Differential expression of proteins in rat mesenchymal stem cells undergoing endothelial differentiation / 中华血液学杂志

<p><b>OBJECTIVE</b>To screen and identify differentially expressed proteins of mesenchymal stem cells (MSC) during endothelial differentiation.</p><p><b>METHODS</b>MSCs were induced to endothelial differentiation with vascular endothelial growth factor (VEGF) and epithelial growth factor (EGF) mixture. The whole cell proteins were extracted and isolated by two-dimensional gel electrophoresis. After gel was analyzed by Imagemaster 5.0 software, differentially expressed proteins were partially selected and identified by MALDI-TOF-MS.</p><p><b>RESULTS</b>The differentiated MSC highly expressed endothelial cells related markers, CD31, CD34 and FVIIIAg were 56.8%, 38.8% and 14.5% respectively by flow cytometer. Compared with the primary cultured MSC, the differentiated cells differentially expressed 91 proteins. Among the 19 identified proteins, 11 up-regulated and 8 down-regulated, which include cytoskeletal proteins, such as myosin, filamin, vimentin and vinculin; cell metabolism enzymes, such as ORP-150, ERO1-α, Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, protein disulfide-isomerase A3, FAS and enolase 3; nuclear factors, such as TAR DNA binding protein, guanine nucleotide binding protein and hypoxia up-regulated protein 1; VEGF receptors, such as KDR and so on.</p><p><b>CONCLUSIONS</b>Cytoskeletal proteins, metabolism enzymes and KDR were all involved in endothelial differentiation of MSC. These proteins may be the regulatory targets for endothelial differentiation of MSC.</p>

Research on induced differentiation of human bone marrow mesenchymal stem cells into vascular endothelial cells / 中国应用生理学杂志

<p><b>AIM</b>To analyze gene expression difference between human mesenchymal stem cells and umbilical vein endothelial cells, to discuss the feasibility of inducing hMSCs to differentiate into endothelial cells through in vitro gene transfection as well as the use and prospective as a seeding cell source of vascular tissue engineering.</p><p><b>METHODS</b>hMSCs and hUVECs were isolated, expanded in culture, and characterized by flow-cytometry, immunocytochemistry, immunofluorescence and transmission electron microscopy (TEM). Differential analysis of gene expression profiles between them was performed by Biostar H-40 cDNA microarrays. The properties of VEGF 165 transfected were also detected with RT-PCR, ELISA et al.</p><p><b>RESULTS</b>Nearly 86% genes were coexpressed in both cells and hMSCs expressed typical endothelial antigen marker of EC. After VEGF165 transfection, hMSCs (or committed hMSCs) were positive for CD31. To different extent, the expression of CD44 was down regulated and CD34, FVIIIAg, Flt-1 up regulated.</p><p><b>CONCLUSION</b>Generation of functional EC or tissue engineered blood vessels from human MSCs is feasible utilizing an in vitro environment gene transfection.</p>

Cloning, expression of soluble VEGFR2 fragment and its effect on tumor angiogenesis / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the anti-tumor angiogenesis effect of soluble VEGF receptor fragment by blocking the combination of VEGF and its receptor in vivo and in vitro.</p><p><b>METHODS</b>RT-PCR technique was used to amplify Flk-1/KDR fragment from embryo mouse liver, which was recombinated to expression vector pET-28b(+) and retrovirus vector PLXSN, which was induced to be expressed, purified and identified with EcoR I and Hind III. Mouse endothelial cells were separated, cultured and identified by immunocytochemistrical staining using VIII factor-related antigen antibody. The expressed product was analyzed about its effect on endothelial cell's growth in vitro with MTT method. The retrovirus vector was transfected to tumor cell lines S180 and B16 by liposome method to observe the biological specificity in vitro after gene transfection.</p><p><b>RESULTS</b>1000 bp size sVEGFR fragment was amplify from E9, E11 embryo mouse liver tissues, which was recombinated to TA clone vector and identified by sequence analysis. This fragment was cloned to expression vector pET-28b(+), the expressed product was purified and identified correctly. The in vitro study showed this expressed product can effectively inhibit endothelial cell(s), growth and proliferation. The fragment was then cloned to retrovirus vector PLXSN and transfected to tumor cell lines S180 and B16 successfully with RT-PCR and SDS-PAGE. The experiments in vivo showed that the weight of tumor smaller, the size decreased significantly, the microvessel density was fewer and Flk1 protein expression were higher in the group of gene transfection than that of control.</p><p><b>CONCLUSION</b>Soluble VEGFR fragment is a kind of effective gene engineer product for anti-tumor angiogenesis gene therapy and the development of anti-tumor drug.</p>

