率低于B组,但明显高于C组,差异有统计学意义（P〈0.0125）。移植2 h后B、C、D组间供心功能评分比较,差异有统计学意义（5 vs.0 vs.3,P〈0.01）,两两比较差异有统计学意义（P〈0.05）。4组间血清肌钙蛋白I（troponin I,Tn I）浓度比较,差异有统计学意义（P〈0.001）,D组高于A、B两组,但低于C组（P〈0.001）。苏木素伊红染色结果显示D组细胞水肿、炎症细胞浸润等组织变化情况较A、B两组明显,但较C组轻。Tunnel染色结果显示D组心肌细胞凋亡较A、B两组明显,但少于C组,差异有统计学意义（P〈0.01）。Western-blot结果显示D组bcl-2的表达上调,高于其余3组,差异有统计学意义（P〈0.05）。结论高压干燥保存可减轻离体供心冷缺血再灌注损伤,维持供心活力,有效延长供心保存时间。

Objectives To investigate the efficacy of preserving mouse hearts with high partial pressure of oxygen and carbon monoxide for transplantation subjected to cold ischemia and reperfusion injury. Methods C57 BL / 6 male mice were used to establish model of heterotopic cervical heart transplantation. Forty-eight donor mice aged 4-6 weeks were randomly divided into 4 equal groups（n=12） and subjected to naive operation（group A）, sham operation（group B）,standard control（group C） and experimental control（group D）. Thirty-six recipient mice aged 6-8 weeks were randomly divided into three equal groups（n =12） and subjected to sham operation, standard control and experimental control. A group ： hearts were isolated; B group ： hearts were isolated and transplanted immediately; C group ： hearts were isolated and preserved in histidine-tryptophan-ketoglutarate（HTK） solution at 4℃ for 24 h before transplantation;D group ： hearts were isolated and preserved in high pressure gas（PO2： 3.2×101.3 k Pa ＋ PCO ： 0.8 ×101.3 k Pa = 4 ×101.3 k Pa） at 4℃ for 24 h before transplantation. Two hours after transplantation, the states of re-beating and cardiac functions were observed in B, C and D group. Twenty-four hours after transplantation, blood test was carried out for recipient mice to compare cardiac enzymes. Pathologic changes were observed through hematoxylin and eosin（HE）staining; cardiac cell apoptosis were detected by Tunnel staining; the expressions of B cell lymphoma / leukemia-2（bcl-2） were observed by Western blotting（WB）. Results Ten transplanted hearts in group B re-beat while the number was 2 in C group and 9 in D group. The difference was significant [83.3%（10 / 12） vs. 16.7%（2 / 12） vs. 75.0%（9 /12）, P =0.0015 ]. The re-beating rate of D group was slightly lower than that of B group, but significantly higher than that of C group（P〈0.0125）. There was significant difference in cardiac function among B, C and D groups two hours after transplantation（5 vs. 0 vs. 3, P〈0.01）. The difference in each pair of the groups was also significant（P〈0.05）.There was significant difference in concentrations of cardiac troponin I（Tn I） among the 4 groups（P〈0.001）. D group had a higher concentration of cardiac enzymes than A and B group, but lower than C group（P〈0.001）. HE staining showed that pathologic changes such as cell edema and inflammatory cell infiltration were more obvious in D group than in A and B group, but less obvious than in C group. Tunnel staining showed significant differences in cardiac cell apoptosis（P〈0.01）. D group had more obvious apoptosis than and A and B group, but less obvious than C group.Western blotting results showed a significant increase of bcl-2 expression in D group than in the other groups（P〈0.05）.Conclusions High-pressure mixed gas preservation can reduce cold ischemia and reperfusion injury of donor heart,maintain myocardial viability and prolong its preservation time.