He Honghui, Wu Tianhui, Xiong Jing, Chen Ke, Mo Zhaohui
Department of Endocrinology, Third Xiangya Hospital, Central South University, Changsha 410013,China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2010 Nov;35(11):1115-22. doi: 10.3969/j.issn.1672-7347.2010.11.001.
To investigate the effect of erythropoietin on the proliferation,differentiation, and apoptosis of the cultured neonatal porcine islet cells in vitro.
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.
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).
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.
探讨促红细胞生成素对体外培养的新生猪胰岛细胞增殖、分化及凋亡的影响。
采用胶原酶消化和组织培养法从新生猪分离纯化胰岛细胞，并检测其活力和纯度。将新生猪胰岛细胞分为对照组和实验组。实验组用促红细胞生成素处理，对照组不做处理，5天后检测胰岛对低、高糖刺激诱导的胰岛素分泌反应性。通过MTT比色法进行细胞计数并测定增殖活性。采用溴化乙锭/吖啶橙（EB/AO）荧光染色和流式细胞术观察细胞凋亡率，并用流式细胞术分析细胞周期。通过RT-PCR检测bcl-2、bax、caspase-3、葡萄糖转运蛋白2（GlUT-2）和胰腺十二指肠同源盒-1（PDX-1）mRNA的表达。
细胞培养中加入促红细胞生成素后，新生猪胰岛细胞形态、功能正常，对葡萄糖刺激的胰岛素分泌反应良好。细胞计数显示实验组细胞数多于对照组（P<0.05）。MTT比色法显示光吸收值随时间呈上升趋势，与对照组相比，实验组光吸收值更高（P<0.05），PDX-1 mRNA表达略有上调（P<0.05）。两组GLUT-2 mRNA表达无差异（P=0.34）。通过流式细胞术和EB/AO荧光染色检测，实验组细胞凋亡率低于对照组（P<0.01），bcl-2 mRNA表达较高。然而，bax mRNA和caspase-3 mRNA明显低于对照组（P<0.01）。
促红细胞生成素可促进新生猪胰岛细胞增殖，但对其体外功能无影响。促红细胞生成素可通过上调bcl-2 mRNA、下调bax和caspase-3 mRNA来保护新生猪胰岛细胞免于凋亡。
