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Objective To compare two sources of mesenchymal stem cells ( MSCs) from human placenta and umbilical cord, and to optimize a technical solution for bench or clinical studies of MSCs.Methods MSCs were isolated from human placenta and umbilical cord and expanded for analysis.The cell morphology was observed under invert microscope, the immunophenotypic feature of MSCs was analyzed with flow cytometer, the cell proliferation ability was determined by cell cycle assay and cell doubling time, the cell differentiation potential was evaluated by osteogenic and adipogenic induction in vitro as well.Results Both sources of MSCs were adherent cells and exhibited fusiform and fibrous morphology. Furthermore, both MSCs high expressed CD90 and CD105, and were negative for the markers of CD34, CD45 and HLA-DR.The population doubling time of MSCs form human placenta and umbilical cord was 39.5 h and 40.8 h separately, and the results of cell cycle analysis showed that the percent of the two sources of MSCs in G0/G1 phase was 52.12%and 57.50% respectively. The above results demonstrated that both sources of MSCs possessed the similar biological characteristics in morphology, phenotype and as well as proliferation ability.In addition, both of them could be induced into osteoblasts and adipocytes in vitro.Conclusion MSCs from human placenta have the similar biological characteristics to these from human umbilical cord, and both of them are better candidates for bench and clinical research.
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Objective To establish an aging model of mesenchymal stem cells (MSCs) and to investigate aging related biological mechanism for the purpose of studying the senesence of MSCs .Methods MSCs were separated and purified from human placenta, and the cells of the third passage(P3-MSCs) were cultured in the medium for 2 hours, then 100,200 and 300 μmol/L hydrogen peroxide ( H2 O2 ) was added to the cells for 2 hours to establish the MSCs aging model in vitro. Biological characteristics of aging MSCs were evaluated by cell cycle assay and senescence associated β-galactosidase staining.The expression of p16,p21 and p53 genes was further measured using quantitative real-time PCR (RT-PCR).Re-sults Compared with the control , the number of MSCs treated with 200μmol/L H2 O2 for 2 hours was significantly decreased and the cells displayed less adipogenic ,osteogenic and chondrogenic differentiation .Moreover ,after exposure to 200 μmol/L H2 O2 , the majority of the cells were in the G 0/G1 phase as showed by cell cycle analysis .The percentage of senescence-associated β-galactosidase-positive cells was increased , and the expression of p 16 , p21 and p53 mRNA and protein was significantly increased.Conclusion The results of this study has demonstrated that the H 2 O2 (200 μmol/L) can be used to establish the aging model of MSCs in vitro, and the cellular phenotypic alteration may attribute to the cell cycle associated gene expression (p16, p21, and p53).
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BACKGROUND:Human umbilical cord mesenchymal stem cells with capabilities for self-renewal and multi-differentiation have attracted widespread attention. OBJECTIVE:To develop an efficient method for isolation and culture of human umbilical cord mesenchymal stem cells, and to analyze the cellbiological features. METHODS:Mesenchymal stem cells were isolated and cultured from human umbilical cord by improved tissue cultivation. Immunophenotype and cellcycle were analyzed by flow cytometry. Growth curve was determined by MTT assay, and differentiation ability was evaluated by in vitro osteogenic and adipogenic induction as wel . RESULTS AND CONCLUSION:Some fusiform cells crawled out from human umbilical cord tissues after cultivation for 5 days and formed colonies about 10 days later. When the removed tissues were further cultured, more cells appeared again within 2 days and formed colonies after 5 days. The isolated cells exhibited similar morphology of fibroblast-like shape after passage. Furthermore, the cells expressed CD90, CD105, but were negative for the markers of CD34, CD45, HLA-DR. Population doubling time of the cells calculated from the result of MTT was about 50 hours and cellcycle analysis showed that 41.24%cells were in the G 2/S phrase. Therefore, the isolated cells had a high prolification ability. In addition, the isolated cells could be induced into osteoblasts and adipocytes in vitro. In a word, the results of this study demonstrated that the cells from the second tissues culture possessed the biological characteristics of mesenchymal stem cells and more primary umbilical cord mesenchymal stem cells were acquired through the improved method.
