RESUMO
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.
RESUMO
BACKGROUND:Placenta is a valuable source of mesenchymal stem cels for stem cel therapy and future application in the field of regenerative medicine. However, conventional methods cannot acquire a large amount of purified human placenta-derived mesenchymal stem cels. Here, we present a new method for isolating human placenta-derived mesenchymal stem cels suitable for banking strategies and for future clinical applications. OBJECTIVE:To analyze the biological characteristics of human placenta-derived mesenchymal stem cels cultured by tissue dissociating and colagenase digestion. METHODS: Human placenta-derived mesenchymal stem cels were obtained from human placenta by tissue dissociating and colagenase digestion method. Immunophenotype was analyzed by flow cytometry. Growth curve was determined by MTT assay, and differentiation ability was evaluated byin vitro adipogenic, osteogenic and chondrogenic induction as wel. RESULTS AND CONCLUSION:Human placenta-derived mesenchymal stem cels could be passaged stablyin vitro. Furthermore, the cels expressed CD73, CD90, CD105, but were negative for the markers of CD11b, CD19, CD34, CD45, and HLA-DR. Human placenta-derived mesenchymal stem cels proliferated actively and began to grow logarithmicaly at days 3-5 folowed by a plateau period at day 6. In addition, the isolated cels could be induced into adipocytes, osteocytes, chondrocytesin vitro. In a word, the results of this study demonstrated that the tissue dissociating and colagenase digestion method is an efficient method for obtaining a large amount of human placenta-derived mesenchymal stem cels that can be stably cultured in vitro and have strong proliferative ability.
RESUMO
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).
RESUMO
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.
RESUMO
BACKGROUND: Many diseases accompany with changes of gene expression which can provide a valuable clue for pathogenesis and therapy of cognitive diseases.OBJECTIVE: To screen expression of different genes in lung tissue of model rats with chronic obstructive pulmonary disease (COPD) by gene expression profiling technique. DESIGN: Open study. SETTING: Affiliated Hospital of Shandong University of Traditional Chinese Medicine; Radiation Medical Institute of Academy of Military Medical Sciences of Chinese PLA; Shangdong University of Traditional Chinese Medicine.MATERIALS: The experiment was carried out in the Central Laboratory of Affiliated Hospital, Shandong University of Traditional Chinese Medicine from April to October 2005. ① Fifteen healthy male Wistar rats of SPF grade and aged 50 days were randomly divided into normal group, model control group and Chinese herb group with 5 in each group. ② Type of gene chip was BiostarR-40S and provided by Shanghai Boxing Gene Chip Company Limited (batch number: G050510010052).METHODS: ① COPD models in model control group and Chinese herb group were established with modified smoking-fumigated method plus adding lipopolysaccharide in windpipe. Pathological changes of airway were observed under optic microscope. Models were established successfully based on the following phenomenon: hyperemia and edema in mucous membrane of airway; degeneration and necrosis of epithelial cells; infiltration of bronchial cavity in lung tissue and surrounding chronic inflammatory cells; proliferation of smooth muscle and fibrocyte surrounding dissepiment; thin and broken interval; amplifying confluence of pulmonary alveolus; thickness of vascular wall of arteriole; decrease of vaso-cavity; infiltration of surrounding inflammatory cells. ② At 30 days after modeling,0.65 g/mL self-made Chinese herb (mahuang, xingren, huangqi, etc.) wasperfused into rats in Chinese herb group with 2 mL each time, once a day,for successive 14 days. Rats in model control group were perfused with 2 mLsaline; however, rats in normal group were untouched. All rats drank freely under the same internal environment. ③ After administration, rats in each group were sacrificed to extract total RNA, label and hybridizated with probe, wash pieces, analyze gray value of clip with clip imaging software,obtain original signal of each gene point (signal values of foreground and background) and determine differently expressed genes. If size of test was more than 2.0, genes were regarded as up-regulation genes; otherwise, if ratio was less than 0.5, they were regarded as down-regulation genes. In this study, based on reliability, if size of test was more than 2.5, they were regarded as up-relation genes; however, if ratio was less than 0.375, they were regarded as down-regulation genes.MAIN OUTCOME MEASURES: ① Comparisons of differently expressed genes in normal and model control groups; ② Comparisons of differently expressed genes in normal and Chinese herb groups; ③Comparisons of differently expressed genes in model control and Chinese herb groups.RESULTS: Fifteen rats were all involved in the final analysis. ① As compared with those in normal group, there were 57 differently expressed genes in model control group, involving in immunity, metabolism, signal transduction, adjusting of gene expression, cell cycle, cell transport, cell migration, fibrosis, etc. ② Gene expression in Chinese herb group reached normal level and there were 11 differently expressed genes as compared with those in normal group, involving in stress, signal transduction, cytoskeleton, etc. ③ As compared with those in model control group, there were 7 differently expressed genes in Chinese herb group, involving in immunity and excretion of neurotransmitter.CONCLUSION: Changes of gene expression may be one of reasons of COPD pathogeneses; moreover, most differently expressed genes can recover the normal level after drug therapy which is beneficial to correct pathological changes of COPD.
RESUMO
Objective To explore the protective effect of HGF on primary cultured spinal neurons in vitro.Methods Cultured neurons were transfected with Ad-GFP or Ad-HGF in different multiplicity of infection(MOI),then flow cytometer and PI-Hoechst double stains were used to assay transfer rate or to determine the status of cells respectively.ELISA was used to detect the expression of HGF in Ad-HGF transfected neurons,and the activity of neurons was judged by neutral red stain,MTT and NSE-ELISA methods.Results After transfection with Ad-GFP in MOI of 50,the transfer rate was high as detected by flow cytometer,and the status of cells was well as judged by PI-Hoechst double stain.The results of ELISA showed that HGF was expressed in medium.After transfection with Ad-HGF,the activity of neurons was better than neurons without treatment(P