[Physicochemical properties and anti-inflammatory and immunomodulatory effects of Shengfupian polysaccharides].

In this study, the crude polysaccharides was extracted from Shengfupian and purified by Sevag deproteinization. Then, the purified neutral polysaccharide fragment was obtained by the DEAE-52 cellulose chromatography column and Sephadex G-100 co-lumn. The structure of polysaccharides was characterized by ultraviolet spectroscopy, infrared spectroscopy, ion chromatography, and gel permeation chromatography. To investigate the anti-inflammatory activity of Shengfupian polysaccharides, LPS was used to induce inflammation in RAW264.7 cells. The expression of the CD86 antibody on surface of M1 cells, the function of macrophages, and the content of NO and IL-6 in the supernatant were examined. An immunodepression model of H22 tumor-bearing mice was established, and the immunomodulatory activity of Shengfupian polysaccharides was evaluated based on the tumor inhibition rate, immune organ index and function, and serum cytokine levels. Research indicated that Shengfupian polysaccharides(80 251 Da) was composed of arabinose, galactose, glucose, and fructose with molar ratio of 0.004∶0.018∶0.913∶0.065. It was smooth and lumpy under the scanning electron microscope. In the concentration range of 25-200 µg·mL~(-1), Shengfupian polysaccharides exhibited little or no toxicity to RAW264.7 cells and could inhibit the polarization of cells to the M1 type and reduce the content of NO and IL-6 in the cell supernatant. It could suppress the phagocytosis of cells at the concentration of 25 µg·mL~(-1), while enhancing the phagocytosis of RAW264.7 cells within the concentration range of 100-200 µg·mL~(-1). The 200 mg·kg~(-1) Shengfupian polysaccharides could alleviate the spleen injury caused by cyclophosphamide, increase the levels of IL-1ß and IL-6, and decrease the level of TNF-α in the serum of mice. In conclusion, Shengfupian polysaccharides has anti-inflammatory effect and weak immunomodulatory effect, which may the material basis of Aconm Lateralis Radix Praeparaia for dispelling cold and relieving pain.

Lipopolysaccharide pretreatment inhibits LPS-induced human umbilical cord mesenchymal stem cell apoptosis via upregulating the expression of cellular FLICE-inhibitory protein.

Mesenchymal stem cell (MSC)-based regenerative therapy is currently regarded as a novel approach with which to repair damaged tissues. However, the efficiency of MSC transplantation is limited due to the low survival rate of engrafted MSCs. Lipopolysaccharide (LPS) production is increased in numerous diseases and serves an essential function in the regulation of apoptosis in a variety of cell types. Previous studies have indicated that low-dose LPS pretreatment contributes to cytoprotection. In the current study, LPS was demonstrated to induce apoptosis in human umbilical cord mesenchymal stem cells (hUCMSCs) via the activation of caspase, in a dose-dependent manner. Low-dose LPS pretreatment may protect hUCMSCs against apoptosis induced by high-dose LPS, by upregulating the expression of cellular FADD-like IL-1ß-converting enzyme-inhibitory protein (c-FLIP). The results of the present study indicate that pretreatment with an appropriate concentration of LPS may alleviate high-dose LPS-induced apoptosis.

[Comparison of different methods for the isolation of human umbilical cord mesenchymal stem cells].

