| Objective: Tissue explant method was used to isolate and culture humanumbilical cord mesenchymal stem cells (hUCMSCs). To explore the effects oflipopolysaccharides (LPS) on proliferation and apoptosis of hUCMSCs and its signalmechanism, hUCMSCs were stimulated with different concentrations of LPS. Theadaptive cytoprotection low concentration of LPS pretreatment on hUCMSCs wasevaluated. The mechanisms were discussed which would lay the theoreticalfoundation for enhancing hUCMSCs endotoxin tolerance.Methods:1hUCMSCs were isolated from healthy full-term fetus umbilical cordusing tissue explant method. The cell morphology was observed under invertedmicroscope. The proliferation ability of3,5and10th-generation cells wasinvestigated by MTT detection. The markers of hUCMSCs were detected byimmunofluorescence staining.2hUCMSCs were stimulated by different concentrations of LPS (0,0.01,0.1,1.0,10.0,20.0,30.0,40.0,50.0μg/ml). The proliferation ability of cells wasinvestigated by MTT detection. Cell apoptosis was detected by AO/EB staining andflow cytometry (FCM). Enzyme-linked immunosorbent assay (ELISA) was used toevaluate levels of Caspase-8, Caspase-3and DNA fragmentation.3hUCMSCs were pretreated by low concentrations of LPS (0.1,1.0,10.0,20.0μg/ml) for12h, and subsequently stimulated by50.0μg/ml LPS for24h.Apoptosis was detected by FCM and Hoechst staining. Cell viability was investigatedby MTT detection.4After pretreatment by low concentrations of LPS (0.1,1.0,10.0,20.0μg/ml) for12h, hUCMSCs were subsequently stimulated by50.0μg/ml LPS with or without siFLIPLtransfection and PDTC cotreatment for24h. Expression of c-FLIPLwasdown-regulated by siRNA. PDTC was used to inhibit NF-κB signal pathway. Theexpression levels of c-FLIPLand NF-κBp65were detected by Western blot. Apoptosiswas evaluated by FCM, and relative activation of Caspase-8, Caspase-3weremeasured by ELISA.Result:1Morphology of cells isolated by tissue explants was similar tofibroblasts, which rendered the adherent and swirling hyperplasia. The3,5and10th-generation cells had fast proliferative activity, whose features in growth curvewere all similar to “S” shape. The cells highly expressed CD105, CD90, CD44, CD73,lowly expressed HLA-Ⅰ, had no expression of CD45, CD31, HLA-DR.2MTT assay showed that A value was higher in the groups treated with0.01-10.0μg/ml LPS (P＜0.05or P＜0.01), especially in the1.0μg/ml LPS-treatedgroup(P＜0.01), and was lower in the groups treated with30.0-50.0μg/ml LPS(P＜0.01), while there was no difference in the20.0μg/ml LPS-treated group(P＞0.05),compared with the control group. The apoptosis of hUCMSCs were observed in20.0-50.0μg/ml LPS-treated groups, but not in the0-10.0μg/ml LPS-treated groups byAO/EB staining. As detected by FCM, the apoptosis rate in the control and1.0μg/ml LPS-treated group〔s(2.68±0.27)%and (3.07±0.35)%〕was similar(P＞0.05);however, the apoptosis rate was significantly increased when the cells were stimulatedwith30.0or50.0μg/ml LPS〔(8.92±3.77)%,(23.46±2.38)%〕(P＜0.05). ELISAshowed that compared with the control group, the levels of Caspase-8, Caspase-3andDNA fragmentation in the50.0μg/ml LPS-treated groups were all significantlyenhanced(P＜0.01), which were also susppressed by Caspase-8inhibitor(z-IETD-FMK)(P＜0.01).3As detected by FCM, the apoptosis rate in the0.1,1.0μg/ml LPS-pretreated groups were significantly decreased(P＜0.01), while there was no difference in the10.0,20.0μg/ml LPS-pretreated groups(P＞0.05), compared with the non-pretreatedgroup. The quantity of apoptotic cells in1.0μg/ml LPS-pretreated group wasremarkably reduced than those in non-pretreated group(P＜0.01). MTT showed thatthe cell viability in1.0μg/ml LPS-pretreated group was more higher than that innon-pretreated group(P＜0.01).4Compared with the control group, the level of c-FLIPLin the0.1,1.0μg/mlLPS-pretreated groups were significantly increased(P＜0.01). The lower level ofc-FLIPLcaused by50.0μg/ml LPS was reversed by1.0μg/ml LPS pretreatment(P＜0.01). By using siRNA of c-FLIPL, the high expression of c-FLIPLinduced by1.0μg/ml LPS pretreatment was significantly decreased(P＜0.01), and the ability of1.0μg/ml LPS pretreatment induced cytoprotection was also significantly weakened(P＜0.01). The high expression of NF-κBp65and c-FLIPLinduced by1.0μg/ml LPSpretreatment was significantly decreased by PDTC(P＜0.01).Conclusion: hUCMSCs were successfully isolated and cultured from humanumbilical cord by tissue explant method. The effect of LPS on hUCMSCsproliferation was in a dose-dependent manner, and exhibits a dual function. LPScould induce apoptosis of hUCMSCs via activating Caspase-8/3signal pathway.Pretreatment with low concentrations of LPS could protect hUCMSCs againstapoptosis induced by subsequent lethal LPS induction, enhance hUCMSCsendotoxin tolerance; the cytoprotection of the low dose LPS pretreatment wasassociated with the up-regulation of c-FLIPLvia NF-κB signal pathway. |
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