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BACKGROUND: Studies have shown that umbilical cord mesenchymal stem cells are ideal seed cells for tissueengineering research.OBJECTIVE: To isolate, culture and identify tree shrew umbilical cord mesenchymal stem cells, in order toestablish a standardized tree shrew umbilical cord mesenchymal stem cell lines.METHODS: Caesarean-isolated tree shrew umbilical cord samples were used to isolate and culture umbilical cordmesenchymal stem cells using tissue explant adherent method. Flow cytometry assay was used to detect cellsurface markers. Osteogenic and adipogenic induction media were used to induce umbilical cord mesenchymalstem cells to differentiate into osteoblasts and adipocytes.RESULTS AND CONCLUSION: The cultured umbilical cord mesenchymal stem cells expressed CD90 and CD105 with the positive rate of 99.9% and 99.8% respectively. Hematopoietic stem cell marker CD34 expression ratewas 0.0% and the endothelial cell marker CD31 expression rate was 0.7%, in line with the characteristics of umbilicalcord mesenchymal stem cell surface markers. Calcium nodules by alizarin red staining and lipid droplets by oil red Ostaining were observed in the induced cells. These experimental findings indicated that umbilical cord mesenchymalstem cells from tree shrews capable of osteogenic and adipogenic differentiation were successfully isolated and cultured.
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BACKGROUND:If an extract can prolong the S phase and reduce the percentage of apoptosis after co-culture with cels, it is proved that the extract is able to promote cel proliferation. OBJECTIVE: To prove the effects of chicken egg-white extracts with different components on the proliferation, cel cycle and apoptosis of 293T cels. METHODS: An ultrafiltration unit was used to separate chicken egg-white extracts into different components that were > 10 ku, 3 ku and < 3 ku to co-culture with cels for 3 days. Then, cel proliferation, cel cycle and cel apoptosis were detected. RESULTS AND CONCLUSION:Chicken egg-white extract components of < 10 ku and < 3 ku could promote cel proliferation, increase the percentage of S-phase cels and reduce the percentage of apoptosis. In conclusion, chicken egg-white extract components that are < 10 ku and < 3 ku can promote cel proliferation, as wel as increase the percentage of S-phase cels and reduce apoptosis percentage.
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Objective To isolate, culture and identify mouse umbilical cord mesenchymal stem cells( mUCMSCs) and to study whether they can be induced to differentiate into adipocytes, chondrocytes and osteoblasts.Methods The mUCMSCs were isolated and expanded by adherent culture from fresh mouse umbilical cord.The morphological characteris-tics of the resulting cells were observed under inverted phase contrast microscope, and their expression of mesenchymal sur-face markers was identified and analyzed by flow cytometry.Then mUCMSCs were induced to differentiate into chondro-cytes, adipocytes and osteoblasts in vitro.Results Fibroblast-like cells could be isolated from the fresh umbilical cord by adherent culture.These adherent cells highly expressed mesenchymal markers including CD29, CD90 and CD105 while low expression of CD34.The cells were successfully induced to differentiate into chondrocytes, adipocytes and osteoblasts. Conclusions The mUCMSCs isolated from fresh mouse umbilical cord by adherent culture have potential of differentiation into chondrocytes, adipocytes and osteoblasts.
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Objective To establish a method for isolation of cynomolgus monkey umbilical cord mesenchymal stem cells.Methods Fresh cynomolgus monkey umbilical cord was directly minced into pasty fine pieces, and the pieces were cultured in tissue flask with DMEM/F12 medium supplemented with 10% fetal bovine serum.The morphological characteristics of the resulting cells were examined, and their expression of mesenchymal cell surface markers were analyzed by flow cytometry.The multidifferentiation potential was examined in vitro, too.Results The fibroblast-like cells were successfully isolated from the fresh umbilical cord by an adherent culture procedure.These adherent cells expressed mesenchymal markers including CD29, CD44, and CD90, and also could be induced to differentiate into adipocytes, osteoblasts and chondrocytes.Conclusion Mesenchymal stem cells can be isolated from fresh cynomolgus monkey umbilical cord by using an adherent culture procedure.
