作　　者： ; ; ;

机构地区： 暨南大学附属第一医院

出　　处： 《热带医学杂志》 2019年第4期426-429,442,527,共6页

摘　　要： 目的分离并纯化培养人羊膜间充质干细胞(hAMSCs),观察脂多糖(LPS)对hAMSCs活性的影响,探讨利用hAMSCs修复胎膜损伤的可行性。方法取无妊娠合并症的足月剖宫产产妇的羊膜组织,采用机械分离联合酶消化法分离并纯化培养hAMSCs,成脂和成骨诱导分化鉴定hAMSCs干细胞特性;LPS(0、1和10μg/mL)处理hAMSCs,分别于处理后第1、3、5天,光镜观察细胞形态变化,MTT法检测细胞活性。结果传代培养至第3代的hAMSCs培养3 d已达到80%～90%融合,均呈规则的长梭形排列,提示纯度增高。在成脂诱导液诱导下,细胞形态逐渐变短变饱满,呈短圆梭形,诱导9 d后可见折光性变强的空泡,提示可能有脂滴形成;随着诱导时间的增加,胞浆内的脂滴逐渐增多,诱导15 d后油红O染色细胞内脂滴被染成红色。在成骨诱导液诱导下,hAMSCs逐渐变细长,诱导20 d后,胞浆内可见钙质沉积和钙结节,茜素红染色可见胞浆被染成红色。MTT法检测hAMSCs细胞OD值,结果显示其细胞OD值从培养后的第3天开始迅速升高至第11天,呈现明显的指数增长;11 d后开始下降进入衰老期。LPS(0、1和10μg/mL)处理hAMSCs 5 d后,三组hAMSCs细胞OD值比较,其中仅10μg/mL LPS处理组低于0μg/mL LPS处理组,差异有统计学意义(P<0.05);1μg/mL LPS处理组的hAMSCs在5 d的培养过程中细胞活性仍在上升、细胞形态无老化现象;10μg/mL LPS处理组的hAMSCs细胞在5 d的培养过程中体积略微增大,细胞活性轻微下降,但第5天与第1天细胞OD值比较,差异无统计学意义(P>0.05)。结论在体外培养的条件下hAMSCs对LPS具有一定的耐受性。 Objective To isolate and purify of human amniotic mesenchymal stem cells(hAMSCs),observe the effect of lipopolysaccharide(LPS)on the activity of hAMSCs,and explore the feasibility of using hAMSCs to repair fetal membrane damage.Methods The amniotic membrane tissues were taken from parturient women with full-term cesarean section without pregnancy complications,and hAMSCs were isolated and purified by mechanical separation combined with enzyme digestion.HAMSCs were induced to differentiate into adipocytes and osteoblasts to identify their characteristics of stem cells.HAMSCs were treated with LPS(0,1 and 10μg/mL),and then at 24 hours(the first day),72 hours(the third day),and120 hours(the fifth day),the morphological changes of cells were observed under light microscope,and cell viability of these cells was detected by MTT assay.Results The hAMSCs cultured for 3 days to the third generation had reached 80%to 90%fusion,all of which were arranged in a regular long fusiform arrangement,suggesting an increase in purity.After treated with adipogenic induction solution,the cell morphology gradually became short and full,showing a short round spindle shape.After 9 days of induction,vacuoles with enhanced refractive power were observed,suggesting that lipid droplets were formed.As the induction time increases,the lipid droplets in the cytosol gradually increased.After 15 days of induction,the lipid droplets were stained red by the oil red 0 stain.Under the induction of osteogenic induction fluid,hAMSCs gradually became slender.After 20 days of induction,calcium deposition and calcium nodules were observed in the cytoplasm,and cytoplasm was stained red by alizarin red staining.The OD value of hAMSCs cells was detected by MTT assay.The results showed that the OD value of the cells increased rapidly from the third day after culture to the 11 th day,showing a significant exponential growth.After 11 days,it began to decline into the senescence phase.After treatment of hAMSCs with LPS(0,1 and 10μg/mL)for 5 days,the OD values

关 键 词： 羊膜间充质干细胞 脂多糖 胎膜早破

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