Role of human umbilical cord mesenchymal stem cells in the maturation and differentiation of dendritic cells in patients with severe acute pancreatitis and its mechanism in the inflammation modulation / 中华胰腺病杂志

ABSTRACT

Objective:To investigate the effect of human umbilical cord mesenchymal stem cells (hUC-MSCs) on the maturation and differentiation of dendritic cells (DCs) and the mechanism involved in the regulation of inflammation in patients with severe acute pancreatitis (SAP).Methods:The full-term fetal umbilical cords(about 4-5 cm) were collected from Zhengzhou Central Hospital Affiliated to Zhengzhou University after cesarean section. hUC-MSCs were isolated and cultured in primary culture. Flow cytometry was used for phenotype identification, adipogenic and osteogenic staining. 20 ml peripheral blood samples from 5 SAP patients were collected, and monocytes were isolated using lymphocyte separation solution and then induced by adding granulocyte macrophage colony-stimulating factor (GM-CSF), IL-4 and tumor necrosis factor (TNF)-α and cultured as DCs. According to different culture methods, DCs were divided into DCs group, hUC-MSCs+ DCs group and hUC-MSCs+ DCs+ NS398 group (NS398 was a specific inhibitor of COX-2, a downstream regulatory gene of NF-κB). The phenotype of DCs was detected by flow cytometry, and the levels of IL-1β, IL-lα, IL-2, IL-6 and IL-10 in the supernatant of cell culture for 24 hours were determined. The expression of toll like receptor (TLR)-4, IKKα and NF-κB-p65 were detected by Western blot.Results:The hUC-MSCs were successfully cultured, and their surface markers CD 90, CD 105 and CD 73 were positively expressed, and they could differentiate into adipocytes and bone cells. With the prolongation of culture time, DCs differentiated from immature to mature cells. Compared with the DCs group, the proportion of regulatory DCs (regDCs) was increased in the hUC-MSCs+ DCs group, and the marker CD 11b was significantly up regulated [(14.26±1.25)% vs (4. 87±0.58)%], CD 1a and CD 11c were significantly down regulated [(2.81±0.34)% vs (13.62±1.52)%, (3.88±0.5)% vs (11. 8±1.22)%]. All the difference were statistically significant ( P<0.05). The expression of IL-1β, INF-γ and IL-6 in culture supernatant were down regulated, but the difference was not statistically significant; The pro-inflammatory factor IL-1α was significantly decreased [(14.91±2.58)ng/L vs (30.19±7.75)ng/L], and the anti-inflammatory factor IL-10 was significantly increased [ (17.03±4.69)ng/L vs (1.83±0.14)ng/L]. The expression levels of NF-κB-p65 and TLR4 were significantly down regulated (0.74±0.02 vs 0.97±0.01, 0.89±0.01 vs 1.72±0.01), and the expression of IKKα protein was significantly up regulated (1.12±0.01 vs 0.21±0.01) in hUC-MSCs-DCs group. All the differences were statistically significant (all P value<0.05). Compared with DCs group and hUC-MSCs+ DCs group, the expression levels of NF-κB-p65 and TLR4 were significantly down regulated (0.34±0.01 vs 0.97±0.01, 0.74±0.02 and 0.14±0.01 vs 1.72±0.01, 0.89±0.01), while the expression of IKKα protein was significantly up regulated (1.68±0.01 vs 0.21±0.01, 1.12±0.01) in hUC-MSCs+ DCS+ NS398 group. All the differences were statistically significant (all P value<0.05). Conclusions:In SAP patients, hUC-MSCs can inhibit the maturation and differentiation of DCs, and induce CD 11bhigh CD 1alow CD 11clowrregDCs to participate in immune regulation, which may play an anti-inflammatory role by inhibiting the inflammatory cascade through TLR4/IKKα/NF-κB/COX-2 pathway.