脂多糖预处理对人脐带间充质干细胞内毒素耐受的影响

Effects of lipopolysaccharide pretreatment on endotoxin tolerance of human umbilical cord mesenchymal stem cells

目的 观察脂多糖预处理对人脐带间充质干细胞（hUCMSCs）内毒素耐受的影响,并探讨其可能机制.方法 将hUCMSCs（1＆#215;104个/孔）分别暴露于0、0.1、1.0、10.0、20.0、30.0、40.0及50.0-μg/ml的脂多糖24 h,采用噻唑蓝（MTT）法检测细胞存活率;以1μg/ml脂多糖为预处理浓度,50 μg/ml脂多糖为诱导凋亡浓度,核因子（NF）-κB特异性抑制剂PDTC浓度为20 μmol/L,处理时间为20 min.使用Stata软件产生随机数字将hUCMSCs随机分为：对照组（A组）;脂多糖诱导组（B组）;预处理＋脂多糖诱导组（C组）;PDTC组（D组）;PDTC＋预处理＋脂多糖诱导组（E组）;预处理组（F组）;PDTC＋预处理组（G组）.利用Hoechst 33258染色及流式细胞术观察细胞凋亡,Western印迹法检测NF-κB p65及细胞型Fas相关死亡区域蛋白样白细胞介素-1β转换酶抑制蛋白（c-FLIP）的表达.结果 0、0.1、1.0、10.0、20.0、30.0、40.0及50.0μg/ml脂多糖刺激组的细胞存活率分别为：100％、（117.0±8.8）％、（134.7±6.9）％、（105.3±8.3）％、（99.2±8.3）％、（84.2±9.3）％、（66.4±6.6）％和（59.2±8.0）％;与0 μg/ml组比较,1.0 μg/ml组hUCMSCs的存活率明显提高（P=0.004）,而40.0、50.0 μg/ml组细胞存活率显著降低（P =0.005、0.002）.A组hUCMSCs染色质分布均匀,为弥散均匀的低强度荧光;B组细胞核呈浓染致密的固缩形态或颗粒状荧光的凋亡细胞明显增多;C组凋亡细胞较B组显著减少.A、B、C、D、E各组细胞凋亡率分别为：（2.8±0.8）％、（29.7±3.4）％、（17.8±3.0）％、（2.9±0.4）％和（23.2±2.6）％;细胞凋亡率B组显著高于A组（P＜0.001）,C组显著低于B组（P ＜0.001）,E组显著高于C组（P=0.015）.F组NF-κB p65、c-FLIP相对表达量（0.851 ±0.031、0.534±0.053）显著高于A组（0.220±0.021、0.049±0.009）（均P＜0.001）和G组（0.418 ±0.007、0.299 ±0.061） （P ＜0.001,P=0.007）.结论 脂多糖预处理能够抵抗内毒素环境引起的hUCMSCs凋亡,增强细胞内毒素耐受性;其机制可能与激活NF-κB信号通路并上调c-FLIP的表达有关.

Objective To explore the effects of lipopolysaccharide （LPS） pretreatment on endotoxin tolerance of human umbilical cord mesenchymal stem cells （hUCMSCs） and its possible mechanism.Methods hUCMSCs （1 ＆#215; 104 cells/well） were exposed to 0,0.1,1.0,10.0,20.0,30.0,40.0,50.0 μg/ml LPS for 24 h respectively.And the cell viability of hUCMSCs was detected by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT）.1 μg/ml and 50.0 μg/ml LPS were used as pretreatment and apoptosis induction concentrations respectively.Pyrrolidine dithiocarbamate （PDTC） （20 μmoL/L,pretreatment for 20 min） was used as a specific inhibitor of nuclear transcription factor NF-κB.hUCMSCs were randomly divided by Stata software into 7 groups：control （A）,LPS induction （B）,pretreatment ＋ LPS induction （C）,PDTC （D）,PDTC ＋ pretreatment ＋ LPS induction （E）,pretreatment （F） and PDTC ＋ pretreatment （G）.The apoptosis of hUCMSCs was measured by Hoechst 33258 staining and flow cytometry （FCM）.The expressions of NF-κB p65 and cellular FLICE-inhibitory protein （c-FLIP） were measured by Western blot.Results The cell viability of 0,0.1,1.0,10.0,20.0,30.0,40.0,50.0 μg/ml LPS groups were 100%,（117.0 ±8.8）%,（134.7 ±6.9）%,（105.3 ±8.3）%,（99.2±8.3）%,（84.2 ±9.3）%,（66.4±6.6）% and （59.2 ±8.0）% respectively.In comparison with 0 μg/ml LPS group,the cell viability of 1.0 μg/ml LPS group increased significantly （P =0.004） while decreased in 40 and 50 μg/ml LPS groups （P =0.005,0.002）.Hoechst 33258 staining indicated that chromatin of hUCMSCs was distributed evenly in group A; the apoptotic cell in group B dramatically increased; and the apoptotic cell in group C significantly decreased in comparison with that in group B.Apoptotic rates of groups A,B,C,D and E were （2.8 ± 0.8） %,（29.7 ± 3.4） %,（17.8 ±3.0）%,（2.9 ± 0.4）% and （23.2 ± 2.6）% respectively.Compared with group A,apoptosis rate significantly increased in group B （P 〈 0.001）.The apoptotic rate in group C significantly decreased than that in group B （P 〈 0.001） while group E was higher than group C （P =0.015）.The levels of NF-κB p65 and c-FLIP in group F （0.851 ± 0.031,0.534 ± 0.053） was higher than that in group A （0.220 ± 0.021,0.049±0.009） （both P〈0.001）,G （0.418 ±0.007,0.299 ±0.061） （P〈0.001,P=0.007）.Conclusions LPS pretreatment can resist LPS-induced hUCMSCs apoptosis and enhance the ability of endotoxin tolerance.And the mechanism may be related with activating the NF-κB signaling pathway and upregulating the expression of c-FLIP.