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BACKGROUND:Systemic lupus erythematosus is an autoimmune disease characterized as an emergence of a variety of autoantibodies in serum and multi-system and multi-organ lesions. Currently, there is a lack of effective treatment options, and umbilical cord mesenchymal stem cel s are a promising therapy for systemic lupus erythematosus based on cel biological roles. OBJECTIVE:To observe the therapeutic efficacy of human umbilical cord mesenchymal stem cel transplantation in the treatment of systemic lupus erythematosus in mice. METHODS:Human umbilical cord mesenchymal stem cel s were isolated and cultured fol owed by labeling with DiR fluorescence. Experimental mice were divided into normal control group (C57BL mice), model control group (C57BL/lpr mice), low-, medium-and high-dose umbilical cord mesenchymal stem cel therapy groups (C57BL/lpr mice), with 10 mice in each group. Mice in the low-, medium-and high-dose groups were respectively injected 0.5×106, 1×106, 2×106 human umbilical cord mesenchymal stem cel s, once a week, for 3 consecutive weeks. At the end of treatment, blood samples were col ected to measure antinuclear antibody, anti-histone antibody, anti-double stranded DNA antibody changes;OPG and Foxp3 gene expression changes were detected by quantitative PCR method. RESULTS AND CONCLUSION:After treatment, the levels of anti-nuclear antibodies, anti-histone antibodies and anti-double stranded DNA antibodies in the peripheral blood of mice were al declined in the low-, medium-and high-dose groups, while the number of peripheral blood CD4+CD25+T cel s was significantly elevated. OPG and Foxp3 gene expression was also increased dramatical y in the low-, medium-and high-dose groups, which was similar to that in the normal control group and significantly different from that in the model control group (P<0.01). Experimental findings demonstrate that after transplantation of human umbilical cord mesenchymal stem cel s, al relevant indicators in C57BL/lpr mice recovered to the normal levels, and the high-dose treatment group had the most obvious effect.
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BACKGROUND:Umbilical cord as childbirth waste has wide variety of sources and can be easily obtained, without any ethical and legal restrictions. Therefore, human umbilical cord mesenchymal stem cells break al the restrictions originated from other sources of mesenchymal stem cells. OBJECTIVE:To review the application of human umbilical cord mesenchymal stem cells in cartilage diseases, neuroglioma, ischemic brain injury, lung disease, liver disease and models of myocardial infarction. METHODS:The PubMed and Wanfang databases were searched by the first author using the keywords of“human umbilical cord mesenchymal stem cells, disease models, celltherapy”in English and Chinese, respectively. Seventy-three articles were searched and final y, 35 articles were included in result analysis. RESULTS AND CONCLUSION:Human umbilical cord mesenchymal stem cells have multilineage differentiation capacity similar to bone marrow mesenchymal stem cells. Compared with bone marrow mesenchymal stem cells, human umbilical cord mesenchymal stem cells have lower immunogenicity. Human umbilical cord mesenchymal stem cells show certain curative effects on cartilage disease, neuroglioma, ischemic brain injury, lung disease, liver disease and myocardial infarction, indicating that human umbilical cord mesenchymal stem cells can be used for celltransplantation to treat various diseases.
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Objective To establish a method of identification of DKO mouse model of Duchenne muscular dystro-phy, and to assess the dystrophin regeneration after stem cell transplantation.Methods Heterozygous mice were mated and the resulting offspring were used to identify their genotype by SSP-PCR.The plasma creatine kinase level was measured by biochemical analyzer and histological changes in the DKO mice were analyzed using HE staining.Human umbilical cord mesenchymal stem cells were prepared and injected into the DKO mice hindlimb muscle, and dystrophin expression was de-tected by immunofluorescence staining at 2 months after injection.Results Mating of heterozygous mice generated three kinds of genotype offsprings, and 21.2%of the offsprings were identified as DKO genotype (285 bp) .DKO mice showed dystrophic symptoms, their plasma creatine kinase level was as high as 16988.52 ±617.48 IU/L, and significant histologi-cal changes including diverse myocyte sizes, numerous centrally nucleated cells and connective tissue proliferation or in-flammatory cells infiltration.Human dystrophin expression was detected in the DKO mouse hindlimb muscle at two months after injection of human umbilical cord mesenchymal stem cells.Conclusion DKO mouse genotype can be identified by SSP-PCR, and DKO mouse is an ideal animal model for studies of stem cell therapy for Duchenne muscular dystrophy.