The expression of Fas, FasL and Bcl-2 on RMA cells during the process of apoptosis induced by chemotherapeutic drugs / 中国实验血液学杂志

The objective of the study is to explore the effect of Fas, FasL and Bcl-2 on the process of apoptosis induced by chemotherapeutic drugs through detecting the expression of Fas, FasL and Bcl-2 on murine lymphoma cell line RMA. Dexamethasone(DEX), etoposide (VP-16), arsenic trioxide As(2)O(3) and all trans-retinoic-acid (ATRA) were added to the RMA cells as well as to the cells preincubated with interleukin-2 (IL-2), interleukin-6 (IL-6) or granulocyte-macrophage colony-stimulating factor (GM-CSF), respectively. The effect on apoptosis was observed and the expression of Fas and FasL mRNA as well as the expression of Fas and Bcl-2 antigen were measured. DEX and VP-16 could promote apoptosis of RMA cells while upregulating the expression of Fas and FasL without affecting the expression of Bcl-2. ATRA downregulated the expression of Bcl-2 without any change of Fas and FasL, and no apoptosis of RMA cells induced by ATRA was observed. Although As(2)O(3) induced apoptosis of RMA cells, it did not affect the expression of Fas, FasL and Bcl-2, which suggested that different drugs induce apoptosis of the same kind of cells by different signal transduction system and apoptosis induced by Fas system needed the coexistence of Fas and FasL. Although IL-2, IL-6 and GM-CSF upregulated the expression of Fas protein when adding to RMA cells separately, none of them induced apoptosis. Apoptosis could be induced by combination of IL-2 and IL-6 along with the upregulation of Fas and FasL. The cytokines facilitated the apoptotic action of chemotherapeutic drugs, the drug concentration for inducing apoptosis decreased and the time period of starting apoptosis shortened. Apoptosis could be observed without the expression of FasL when anti-Fas-antibody was added to RMA cells. The results demonstrated that there was synergistic effect of chemotherapeutic drugs and some cytokines for induction of apoptosis. Fas-FasL system participated in the apoptosis induced by DEX and VP-16; different drugs induce apoptosis by different pathway of signal transduction.

Comparative study on activated immunocytes of human bone marrow and peripheral blood by cytokines / 中国实验血液学杂志

To study immunophenotype and cytotoxicity of the immunocytes in bone marrow and peripheral blood after activation by combined cytokines, mononuclear cells (MNC) of bone marrow and peripheral blood were activated by IFN-gamma, IL-1, IL-2 and McAb-CD3 in vitro. The cell amount and morphology during culture were observed. Cytochemical staining and immunophenotype analysis were done before and after culture in two groups of MNC. Cytotoxicity was tested by MTT method. The results showed that the cell number of two groups increased obviously in culture (P < 0.05), while the peripheral blood mononuclear cells increased more markedly (P < 0.05). The cytochemical staining showed POX decrease, but PAS increase in two groups. The positive ratios of CD3(+), CD56(+) and CD38(+) cells in two groups increased obviously after culture (P < 0.05), but there was no significant difference between those two groups. CD3(+) CD56(+) cells increased obviously in peripheral blood mononuclear cells activated by cytokines (P < 0.05), but CD3(+) CD56(+) cells did not increase in bone marrow mononuclear cells. There was no significant difference between two groups' cytotoxicity. It was concluded that IFN-gamma, IL-1, IL-2 and McAb-C D3 increased cell number and cytotoxicity of both bone marrow and peripheral blood mononuclear cells that can be used in cell immunotherapy.