Effects of endothelial progenitor cells on proliferation and biological function of hepatic stellate cells under shear stress / 中国应用生理学杂志

OBJECTIVE@#To investigate the effects of endothelial progenitor cells (EPCs) under shear stress on the biological function such as proliferation, adhesion, migration, apoptosis and expression of α-smooth muscle actin (α-SMA), collagen-I and collagen-Ⅲ of hepatic stellate cells (HSCs).@*METHODS@#HSCs and EPCs were inoculated into the upper and lower layers of the co-culture chamber respectively and co-incubated for 24 hours. Then, 12 dyne/cm shear stress was applied to EPCs cells for another 24 hours. After that, proliferation, adhesion, migration and apoptosis of HSCs were detected by cell counting kit-8 (CCK-8) kit, cell adherent assay, Boyden cell migration assay and flow cytometry respectively. Fluorescence quantitative PCR and Western blot were used to detect the mRNA and protein expression of alpha -SMA, collagen I and collagen-Ⅲ in HSCs.@*RESULTS@#Under shear stress, EPCs ecological niche could obviously inhibit the proliferation, adhesion and migration of HSCs, promote the apoptosis of HSCs, and down-regulate the mRNA and protein expression of collagen-I, collagen-Ⅲ in HSC cells.@*CONCLUSIONS@#Under shear stress, EPCs ecological niche could inhibit the fibrosis development of HSCs to a certain extent.

Effects of inward rectifier potassium channel blockers on EPCs function / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the effects of inward rectifier potassium channel blockers (BaCl2, CsCl) on the functions of endothelial progenitor cells (EPCs).</p><p><b>METHODS</b>Density gradient centrifugation-isolated rat hone marrow mononuclear cells were cultured in vitro. EPCs were harvested and seeded on six culture dish when cells grew to 3-5 passages. Before testing the EPCs were synchronized with M199, which contain 2% fetal calf serum. In the end, EPCs were treated with different intervention. The experiment mainly included two parts: (1) BaCl2 (100 micromol/L) and free BaC2 of Tyrodes solution; (2) CsCl (1 mmol/L) and control. Cell pretreated with blockers above mentioned for 12 h, then the gene expression of stromal cell-derived factor-1 (SDF-1), epoprotenol (PGI2) were assessed, beyond that the ability of adhesion, migration were assayed with different tests. In addition, the medium was collected when EPCs were treated for 3 days. The levels of SDF-1 were measured by sandwich enzyme-linked immunosorbent assay (ELISA). Going even further, EPCs were treated with the signal pathway blockers in advance, after repeat the above steps, in order to analyze the change of SDF-1 and then discuss its mechanism.</p><p><b>RESULTS</b>Compared with control group, BaCl2, CsCl could increase EPC adhesion and migration to same extent. Moreover, the gene expression of SDF-1, PGI2 was significantly up-regulated and the production of SDF-1 increased evidently. Furthermore, the mechanism of SDF-1 secretion increasing mainly was associated with eNOS signaling pathways.</p><p><b>CONCLUSION</b>Ba2+ and Cs+ play important roles in increasing EPCs functions, such as adhesion, migration and secretion.</p>

Effects of shear stress on late endothelial progenitor cell functions / 医用生物力学

Objective To investigate the effects of shear stress on late endothelial progenitor cells (EPCs) functions in vitro and in vivo. Methods Density gradient centrifugation-isolated rat bone marrow mononuclear cells were cultured in EGM-2MV and induced into EPCs. The 3rd~4th generation of EPCs, namely late EPCs, were treated with shear stress (1.2 Pa). Then cell biological functions, such as proliferation, adhesion, migration and ability of tube formation, were assayed with EdU incorporation assay, adhesion testing, Boyden chamber assay and Matrigel, respectively. The gene expression of VEFG was analyzed by real time RT-PCR. The apoptosis and aging situation of late EPCs were assayed by FACS and senescence-associated β-galactosidase (SA-β-gal) staining. The reendothelialization capacity of late EPCs treated by shear stress was evaluated by establishing models of freshly balloon-injured carotid arteries of rats and cell transplantation in situ. Results Shear stress could increase proliferation, adhesion, migration and tube formation of late EPCs (P＜0.05), upregulate the gene expression of VEGF, inhibit EPC apoptosis and delayed EPC aging (P＜0.05). Transplantation of late EPCs treated by shear stress facilitated in vivo reendothelialization in the injured arterial segment and inhibited neointima formation. Conclusions Shear stress within the physiological range can improve the functions of late EPCs and enhance their therapeutic ability of repairing vascular endothelial injury, which provides experimental basis for the clinic application of EPCs and shear stress-mediated cell therapy.

