流式细胞仪分选大鼠胰岛β细胞方法的建立及应用

Preparation and evaluation of rat islet β cell fraction by flow cytometry

目的:应用流式细胞仪(FCM)结合侧向角散射光(SSC)和细胞自发荧光强度分选大鼠胰岛β细胞。方法:选取正常雄性SD大鼠,胶原酶消化显微镜下手捡纯化胰岛。胰岛以胰蛋白酶和DNA酶消化成单细胞悬液。依据细胞自发荧光强度和SSC的差异通过流式细胞仪进行细胞分选。细胞存活率用PI/HO双染法鉴定,β细胞的纯度用细胞免疫组化鉴定。胰岛素测定采用酶联免疫吸附法。结果:自发荧光强度和SSC均高的细胞群中胰岛素染色阳性的细胞百分比为(94.5±0.95)%,较分选前单细胞中胰岛素染色阳性细胞百分比(81.2±1.12)%增高,为β细胞富集群;自发荧光强度和SSC均较低的细胞群中胰岛素染色阳性的细胞百分比为(10.0±0.82)%,为non-β细胞群。分选出的β细胞群死亡率低(<5%),其胰岛素分泌随葡萄糖刺激浓度的增加而增加(r=0.8,P<0.001),其中葡萄糖浓度在20 mmol/L时β细胞分泌的胰岛素明显大于浓度为2.8 mmol/L时的分泌量(P<0.001)。此外,Forskolin(FK)和Exendin-4(EX4)可以显著增强β细胞胰岛素的分泌(PFK<0.001,PEX4<0.05)。结论:根据β细胞自发荧光和SSC与non-β细胞的差异进行纯化的β细胞死亡率低、纯度高,胰岛素分泌功能较好,是一种值得推广的分选β细胞的方法。

Objective To establish a method for purifying rat pancreatic β cells by flow cytometry.Methods Rat islets were digested by collagenase and fished under microscopy.After dissociated with trypsin and deoxyribonuclease（DNase）,single islet cells were analyzed and sorted according to their autofluorescence intensity and side scatter（SSC） property by flow cytometry.Cell viability was detected by Hoechst 33342 and propidium iodide.β cell purity was identified by immunocytochemistry.Insulin was measured with enzyme-linked immunosorbent assay.Results β cell enriched-population had higher autofluorescence intensity and SSC than non-β cell enriched-population.The proportion of insulin positive staining cells in β cell enriched-population was（94.5±0.95）%,while that in non-β cell enriched population was（10.0±0.82）%.The death rate of β cell enriched-population was low（5%）.Insulin release from β cell enriched-population increased with the increase of glucose concentration from 2.8-20mmol/L（r=0.8,P0.001）,and statistical significance was found when 20mmol/L glucose was compared with 2.8mmol/L glucose（P0.001）.Moreover,Forskolin（FK） and Exendin-4（EX4） significantly enhanced the secretion of insulin（PFK 0.001,PEX40.05）.Conclusions β cell fraction purified by flow cytometry according to cell autofluorescence and side scatter has low mortality,high purity and good function.