| The morbidity and mortality in critically ill patients is very high, especially in sepsis with acute lung injury (ALI) and its severe form acute respiratory distress syndrome (ARDS). At present, most of research of ALI focus on ALI related inflammatory cells, cytokines, adhesion molecule interactions, and expression regulation, etc. Although some achievements have been made, the pathogenesis of ALI has yet to clarify. The method of ALI therapy is mainly supportive treatment. The mortality rate of ALI is up to40%because of the short of specific treatment measures. There is a urgent need to explore new methods of treatment to overcome this significant medical problem. Recent study found that bone marrow mesenchymal stem cells (BM-MSCs) have multilineage differentiation ability, immunomodulatory and paracrine features. MSCs could suppress the immune response and differentiate into a variety of tissue cells after homing to the damaged tissue, have broad application prospects in the field of organizational functions regeneration, reconstruction, and immunomodulatory therapy. Many animal studies showed that BM-MSCs can reduce endotoxin-induced ALI, improve lung function and the survival rate by anti-inflammatory, anti-apoptotic and immunomodulatory. Thus MSCs therapy has provided a new methods and ideas in the field of ALI.However, the collection of BM-MSCs faced many difficulties because the quality of MSCs decreased in the aged. The proliferation ability of MSCs is also reduced along with the passage time in culture. The operating process of bone marrow aspiration is easily lead to infection and pain. Thus to find a new ideal source of mesenchymal stem cells (MSCs) is very necessary. The MSCs is survival in almost all tissues and organs. Its advantages in immune regulation and low immunogenicity has nothing to do with their origin. Compared to BM-MSCs, umbilical cord MSCs (UC-MSCs) have more utility on the bisis of convenient accessibility, easy extracting, high proliferation rate, and more safety. There is no treatment of UC-MSCs in endotoxin induced ALI reported. In this study, we compared cell morphology, cell phenotype, differentiation capacity and immunomodulatory ability between the BM-MSCs and UC-MSCs first. Then Gene expression and small RNA expression were compared through the human genome-wide expression chip and GO term functional cluster analysis. Finally, we verify whether UC-MSCs transplanting can inhibit the endotoxin-induced ALI, reduce oxidative stress, and improve survival rate by inhibiting the inflammatory response and oxidative stress.Method:1) The primary culture and identification of MSCs:human BM-MSCs and UC-MSCs were primary cultured. The general characteristics of these two cell were identified through morphological observation and flow cytometry. Using chemical induction method such as β-glycerophosphate, TGF-β and insulin, these two cells were induced into osteoblasts, cartilage and adipocytes. Functional differentiations were evaluated using alizarin Red S, oil Red O, and toluidine blue staining method.2) MSCs gene expression analysis using microarray:Total RNA was extract from BM-MSCs and UC-MSCs groups. Gene expression and small RNA expression were compared through the human genome-wide expression chip and GO term functional cluster analysis.3) Rat ALI model making:60SD rats were selected and randomly divided into four groups:control group, LPS group, Fibroblast+LPS group and MSC+LPS group(n=15for each group). Rats in the latter three groups were intraperitoneal injected with10mg/kg (body weight) of LPS to produce a sepsis ALI model. Routine biopsy, bronchoalveolar lavage, lung wet-to-dry ratio and serum concentration of inflammary factors such as TNF-α, IL-1β, and IL-6, etc. were analyzed.4) UC-MSCs infusion treatment of lung injury:After LPS injection induced lung injury, saline containing5×105UC-MSCs in300μl were injected through tail vein in MSC+LPS group. Total300μl saline containing5×105fibroblasts (MRC-5cell line) were injected in Fibroblast+LPS group, while the other two groups were injected with normal saline volume. Each group were sacrificed3-5rats at6hours,24hours and48hours after infusion therapy respectively. The plasma and lung tissue were collected for routine biopsy, bronchoalveolar lavage fluid neutrophil