The Treatment And Mechanisms Of UC-MSCS On Ali Mice

PART I THE THERAPY OF UC-MSCS ON ALI MODELObjective:To confirm the expression of Embryonic Stem cell-related genes in UC-MSCs and to explore UC-MSCs treatment on LPS-induced ALI model.Methods:The expressions of Embryonic Stem cell-related genes OCT-4, SOX-2, NANOG, SSEA-4 in UC-MSCs were detected by Fluorescence quantitative PCR. C57BL/6 (or BALB/C) mice were randomly divided into 3 groups:Control (PBS+PBS), ALI (LPS+PBS) and UC-MSCs treated ALI (LPS+UC-MSCs) respectively. After intraperitoneal injection of anesthesia for tracheal intubation, ALI mice and ALI+ UC-MSCs mice were administrated by 30μl LPS (5mg/kg) and Ctrl mice were administrated by 30μl PBS. After 1h, ALI+UC-MSCs mice were treated with 80μl UC-MSCs cell suspension (cell number 0.5xl06), and both of ALI group and Ctrl group were treated with PBS (80μl). Mice were observed everyday for the change of weight and survival rate, and were killed at 24d,72h, and 120h respectively. Mice lungs were used for lunge pathological analysis, and bronchoalveolar lavage fluid (BALF) were harvested for the detection of total cell number count, classified count, protein concentration and MPO activity.Results:The expression of embryonic stem related genes SSEA-4 was highly expressed in UC-MSCs, approaching one quarter of SSEA-4 in embryonic stem cells. However, the expression of other embryonic stem related genes OCT-4、SOX-2、NANOG couldn’t detected by Fluorescence quantitative PCR. The animal experiments showed that the therapy of UC-MSCs not only could protect the C57BL/6 ALI mice, but also treat BALB/C ALI mice. After administering of UC-MSCs, both of their weight and survive rate were significantly improved. Meanwhile, the lung tissue injury were attenuated, and the total cell count and neutrophil count and protein and MPO activity in BALF were decreased significantly and the difference was statistically significant (p<0.05).Conclusion:Embryonic stem related genes SSEA-4 was highly expressed in UC-MSCs. The protection of UC-MSCs to ALI mice could be observed on two different background mice (BALB/C and C57L/6), to confirm the universal treatment effects of UC-MSCs on the ALI mice.PART Ⅱ THE REGULATION OF UC-MSCS ON ALI MODEL IMMUNE RESPONSEObjective:To explore the therapy of UC-MSCs on ALI mice, and to illustrate the molecular mechanisms of UC-MSCs effects on ALI mice.Methods:Protein chip was applied for detection the change of BALF proteins of ALI model after 72h UC-MSCs treatment. GO-Term and KEEY-pathway on DAVID database were used for analyzing the protein function cluster and pathway cluster.Fluorescence quantitative PCR and ELISA were used to validate the expression of TNF-a, IL-1β,IL-6, IL-10, CCL17 and CCL22, the macrophage associated factors. The pulmonary alveolar macrophage and IL-10 postive alveolar macrophage changes were detected by flow cytometry. In vitro cell experiment, LPS-stimulted macrophages were cultured with UC-MSCs, to observe the regulation of UC-MSCs on its morphology and expression of TNF-a and IL-10.Result:The protein chip showed that after the administration of UC-MSCs,22 kind proteins were significantly down-regulated and 8 kinds were significantly up-regulated in ALI mice BALF. Analysis methods of GO-Term and KEEY-pathway showed that the function cluster of these significantly changed proteins focused on the immune response and were specialy related with the immune function of macrophages, including TNF-a, IL-1β, IL-6, IL-10, CCL17 and CCL22. Fluorescence quantitative PCR and ELISA results validated that after UC-MSCs treatment, TNF-α, IL-1β, IL-6 in ALI mice lung were decreased significantly and IL-10, CCL17 and CCL22 were increased