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目的研究恒河猴BMSCs在缺氧再给氧环境下对新生猪胰岛活性和功能的保护作用。方法取健康成年恒河猴5只,体重6～10 kg,取骨髓采用骨髓单核细胞贴壁培养法筛选获得BMSCs;取3～5日龄新生长白猪5只,取胰腺以Ⅴ型胶原酶消化制备新生猪胰岛。将实验分为胰岛正常组(A组)、胰岛加BMSCs正常组(B组)、胰岛缺氧再给氧组(C组)、胰岛加BMSCs缺氧再给氧组(D组),观察比较常规培养和缺氧(1%O2)12 h再给氧24 h或48 h条件下,胰岛的功能活性变化:二乙酸荧光素/碘化丙啶(propidium iodide,PI)染色计算胰岛成活率;细胞计数试剂盒8法检测新生猪胰岛的代谢活性;膜联蛋白V-FITC/PI标记后流式细胞仪检测胰岛凋亡率;葡萄糖刺激法分析胰岛功能糖刺激指数(stimulation index,SI)。结果 C组较A、B组胰岛团更为分散,死亡细胞增多;D组较C组胰岛团完整,成活率更高。A、B、C、D组胰岛成活率分别为90.2%±9.1%、88.3%±5.9%、52.3%±12.1%、71.4%±11.5%,C、D组显著低于A、B组(P<0.05),D组显著高于C组(P<0.05)。D组BMSCs以1∶10和1∶20比例与胰岛缺氧再给氧共培养后,代谢活性显著高于C组(P<0.05)。A、B、C、D组胰岛凋亡率分别为27.1%±3.2%、24.0%±1.0%、64.3%±1.8%、46.2%±1.4%,C、D组显著高于A、B组(P<0.05),但D组显著低于C组(P<0.05)。各时间点A、B组SI比较差异均无统计学意义(P>0.05),C组显著低于A、B组(P<0.05),在24 h和72 h时D组显著高于C组(P<0.05)。结论 BMSCs对缺氧再给氧诱导的新生猪胰岛活性和功能损伤具有保护作用。
Objective To study the protective effect of rhesus monkey BMSCs on islet activity and function in neonatal piglets under anoxic and reoxygenation conditions. Methods Five adult Rhesus macaques weighing 6 to 10 kg were obtained. BMSCs were obtained from bone marrow by adherent culture of bone marrow mononuclear cells. Five new-born white pigs were cultured at 3 to 5 days old. The pancreas was treated with type Ⅴ collagenase Digestion to prepare newborn pig islets. The experiment was divided into normal group (group A), islet plus normal BMSCs (group B), islet hypoxia reoxygenation group (group C), islet plus BMSCs hypoxia reoxygenation group (group D) The changes of functional activities of islets under routine culture and hypoxia (1% O2) for 12 h and then oxygen for 24 h or 48 h were measured by PI staining and propidium iodide (PI) staining. Cell counting kit 8 method was used to detect the metabolic activity of islets of newborn pigs; apoptosis rate of islets was detected by flow cytometry after Annexin V-FITC / PI labeling; and stimulation index (SI) of islet function was analyzed by glucose stimulation. Results Compared with group A and group B, pancreatic islets were more dispersed and the number of dead cells was increased in group C than those in group C. The survival rate was higher in group D than in group C. The survival rates of islets in groups A, B, C and D were 90.2% ± 9.1%, 88.3% ± 5.9%, 52.3% ± 12.1%, 71.4% ± 11.5% <0.05), D group was significantly higher than C group (P <0.05). The metabolic activity of BMSCs in D group was significantly higher than that in C group (P <0.05) after 1:10 and 1:20 co-culture with islet hypoxia and reoxygenation. The apoptotic rates of islets in groups A, B, C and D were 27.1% ± 3.2%, 24.0% ± 1.0%, 64.3% ± 1.8%, 46.2% ± 1.4% P <0.05), but D group was significantly lower than C group (P <0.05). There was no significant difference in SI between groups A and B at each time point (P> 0.05), while in group C was significantly lower than that in group A and B (P <0.05), and at 24 and 72 h, (P <0.05). Conclusion BMSCs have a protective effect on hypoxic reoxygenation-induced neonatal pig islet activity and functional injury.