促红细胞生成素对新生猪胰岛细胞增殖分化和凋亡的影响

［摘要］目的：研究促红细胞生成素在体外对新生猪胰岛细胞增殖分化和凋亡的影响。方法：采用胶原酶消化和组织培养方法分离纯化新生猪胰岛细胞,检测其纯度和活性,再分为实验组和对照组。实验组培养基中添加促红细胞生成素（10.0 μmol/L）,对照组不添加促红细胞生成素,培养5 d后行低糖和高糖刺激胰岛素释放试验;并行细胞计数及MTT比色法测细胞增殖活性;流式细胞仪和EB/AO染色荧光检测细胞凋亡百分率,流式细胞仪分析细胞周期,RT-PCR法检测胰岛细胞胰岛素促进因子-1(PDX-1),葡萄糖转运载体2（GlUT-2）,bcl-2,bax,caspase-3 mRNA的表达。结果：促红细胞生成素干预后新生猪胰岛细胞不改变形态和功能,对葡萄糖刺激胰岛素分泌的反应正常;细胞计数示实验组新生猪胰岛细胞数目均比对照组增多（P＜0.05）;随着培养时间延长,实验组和对照组吸光度值均呈上升趋势,且实验组的吸光度值均高于对照组（P＜0.05）,实验组PDX-1 mRNA表达稍有上调（P＜0.05）,而GlUT-2 mRNA表达无明显差别（P=0.34）。流式细胞仪和EB/AO染色荧光检测实验组的凋亡百分率均较对照组降低（P＜0.01）。RT-PCR示实验组bcl-2 mRNA较对照组上调,而bax和 caspase-3 mRNA明显下调（P＜0.01）。结论：促红细胞生成素在体外能促进新生猪胰岛细胞的增殖分化,对形态和功能无影响。促红细胞生成素对新生猪胰岛细胞有抗凋亡保护作用,可能是通过上调bcl-2 mRNA的表达,下调bax, caspase-3 mRNA的表达来实现的。

Effect of erythropoietin on the proliferation and apoptosis of neonatal porcine islet cells

Objective To investigate the effect of erythropoietin on the proliferation,differentiation,and apoptosis of the cultured neonatal porcine islet cells in vitro.Methods Neonatal porcine islet cells were separated and pured from neonatal pigs with collagenase digestion and tissue culture,and their viability and purity were tested. The neonatal porcine islet cells were divided into a control group and an experimental group.The experimental group was treated with erythropoietin but not the control group,and the insulin secretion responsiveness induced by low and high glucose stimulation in the islet was tested after 5 days. Cells were counted and the activation of amplification was determined by MTT chromatometry. The rates of cell apoptosis were observed by ethidium bromide/acridine orange (EB/AO) of fluorescent light staining and flow cytometry,and the cell cycle was analyzed by flow cytometry. The expression of bcl-2,bax,caspase-3,glucose transporter 2 (GlUT-2),and pancreatic duodenal homeobox-1 (PDX-1) mRNA was tested by RT-PCR.Results After erythropoietin was treated in the cell culture,the neonatal porcine islet cells had normal morphology,function,and reaction of insulin secretion to the glucose stimulation. Cell count showed more cells in the experimental group than in the control group (P＜0.05). MTT chromatometry showed the optical absorbance tended to increase with time,and compared with the control group,the optical absorbance was higher in the experimental group (P＜0.05),the expression of PDX-1 mRNA was slightly up-regulated (P＜0.05). The expression of GLUT-2 mRNA had no difference in the 2 groups (P=0.34). In the experimental group,the apoptisis rate was lower than that in the control group by flow cytometry and EB/AO fluoscence staining (P＜0.01),and the expression of bcl-2 mRNA was higher. Howerer bax mRNA and caspase-3 mRNA were obviously lower than those in the control group (P＜0.01).Conclusion Erythropoietin can promote the proliferation but has no effect on the function of neonatal porcine islet cells in vitro. Erythropoietin can protect neonatal porcine islet cells from apoptosis through up-regulating bcl-2 mRNA and downreguling bax and caspase-3 mRNA.