significantly. And in vivo flow cytometry showed that UC-MSCs can reduce the number of ALI mice lung macrophages but increase its’ expression of IL-10. In vitro when LPS-stimulated macrophages were cultured with UC-MSCs, which could improve morphology of macrophage and reduce the macrophage’expression of TNF-a, and increase the expression of IL-10.Conclusion:UC-MSCs were proved to effectively regulate the function of alveolar macrophages of ALI mice, to inhibit the inflammatory reaction after LPS stimulation, and to promote the secretion of more IL-10, which need the foundation for in-depth study of UC-MSCs effction.PART Ⅲ THE PARACRINE EFFECTS OF UC-MSCS ON ALI MODELObjective:To explore the therapy of UC-MSCs on ALI by paracrine and its molecular mechanisms.Methods:The BABL/C mice were randomly divided into ALI, ALI+CM (UC-MSCs), ALI+CM (Fibroblast) groups. After administering of LPS 1h, ALI+CM (UC-MSCs), ALI+CM (Fibroblast) and ALI group were treated with 80ul concentrated culture medium of UC-MSCs and Fibroblast and the 80ul PBS.Mice were killed at 24h,72h and 120h. The lung issues were collected, the weight, survival rate of mice, and the changes of pathologic were observed. In vitro cell experiments, UC-MSCs were co-cultured with LPS-stimulated macrophages and the supernatant was collected for detection PGE2, IL-4, IL-13secrted by UC-MSCs. In vitro experiments, Celecoxib, the specific inhibitor of PGE2 key synthesis enzyme, was used to interve UC-MSCs, and after that UC-MSCs and its’ conditional medium were co-cultured with model of LPS-stimulated macrophage whose TNF-a and IL-10 were detected by ELISA. And in vivo Mice were randomly divided into Ctrl, ALI, ALI+UC-MSCs, ALI+UC-MSCs (-PGE2), ALI+CM, ALI+CM (-PGE2) respectively. After intratracheal instillation LPS 1h, ALI+UC-MSCs and ALI+CM mice were respectively given UC-MSCs suspension and concentrated culture medium without Celecoxib intervention, ALI+UC-MSCs (-PGE2) and ALI+CM (-PGE2) mice were were respectively given UC-MSCs suspension and concentrated culture medium with Celecoxib intervention. Ctrl and ALI group were treated with the same volume of PBS. Mice were killed at 72h and collected samples to detect the lung pathological changes, and the expression of TNF-a and IL-10 in BALF by ELISA.Results:After the treatment of UC-MSCs conditional medium, compared with Ctrl group, the survival rate and weight of ALI mice were increased and lung injury score were down-regulated significantly. In vitro cell results, when UC-MSCs were co-cultured with LPS-stimulated macrophage for 72h, the expression of PGE2 secreted by UC-MSCs was significantly increased (p<0.05), but the expression of IL-13 and IL-4 was not altered. In vitro experiments after the intervention of PGE2 by Celecoxib, the regulation capacity of LPS-stimulated macrophage by UC-MSCs and its culture conditions were decreased, respectively compared with Mac+LPS+UC-MSCs group, Mac+LPS+CM group, in Mac+LPS+UC-MSCs (-PGE2) group and Mac+LPS+CM (-PGE2) group the expression of TNF-a was increased and expression of IL-10 was decreased (p<0.05) And in vivo, after the intervention of PGE2 by Celecoxib, the protection to ALI mice by UC-MSCs and its culture conditions were decreased, respectively compared with ALI+CM and ALI+UC-MSCs, weight and survival rate of ALI+UC-MSCs (-PGE2) and ALI+CM (-PGE2) were down-regulated, and lung tissue injury was increased, and the expression of TNF-a in BALF was significantly increased and the expression of IL-10 was decreased (p<0.05).Conclusion:The effects of UC-MSCs on ALI mice are mainly by paracrine function, especially by secreting with PGE2, which provides a novel evidence for clinical treatment of ALI/ARDS by using UC-MSCs.