人脂肪源性间充质干细胞修复放化疗所致胸腺损伤机制研究

Mechanism of human adipogenic mesenchymal stem cells in repairing thymus damaged by radiotherapy and chemotherapy

目的观察体外人脂肪源性间充质干细胞对放化疗损伤胸腺上皮细胞增殖作用并进行机制探索。方法无菌环境下分离提取人脂肪源性间充质干细胞;以20、30、50及100μmol/L KGF-si RNA作用于脂肪源间充质干细胞,筛选最佳KGF-siRNA基因沉默作用浓度;比较KGF基因沉默干预与非KGF基因沉默干预对胸腺上皮增殖影响。结果人脂肪源性间充质干细胞培养6~8 d呈典型纤维状,流式细胞检测细胞表面抗原CD44阳性93.7%,CD34阳性率0.405%。不同浓度20、30、50及100μmol/L的KGF-siRNA转染作用48 h后人脂肪源间充质干细胞mRNA水平下降33.22%、46.40%、81.06%及42.35%,50μmol/L作用48 h效果明显,差异有统计学意义(P<0.05)。48 h基因沉默组、空白对照组及空质粒组角质细胞生长因子表达发现沉默组与空白对照及空质粒组存在显著差异(P<0.05),后二者间无显著差异(P>0.05)。应用Brdu增殖实验连续3 d分别测定基因沉默组、正常组、单纯人胸腺上皮细胞增殖情况及基因沉默组、正常组及单纯鼠胸腺上皮细胞增殖情况发现基因正常组显著高于基因干预组,差异有统计学意义(P<0.05)。应用PI单染测定基因沉默组、正常组、单纯人胸腺上皮细胞及基因沉默组、正常组、单纯鼠胸腺上皮细胞周期计算细胞增殖密切相关S期细胞所占比,证实正常对照组增殖显著高于况默组及单纯小鼠胸腺上皮细胞组增殖,与其他组比较差异有统计学意义(P<0.05)。结论脂肪源性间充质干细胞可通过分泌角质细胞生长因子完成胸腺上皮细胞增殖;脂肪源性间充质干细胞对放化疗损伤胸腺有促进修复的作用。

Objective To observe and explore the proliferative effects and action mechanism of human adipogenic mesenchymal stem cells(MSCs)in repairing thymus damaged by radiotherapy and chemotherapy in vitro.Methods The human adipogenic MSCs were isolated and extracted in sterile environment,then the best concentration of KGF siRNA gene silencing was screened by using 20μmol/L,30μmol/L,50μmol/L and 100μmol/L KGF Si RNA.The effects of KGF gene silencing and non KGF gene silencing on thymocyte proliferation were compared.Results Human adipose-derived mesenchymal stem cells showed typical fibrous shape after 6-8 days of culture,and the positive rate of surface antigen CD44 detected via flow cytometry was 93.7%and the positive rate of CD34 was 0.405%.The mRNA level of human adipose-derived mesenchymal stem cells decreased by 33.22%,46.40%,81.06%and 42.35%after transfection with KGF-siRNA at different concentrations of 20,30,50 and 100μmol/L for 48 h,and the effect of 50μmol/L in 48 h was evident,and the difference was statistically significant(P<0.05).The expression of keratinocyte growth factor in the 48 h gene silencing group,blank control group and empty plasmid group was significantly different from that of blank control and empty plasmid group(P<0.05),and no significant difference was found in such indexes between the latter two groups(P>0.05).Brdu proliferation assay was used to measure the proliferations of gene silencing group,normal group,pure human thymic epithelial cells and gene silencing group,normal group and pure mouse thymic epithelial cells respectively for 3 consecutive days.It was found that the gene normal group was significantly higher than the gene intervention group.The differences were statistically significance(P<0.05).PI single staining was applied to determine the proportion of S-phase cells closely related to cell proliferation in gene silencing group,normal group,pure human thymic epithelial cells and gene silencing group,normal group and pure mouse thymic epithelial cell cycle,and it was confirmed that the proliferation of normal control group was significantly higher.The proliferation of thymic epithelial cells in the Kuangmo group and the pure mouse thymic epithelial cell group was significantly different from other groups(P<0.05).Conclusion The human adipogenic MSCs is capable of completing the proliferation of thymocytes by secreting keratinocyte growth factor,and human adipogenic MSCs have the effects of promoting the repair of thymus after radiotherapy and chemotherapy.