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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 sort rat pancreatic β-cells by flow cytometry (FCM) combined with lateral angle scattered light (SSC) and cell autofluorescence intensity. Methods: Normal male SD rats were selected and purified by collagenase digestion microscope. Islet is digested with trypsin and DNase into a single cell suspension. Cell sorting was performed by flow cytometry based on the differences in autofluorescence intensity and SSC. The cell viability was identified by PI / HO double staining, and the purity of β cells was identified by immunohistochemistry. Insulin determination by enzyme-linked immunosorbent assay. Results: The percentage of cells stained with insulin in positive cell group with high autofluorescence intensity and SSC was (94.5 ± 0.95)%, which was higher than that of single cell before immunization (81.2 ± 1.12)%, which was β cell (10.0 ± 0.82)% of cells with positive autofluorescence intensity and low SSC group were non-β cell population. The mortality of the sorted β cell population was low (<5%), and the insulin secretion increased with the increase of the glucose stimulated concentration (r = 0.8, P <0.001). The glucose secreted by the β cells at a glucose concentration of 20 mmol / L Insulin was significantly greater than at 2.8 mmol / L (P <0.001). In addition, Forskolin (FK) and Exendin-4 (EX4) significantly enhanced β-cell insulin secretion (PFK <0.001, PEX4 <0.05). Conclusion: According to the autofluorescence of β cells and the difference of SSC and non-β cells, the purified β cells have low mortality, high purity and good insulin secretion function, which is a worthy method to differentiate β cells.