Effects of extracellular matrix on biological characteristics of late endothelial progenitor cells / 生理学报

The present study was designed to investigate the effects of various extracellular matrix (ECM) proteins on the biological characteristics of late endothelial progenitor cells (EPCs). Density gradient centrifugation-isolated rat bone marrow mononuclear cells were cultured in complete M199 medium, which contained 15% fetal calf serum, 10 μg/L vascular endothelial growth factor (VEGF) and 5 μg/L basic fibroblast growth factor (bFGF). EPCs were plated on substrates containing fibronectin (Fn), laminin (Ln) or rat tail tendon collagen (Col), and the corresponding cells were defined as Fn, Ln and Col groups. The 3rd generation EPCs, namely late EPCs, were harvested. The proliferation, adhesion, migration and the ability of forming tubes were assayed using CCK-8, adhesion test, wound healing assay and Matrigel, respectively. The mRNA expressions of endothelial cell differentiation markers, vWF and CD31, were analyzed by real time RT-PCR. The apoptosis was assayed by flow cytometry (FCM). The results showed that cell proliferation ability of Fn and Col groups were higher than that of Ln group; Fn group showed increased adhesion compared to Col and Ln groups (P < 0.01); The migration ability of Fn and Col groups were higher than that of Ln group. Moreover, Fn group showed increased tube formation abilities compared to Col and Ln groups (P < 0.05). Although 24-hour free-serum-induced apoptosis in Ln group was the highest, there was no difference of auto-apoptosis among the three groups. Furthermore, the mRNA expressions of vWF and CD31 exhibited no difference among the three groups. These results suggest the ECM affects the biological functions of late EPCs, which would have a high probability of providing new directions that lead to the development of artificial heart and blood vessels.

Role of F-actin cytoskeleton in differentiation of endothelial progenitor cells induced by laminar shear stress / 医用生物力学

Objective To investigate effects of F-actin cytoskeleton on differentiation of endothelial progenitor cells (EPCs) under laminar shear stress. MethodsEPCs isolated from rat bone marrow were treated with laminar shear stress (1.2 Pa). Then the gene and protein expressions of the endothelial cell differentiation markers, such as vWF and CD31, were assayed with real time RT-PCR and Flow Cytometry. The effects of laminar shear stress on F-actin cytoskeleton and Ras activity were investigated by immunofluorescence technique and Pull-down assay. Results Compared with the untreated group, the expressions of vWF and CD31 were obviously increased in the group treated with laminar shear stress (P<0.05). Moreover, exposure of EPCs to laminar shear stress led to the reorganization of cytoskeleton and enhanced the activity of Ras in EPCs. The treatment to EPCs with either F-actin stabilizer jasplakinolide or depolymerizers cytochalasin D inhibited the cytoskeleton reorganization induced with laminar shear stress, the activity of Ras and the up-regulation of the vWF and CD31 genes. However, over-expression of Ras augmented the up-regulation of the vWF and CD31 genes induced by laminar shear stress in EPCs.Conclusions The mechanism that laminar shear stress accelerates the differentiation of EPCs may be related with the laminar shear stress-induced cytoskeleton rearrangement and Ras activation. This study is of significance in revealing the mechanism of vascular endothelial repair which could be useful for the prevention and treatment of atherosclerosis.

Chemical constituents of Scirpus yagara Ohwi / 中国药学杂志

OBJECTIVE: To study the chemical constituents of Scirpus yagara Ohwi. METHODS: Column chromatography techniques were applied to isolate the constituents. The chemical structures of the constituents were elucidated on the basis of physicochemical properties and spectral data. RESULTS: Forteen compounds were isolated and identified as sanleng diphenyllactone (1), β-sitosterol (2), ferulic acid(3), succinic acid(4), azelaic acid(5), docosanoic acid (6), 6, 7, 10-trihydroxy-8-octadecenoic acid(7), 4, 4-dime-thyl-1, 7-pimelic acid(8), vanillic acid(9), 3, 5-dihydroxy-4-methoxy-benzoic acid(10), p-hydroxybenzaldehyde(II), 3, 4-dihydroxy-benzoic acid(12), 2, 7-dihydroxy xanthone(13) and daucosterol(14). CONCLUSION: Compounds 1-10 and 12-14 are obtained from this plant for the first time, and compounds 1 and 13 were obtained from the genus Scirpus for the first time.