OBJECTIVE: To explore the most appropriate method for the isolation of human umbilical cord mesenchymal stem cells (MSCs) through a comparison of different methods. METHODS: Fifteen umbilical cord specimens from full-term healthy fetus with caesarean birth were completely rinsed with phosphate buffer saline (PBS) and sliced into 1 mm(3) tissue blocks after removal of umbilical vessels and external membrane. These tissue blocks were averagely divided into 4 groups after washing and centrifuge. Then four methods for the isolation of human umbilical cord MSCs were compared: an explant culture and three enzymatic methods of collagenaseII, collagenaseII/trypsin and collagenaseII/hyaluronidase. The count of living cells was evaluated by trypan blue dye exclusion test. Cell morphology was observed under inverted microscope. The expressions of cell surface markers CD105, CD90, CD73, CD31, CD44, CD45, human leukocyte antigen-I (HLA-I) and human leukocyte antigen class IImolecules (HLA-DR) were detected by immunofluorescent staining. Cell proliferation was assayed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). RESULTS: The human umbilical cord MSCs were successfully isolated by four isolated methods. However the isolation method used profoundly altered the cell number and proliferation capacity of isolated cells. Isolated cells using four methods were counted at (5.44 ± 0.21)×10(5), (4.03 ± 0.24)×10(5), (4.91 ± 0.33)×10(5) and (5.94 ± 0.40)×10(5) respectively. More cells were obtained with collagenaseII/hyaluronidase than other three methods (all P < 0.05). Cells out of tissue blocks were observed at Day 9-11 and cells were observed at Day 2 with three types of enzyme digestion. The fusion time of cells were (18.5 ± 3.5), (8.0 ± 1.0), (7.5 ± 1.5) and (3.5 ± 0.5) days respectively. The fusion time of cells obtained with collagenaseII/hyaluronidase was lower than other methods (all P < 0.05). Cell morphology: polygonal, irregular and of large volume for explant culture; relatively short and small for collagenaseII and collagenaseII/trypsin methods; thin spindle for collagenaseII/hyaluronidase method. Immunofluorescent staining revealed that CD105, CD73, CD90 and CD44 were expressed in all groups while there was no expression of CD31, CD45 or HLA-DR. And the cells obtained with collagenaseII/hyaluronidase method were in a higher cell proliferation rate and activity compared to other methods. CONCLUSION: The collagenaseII/hyaluronidase method is optimal for the isolation of human umbilical cord MSCs than other methods.

Basic fibroblast growth factor/vascular endothelial growth factor in the serum from severe burn patients stimulates the proliferation of cultured human umbilical cord mesenchymal stem cells via activation of Notch signaling pathways.

BACKGROUND: Mesenchymal stem cells (MSCs) are the leading cellular constituents used in regenerative medicine. MSCs repair and reconstruct wounds of acute traumata and radiation-induced burns through proliferation, differentiation, and trophic activity. However, repair effect of MSCs on severe burn wounds remain to be clarified because severe burns are much more complex traumata than radiation-induced burns. Survival and proliferation of MSCs in microenvironments affected by severe burns are very important for improving wound repair/regeneration. This study aimed to elucidate the survival and proliferation effects and the potential proliferation mechanism of serum from severe burn patients (BPS) on human umbilical cord MSCs (hUCMSCs) in vitro. METHODS: The hUCMSCs were isolated, cultured, and identified. Next, we evaluated the effects of BPS on cell numbers, cell cycle progression, cyclin D expression, and key proteins and genes of the Notch signaling pathway. Putative mechanisms underlying the proliferation of hUCMSCs were investigated. RESULTS: BPS markedly increased the number of hUCMSCs, and the results of the cell cycle studies indicated that BPS induced cell cycle progression into the M phase. Cyclin D expression was higher with BPS than in the control group. Moreover, Notch-1, a key determinant of hUCMSC activation and proliferation, and its target gene Hes-1 were overexpressed after BPS treatment. Proliferation numbers of hUCMSC, rate of proliferation period (G2/M+S), and the expression of cyclin D, Notch-1, and Hes-1 were markedly decreased by Notch signaling inhibitors (DAPT/GSI). In the case of BPS, basic fibroblast growth factor and vascular endothelial growth factor were the key factors that promoted hUCMSC proliferation. CONCLUSION: This study provides novel evidence for the role of BPS in the survival and rapid proliferation of hUCMSCs and suggests that these cells could be used for cell therapy-based clinical applications for treating severe burns. Furthermore, hUCMSC proliferation was induced by basic fibroblast growth factor/vascular endothelial growth factor in BPS through activation of Notch signal.