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To improve thrombolytic effect, a fusion protein SFH composed of staphylokinase (SAK) and hirudin (HV) with blood coagulation factor Xa (FXa) recognition peptide as a linker, was designed. SFH showed improved thrombolytic effect and low bleeding in vivo. Two thrombus-targeting mechanisms might account for the above features of SFH. This study was designed to study the two thrombus-targeting mechanisms of SFH. ELISA and immunohistochemistry assay were used to study the improved thrombus selectivity of SFH and the results showed that SFH, compared with SAK, displayed higher affinity for thrombin and thrombin-rich thrombus. To verify the thrombus-targeting release of anticoagulant activity of SFH, FH-a derivative of HV with only FXa recognition sequence at N terminus of HV was designed and used in animal tests. In inferior vena cava thrombosis model, FH showed equal antithrombotic effect as HV, indicating that HV could be successfully released from FH by FXa cleavage in vivo. More importantly, no prolongation of plasma TT, APTT and PT were found in FH group, but significant prolongations were discovered in HV group. This revealed that the anticoagulant activity of FH was released in thrombus-targeting way and limited in the vicinity of the thrombus, and this could be extrapolated to SFH. In conclusion, the high thrombus affinity and thrombus-targeting release of anticoagulant activity of SFH assigned low bleeding risk to SFH.
Subject(s)
Animals , Mice , Rats , Anticoagulants , Pharmacology , Factor X , Pharmacology , Hirudins , Genetics , Metalloendopeptidases , Genetics , Recombinant Fusion Proteins , Genetics , Pharmacology , Thrombolytic Therapy , Methods , Thrombosis , Drug Therapy , Vena Cava, InferiorABSTRACT
AIM:To prepare a compound as the chemical reference substance of Guangjinqiancao Zonghuangtong Capsule.METHODS:To apply general column chromatography combined with preparative HPLC to isolate the target compound,to use analytic HPLC to determine the purity,stability and its content in the capsule,and to employ spectroscopic analysis (UV,IR,ESI-MS,1H-NMR,13C-NMR,DEPT,1H-13CCOSY,1H-1HCOSY,1DHOHAHA.1D.NOE,HMBC) to elucidate the structure of the isolated compound.RESULTS:The obtained compound was identified as isoschaftoside with the purity of over 99%, which was stable within 3 months at ambient temperature.As for isosehaftoside solution.it was stable within 8 h at ambient temperature.Its content in the capsule was above 3.0%.CONCLUSION:Isoschaftoside is a qualified reference substance for analytic assay ofGuangjinqiancao Zonghuangtong Capsule,and can be isolated from Desmodium styracifolium(Osb.)Merr.
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To observe the expression of human wild-type p53, B7-1 and GM-CSF genes mediated by recom-binant adenovirus and their effects of inducing apoptosis on lung cancer cells. Methods; Human wild-type p53, B7-1 and GM-CSF genes were transfected into lung cancer cells mediated by recombinant adenovirus. The expression products of these genes were detected by immunohistochemistry assay, flow cytometric analysis and ELISA. Cell growth assay was carried out by counting alive cells after trypan blue exclusion. Cell apoptosis was detected by TdT assay of DNA fragmentation . Results: The multi-genes could be efficiently expressed in lung cancer cells mediated by recombinant adenovirus, which could suppress lung cancer cell growth and induce their apoptosis. Conclusion: These results suggest the feasibility of muti-gene therapy for lung cancer.
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AIM: To develop an RP-HPLC method for preparing the reference substanc of vicenin-2 in Desmodium styracifolium. METHODS: Ethanol-extract of desmodium styracifolium was isolated and purified by RP-HPLC combining solvent extraction with column chromatography and recrystalliztion.The purity and content of vicenin-2 were identified by HPLC. RESULTS: The flvonoids were completely separated under this chromatographic condition.The purity of the reference substance was 99.0% or above. CONCLUSION: The method is simple,accurate,better on repeatability,and effective to yield high-purity product.It can be used as reference substance for the research of herbal medicine.
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With techniques of biochemical isolation and pruification, a semi-purified preparation of new born calf liver (BLS) with the peculiarity of low molecular weight has been found to have tumor surppressor activity inhibiting selectively the growth of murine myeloid leukemic cell (L801). But its effect is less marked against normal bone marrow granulocyte / macrophage progenitors in in vitro liquid or agar culture. Mice after inoculation of L801 cells died within 15 days due to development of leukemia. After consecutive injections of BLS, 25% - 53.8% of treated mice survived. Furthermore, it has been found that the survival rate was raised from 50% to 66% when Cyclophosphamide was first injected in the dose of 300?g / kg body weight once, followed by the consecutive injections of BLS. NO pathological changes have been observed in the mice which survived longer than 100 days after inoculation of L801 leukemic cells.