CAJ | 학술논문

目的：观察曲古抑菌素A（ TSA）诱导小鼠骨髓间充质干细胞分化为胰岛素分泌细胞的适宜浓度。方法：C57BL／6小鼠骨髓间充质干细胞系体外培养。实验分为5组：对照组（ DMSO，A组）和TSA干预组（25 nmol／L，B组；50 nmol／L，C组；100 nmol／L，D组；200 nmol／L，E组）。各组分两阶段用相应的培养基培养10 d后进行检测。双硫腙染色鉴定各组的胰岛素分泌细胞，结果进行半定量分析。免疫荧光染色鉴定各组的胰岛素分泌，比较各组胰岛素平均荧光强度。酶联免疫吸附试验检测各组胰岛素分泌细胞胰岛素的分泌量。结果： TSA作用10 d能够诱导C57BL／6小鼠骨髓间充质干细胞分化成为胰岛素分泌细胞并分泌胰岛素。B组胰岛素染色阳性面积、阳性率、胰岛素染色积分吸光度以及胰岛素平均荧光强度显著高于其它干预组，差异有统计学意义（均P＜0．05）。随着各干预组TSA浓度的升高，胰岛素的分泌量减少。 B组胰岛素含量显著高于其它干预组，差异有统计学意义（均P＜0．05）。结论：TSA处理10 d能诱导C57BL／6小鼠骨髓间充质干细胞分化成为胰岛素分泌细胞，并且25 nmol／L为TSA的适宜浓度。
목적：관찰곡고억균소A（ TSA）유도소서골수간충질간세포분화위이도소분비세포적괄의농도。방법：C57BL／6소서골수간충질간세포계체외배양。실험분위5조：대조조（ DMSO，A조）화TSA간예조（25 nmol／L，B조；50 nmol／L，C조；100 nmol／L，D조；200 nmol／L，E조）。각조분량계단용상응적배양기배양10 d후진행검측。쌍류종염색감정각조적이도소분비세포，결과진행반정량분석。면역형광염색감정각조적이도소분비，비교각조이도소평균형광강도。매련면역흡부시험검측각조이도소분비세포이도소적분비량。결과： TSA작용10 d능구유도C57BL／6소서골수간충질간세포분화성위이도소분비세포병분비이도소。B조이도소염색양성면적、양성솔、이도소염색적분흡광도이급이도소평균형광강도현저고우기타간예조，차이유통계학의의（균P＜0．05）。수착각간예조TSA농도적승고，이도소적분비량감소。 B조이도소함량현저고우기타간예조，차이유통계학의의（균P＜0．05）。결론：TSA처리10 d능유도C57BL／6소서골수간충질간세포분화성위이도소분비세포，병차25 nmol／L위TSA적괄의농도。
AIM:To investigate whether trichostatin A ( TSA) , a new revulsant ,can induce mouse mesenchy-mal stem cells to differentiate into insulin-secreting cells and to explore the appropriate concentration of TSA .METHODS:The mesenchymal stem cell line from C57BL/6 mice was cultured in vitro and divided into 5 groups before treated with dif-ferent concentrations of TSA , ( group A:DMSO;group B~E:treated with 25 nmol/L, 50 nmol/L, 100 nmol/L and 200 nmol/L of TSA, respectively).After exposed to different cultured media for 10 d during the 2 stages, the cells were detec-ted by the following methods:the insulin-secreting cells in each group were identified by dithizone staining and the results were calculated with immunohistochemical half quantitative analysis .The insulin secreted by insulin-secreting cells in each group was identified by immunofluorescence , and the mean fluorescence intensity of insulin was compared .The content of insulin in each group was quantified by ELISA .The appropriate concentration of TSA was determined according to the above results .RESULTS:TSA treatment for 10 d promoted the mouse bone marrow mesenchymal stem cells to differenti-ate into insulin-secreting cells which produced insulin .The immunohistochemistry and immunofluorescence imaging analysis of insulin-secreting cells showed that the insulin staining positive area , positive ratio , total density of insulin expression and mean fluorescence intensity of insulin in group B were significantly higher than those in the other TSA -treated groups .When the concentrations of TSA gradually increased , the content of insulin reduced accordingly .The content of insulin in group B was significantly higher than that in the other TSA-treated groups .CONCLUSION:TSA treatment for 10 d promotes bone marrow mesenchymal stem cells from C57BL/6 mice to differentiate into insulin-secreting cells and the appropriate concen-tration of TSA is 25 nmol/L.