count, protein quantification, lung wet-to-dry ratio and serum inflammatory cytokines TNF-α, IL-1β, IL-6ELISA analysis. MDA content, HO-1enzyme activity and protein quantitative analysis were selected as indicators of lung tissue oxidative damage levels.Results: 1) Both of MSCs derived from bone marrow and umbilical cord can be exuberant proliferated with typical fibroblast cell morphology. They can express typical MSC cell surface antigen such as CD29, CD44, CD73, CD90, CD105and CD166. No hematopoietic or endothelial cell markers such as CD31, CD34, and CD45can be detected. Osteoblasts, cartilage and adipocytes differentiation can be achieved after induction with the expression of specific markers respectively.2) Data from gene expression chip and microRNA chip were analyzed using bioinformatics software. Compared with UC-MSCs, BM-MSCs express more immune response genes. While UC-MSCs express more genes related to organ development and early developmental genes. KEGG signaling pathway analysis also demonstrated these results. The top three miRNAs up-regulated in UC-MSCs were miR-519e*, miR-518a-5p and miR-520d-5p. MicroRNAs highly expressed in BM-MSCs were miR-373*, miR-492, miR-498and miR-409-5p. all of these seven miRNAs target genes associated with oncogenesis.3)Six hours after intraperitoneal injection of LPS, the capillaries in the lung tissue expanded and became congested by a significant increase in neutrophils. These events peaked at the24hour time-point. In addition, the lung septae obviously thickened and did not show any improvement48hours later. The lung wet-dry weight ratio was significantly higher at24h after LPS administration and slightly reduced at48h. LPS caused significant increase of neutrophil counts in the BALF and MPO activity in the lung tissue at24and48h. LPS caused a significant acute systemic inflammatory response as evidenced by the increased serum concentrations of the pro-inflammatory mediators TNF-α, IL-1β, and IL-6. The response of pro-inflammatory cytokines reached the peak at6hours after injection of LPS, and decreased gradually at the24and48hour time-points.4) MSC+LPS rats also displayed moderate injury, but the severity was significantly less compared to the LPS group at all three time points. In contrast, rats given injection of human fibroblast cell line, MRC-5, had no improvement in lung injury UC-MSCs decreased wet-dry weight ratio significantly. In terms of endothelial and epithelial permeability, the MSC+LPS group exhibited relatively lower BALF protein concentrations, but this difference was not statistically significant. The presence of UC-MSCs reduced the increase of TNF-α, IL-1β, and IL-6at each of the time point. LPS also caused an increase of the serum concentration of the anti-inflammatory cytokine IL-10. This change in IL-10concentration was not altered by intravenous administration of UC-MSCs.5) Lung MDA levels increased markedly in the LPS group compared with the control group at each time point, whereas the increase was significantly attenuated in the MSC+LPS group. In the control group, the lung expression of HO-1was very weak at24h. However, HO-1expression was found to be markedly enhanced in the LPS group and even higher in the MSC+LPS group. A similar observation was made in terms of lung HO-1activity6) Rats that received UC-MSCs had significantly higher rate of survival versus the LPS group (87%vs.60%;), in contrast, rats given fibroblast cells have no improvement in survival compared with LPS group. Treatment with UC-MSCs increased the survival rate about20%over48hours. p<0.05.Conclusion:1) MSCs can be derived from both of bone marrow and umbilical cord and in line with international standard for MSCs. The proliferation of UC-MSCs was markedly higher than BM-MSCs, and UC-MSCs also has the ability to multi-differentiation. Compared with UC-MSCs, BM-MSCs mediated more immune inhibition but without significant difference.2) Intravenous injection of UC-MSCs clearly increased the survival rate of rats suffering from LPS-induced lung injuries and significantly reduced systemic and pulmonary inflammation. Promoting anti-inflammatory homeostasis and reducing oxidative stress may be the therapeutic basis of UC-MSCs for this disease model. |
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