CAJ | 학술논문

目的探讨体外诱导人羊膜间充质干细胞(hAD-MSCs)向胰岛素分泌细胞分化潜能.方法采用胰蛋白酶-胶原酶消化法分离提取hAD-MSCs,流式细胞术分析和免疫细胞化学染色行表型鉴定.取第3代按2.5× 106个/ml或5×105个/ml细胞密度接种6孔培养板或预置盖玻片的24孔培养板,在含10mmol/L尼克酰胺和N2补充物的无血清HG-DMEM培养基培养,未诱导组为含10％胎牛血清的LG-DMEM基础培养基.分别于体外诱导第7、14、21天采用免疫细胞化学法检测胰岛素和β2微球蛋白的表达,采用放射免疫法检测上清液中胰岛素含量,采用RT-PCR检测胰岛素mRNA和胰十二指肠同源异型盒因子1(PDX-1)mRNA的表达.结果 (1)hAD-MSCs高表达间充质干细胞表面标志CD29、CD44、CD73、CD166及波形蛋白；(2) hAD-MSCs诱导第7、14、21天胰岛素阳性细胞百分率分别为74.67％±1.53％、75.00％±1.00％、74.33％±1.53％,培养物上清液中胰岛素含量分别为(331.62±1.76)、(330.50±1.22)和(331.65±0.48) μIU/ml,各时点间比较无显著性差异(均P＞0.05),而未诱导组仍未见胰岛素阳性细胞；培养上清也未检测到胰岛素；(3) hAD-MSCs诱导前后均有PDX-1 mRNA和蛋白表达,胰岛素基因mRNA表达仅见于诱导组；(4) hAD-MSCs诱导组和未诱导组各时点均有β2微球蛋白表达,其阳性细胞百分率组间差异无显著性(均P＞0.05).结论 hAD-MSCs具有向胰岛素分泌细胞分化的能力,可能成为1型糖尿病细胞移植治疗的新的细胞供源.
목적 탐토체외유도인양막간충질간세포(hAD-MSCs)향이도소분비세포분화잠능.방법 채용이단백매-효원매소화법분리제취hAD-MSCs,류식세포술분석화면역세포화학염색행표형감정.취제3대안2.5× 106개/ml혹5×105개/ml세포밀도접충6공배양판혹예치개파편적24공배양판,재함10mmol/L니극선알화N2보충물적무혈청HG-DMEM배양기배양,미유도조위함10％태우혈청적LG-DMEM기출배양기.분별우체외유도제7、14、21천채용면역세포화학법검측이도소화β2미구단백적표체,채용방사면역법검측상청액중이도소함량,채용RT-PCR검측이도소mRNA화이십이지장동원이형합인자1(PDX-1)mRNA적표체.결과 (1)hAD-MSCs고표체간충질간세포표면표지CD29、CD44、CD73、CD166급파형단백；(2) hAD-MSCs유도제7、14、21천이도소양성세포백분솔분별위74.67％±1.53％、75.00％±1.00％、74.33％±1.53％,배양물상청액중이도소함량분별위(331.62±1.76)、(330.50±1.22)화(331.65±0.48) μIU/ml,각시점간비교무현저성차이(균P＞0.05),이미유도조잉미견이도소양성세포；배양상청야미검측도이도소；(3) hAD-MSCs유도전후균유PDX-1 mRNA화단백표체,이도소기인mRNA표체부견우유도조；(4) hAD-MSCs유도조화미유도조각시점균유β2미구단백표체,기양성세포백분솔조간차이무현저성(균P＞0.05).결론 hAD-MSCs구유향이도소분비세포분화적능력,가능성위1형당뇨병세포이식치료적신적세포공원.
Objective To investigate the potential of human amnion-derived mesenchymal stem cells to differentiate into insulin secreting cells in vitro.Methods The hAD-MSCs were isolated from human amnion by trypsin-collagenase digestion.The phenotype of the isolated cells was identified by flow cytometry and immunocytochemical staining.The 3rd generation cells were inoculated at density of 2.5 × 106 unit/ml or 5 × 105 unit/ml in 6 well plates or preset coverslip 24 well plates.Induced groups were treated in serum-free HG-DMEM with 10 mmol/L nicotinamide and N2 supplement.The cells in the non-induced groups were incubated in LG-DMEM supplemented with 10％ fetal bovine serum.At days 7,14 and 21 after induction,insulin and β2 microglobulin was determined by immunocytochemical stain,the content of insulin in the culture supernatant was assayed by radioimmunoassay,and insulin mRNA and PDX-1 mRNA were detected by reverse transcriptase-polymerase chain reaction.Results ( 1 ) The hAD-MSCs highly expressed CD29,CD44,CD73,CD166,and vimentin.( 2 ) At 7,14,and 21 days,the percentages of insulin-positive cells in hAD-MSCs induced groups were 74.67％ ± 1.53％,75.00％:1.00％,and 74.33％ ±1.53％,respectively.Contents of insulin in the supematant of hAD-MSCs induced groups were ( 331.62 ± 1.76 ),( 330.50 ± 1.22 ),and ( 331.65 ± 0.48 ) μIU/ml,respectively,but non-induced groups were negative.(3) PDX-1 mRNA and PDX-1 protein were expressed before and after the induction of hADMSCs,but insulin mRNA was expressed only in the induced groups.( 4 ) Both hAD-MSCs induced groups and non-induced groups expressed β2 microglobulin ( all P ＞ 0.05 ).Conclusion The hAD-MSCs have a potential of differentiating into ISCs and thus may become a new cell source of therapy for type 1 diabetes.