外源性ATP对大鼠移植胰腺的作用及机制(一)
详细内容
【关键词】 胰十二指肠移植；三磷酸腺苷；细胞凋亡；细胞胀亡
Protective effect of exogenous ATP on transplanted pancreas in rats and its mechanism
【Abstract】 AIM: To study the protective effect of exogenous adenosine triphosphate(ATP) on transplanted pancreas in rats and its mechanism. METHODS: The allogeneic male SD rat models of heterotopic whole pancreaticoduodenal transplantation were established. The isolated pancreas of rats was perfused and stored with calparine balanced solution in which ATP was added as an additive agent in ATP group. No ATP was added to calparine balanced solution in control group. The ans were stored at 0-4℃ for 60 min in both groups. Blood sugar，plasma concentrations of amylase and lipase were examined and histological observation was performed at 0, 1, 6 and 12 h after reperfusion. The concentration of ATP in pancreatic tissue was determined. The percentages of normal, apoptotic and oncotic cells were measured. RESULTS: Light microscopic observation showed that histomorphological changes of pancreas in ATP group were much milder than those in control group. The blood sugar concent, plasma concentrations of amylase and lipase were significantly lower in ATP group than those in control group at 6, 12 h after reperfusion(blood sugar, lipase, P0.05; amylase, P0.01). Tissue concentration of ATP in ATP group was higher than that in control group at 0, 1 h after reperfusion(P0.01). The percentage of oncotic cells was significantly lower in ATP group than that in control group at all time points after reperfusion (0, 1, 6 h, P0.01; 12 h, P0.05). Tissue concentration of ATP had a significant negative correlation with the percentage of oncotic cells(r=-0.756, P0.01), but no evident correlation with the percentage of apoptotic cells. CONCLUSION: Exogenous ATP has a protective effect on transplanted pancreas in rats. The mechanism of the protective effect maybe lies in that ATP can decrease the percentage of oncotic cells.
【Keywords】 pancreaticoduodenal transplantation; adenosine triphosphate; apoptosis; oncosis
【摘要】目的： 探讨外源性三磷酸腺苷(ATP)对大鼠移植胰腺的作用和机制. 方法： 建立SD大鼠同种异位全胰十二指肠移植模型，供体胰腺分别用肝素平衡盐液或肝素平衡盐添加ATP液进行低温(0~4℃)灌注并保存60 min后行移植手术，检测再灌注0, 1, 6, 12 h时血糖、脂肪酶、淀粉酶含量，同时检查胰腺组织病理学变化，ATP含量及凋亡、胀亡细胞百分数. 结果： ATP组胰腺组织结构损伤明显轻于对照组，ATP 6, 12 h组血糖、血清淀粉酶、脂肪酶含量明显低于同时间点对照组 (血糖，脂肪酶，P0.05；淀粉酶，P0.01)，ATP 0, 1 h组胰腺组织ATP含量明显高于同时间点对照组（P0.01），ATP组各时间点胀亡细胞百分数明显小于同时间点对照组(0, 1, 6 h, P0.01; 12 h, P0.05)， ATP含量与胀亡细胞百分数明显负相关（r=-0.756, P0.01）,与凋亡细胞百分数无明显相关. 结论： 外源性ATP对移植胰腺有保护作用，减少胰腺组织细胞胀亡的发生可能是其重要的保护机制.
【关键词】 胰十二指肠移植；三磷酸腺苷；细胞凋亡；细胞胀亡
0引言
提高移植物低温保存质量，减少移植物细胞死亡是移植成功的重要因素〔1-2〕. 有资料显示，外源性三磷酸腺苷(ATP)能够进入低温保存的胰腺细胞内〔3〕. 我们通过补充外源性ATP以提高移植器官内ATP的储备，研究其对胰腺组织中能量代谢以及细胞死亡方式的影响，探讨外源性ATP对移植胰腺的作用及机制.
1材料和方法
1.1材料采用健康清洁级、近交系封闭群SD大鼠96只，均为雄性，体质量250~300 g ，由徐州医学院实验动物中心提供. 实验前1 wk受体大鼠经阴茎背静脉按55 mg/kg 一次注入链脲霉素(美国Sigma公司)制备非空腹血糖>19.4 mmol/L的糖尿病模型. 实验分为2组： 对照组，供体胰十二指肠单纯用4℃肝素平衡盐液（150 ku/L, 15~20 mL)灌注并保存60 min后行移植手术；ATP组，供体胰十二指肠用4℃肝素平衡盐液添加ATP（5 mmol/L, 15～20 mL）进行灌注并保存60 min后行移植手术. 两组按移植后再灌注时间各分为再灌注0, 1, 6, 12 h亚组（每亚组12只大鼠）.
1.2方法采用全胰十二指肠移植模型，供体胰十二指肠根据分组分别用4℃肝素平衡盐液和添加了ATP的肝素平衡盐液灌注并保存60 min. 切除受体左肾,将供体带腹腔动脉和肠系膜上动脉的腹主动脉段与受体腹主动脉端侧吻合，供体门静脉与受体左肾静脉行端端吻合，供体的十二指肠近端关闭，远端与受体Treitz韧带下2 cm空肠作端侧吻合. 分别于设计时间点经下腔静脉采血后处死动物并切取移植胰腺行各项检查. 血糖测定采用美国强生血糖仪，淀粉酶、脂肪酶测定采用日立7600010型全自动生化仪. 切取部分胰腺标本制成石蜡切片后行HE染色，光镜下观察胰腺组织病理学改变. 切取胰腺组织约0.5 cm×0.5 cm×0.5 cm，应用岛津SCL10AVP高效液相色谱仪以反相高效液相色谱法测定胰腺组织ATP含量. 取胰腺组织1 g，生理盐水洗净，剪碎，振荡后300目筛网过滤，收集细胞制成细胞悬液，调整细胞密度至1×109个/L，加标记有异硫氰酸荧光素的联结素V(AnnexinVFITC，上海晶美生物工程有限公司)与碘化丙锭(PI，武汉博士德生物工程有限公司)，混匀双染色. 避光冰浴10 min，使用流式细胞仪上机检测各类细胞百分数. 结果判断：正常活细胞不着色（Annexin V-, PI-）；胀亡细胞能与Annexin V, PI结合，呈强绿色和红色荧光（Annexin V＋, PI＋）；凋亡细胞不与PI结合但与Annexin V结合，显示绿色荧光（Annexin V＋, PI-）.
统计学处理： 数据以x±s表示，采用t检验及相关分析，P0.05为有统计学差异.