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BACKGROUND:Reprogramming somatic cells to generate pluripotent stem cells has a wide application in biomedical research. OBJECTIVE:To analyze the effect of different molecular weight proteins in chicken egg-white extract to elevate expression of pluripotent genes Oct-3/4 and Nanog in 293T cells. METHODS:The extracts of chicken egg-white were separated into more than 3 ku and less than 3 ku ingredients to be used for co-culture with 293T cells. There were four groups, 1×105 293T cells per wel , total 500μL. In the control group, 500μL culture medium was added;in the other three groups, 500μL chicken egg-white extract, more than 3 ku and less than 3 ku ingredients were respectively added. Quantitative PCR was used to determine the relative expression levels of pluripotent genes Nanog and Oct-3/4 in 293T cells. RESULTS AND CONCLUSION:By using co-culture method, more than 3 ku ingredients have a role to increase the expression of pluripotent genes Oct-3/4 and Nanog, but less than 3 ku ingredients cannot elevate the expression of pluripotent genes. This indicates that the ingredient of chicken egg-white extract to elevate the expression of pluripotent genes is more than 3 ku proteins.
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The culture of umbilical cord-derived mesenchymal stem cells is extremely important for studies on umbilical cord mesenchymal stem cells. Optimization of cellculture technology is crucial for clinical application of mesenchymal stem cells and even celltherapy. Meanwhile, the labeling and tracer technique of umbilical cord-derived mesenchymal stem cells is a hotspot in stem celltransplantation. OBJECTIVE:To review the research and development of the cellmarkers and tracer methods of umbilical cord-derived mesenchymal stem cells. METHODS:A computer-based search of VIP, CNKI, Medline, Highwire and Foreign Journals Integration System databases was performed for articles concerning culture and labeling of umbilical cord-derived mesenchymal stem cells published from January 2001 to October 2013. The keywords were“stem cells, mesenchymal stem cells, umbilical cord-derived mesenchymalstem cells, cellculture, labeling methods”in Chinese and English, respectively. Final y, 35 articles were included in result analysis. RESULTS AND CONCLUSION:Umbilical cord-derived mesenchymal stem cells have not yet been widely used, mainly because of the immature isolation, culture and staining techniques of umbilical cord-derived mesenchymal stem cells. These techniques are worthy of further optimization studies. Although in recent years, cellmarkers and tracer technology of umbilical cord-derived mesenchymal stem cell s have made great progress, there are stil many problems need to be solved.
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BACKGROUND:There are less studies addressing whether bone marrow mesenchymal stem cells from systemic lupus erythematosus patients are different from healthy people. OBJECTIVE:To compare the multi-differentiation capacity of bone marrow mesenchymal stem cells isolated from systemic lupus erythematosus model mice and normal control mice. METHODS:Bone marrow mesenchymal stem cells of systemic lupus erythematosus model mice and C57BL mice were isolated and cultured fol owed by osteogenic and adipogenic differentiations, respectively. Differentiation abilities of two kinds of mouse bone marrow mesenchymal stem cells were observed. RESULTS AND CONCLUSION:Passaged bone marrow mesenchymal stem cells from C57BL mice were long spindle-shaped and evenly distributed, while bone marrow mesenchymal stem cells from systemic lupus erythematosus model mice showed slow growth and were relatively smal er than those from C57BL mice. After osteogenic induction, the amount of calcium salt and calcium nodules were significantly less in the bone marrow mesenchymal stem cells from systemic lupus erythematosus model mice than from normal control mice. PCR detection showed that expressions of Runx2, alkaline phosphatase and osteocalcin were also significantly decreased in the bone marrow mesenchymal stem cells from systemic lupus erythematosus model mice. After adipogenic induction, the number of lipid droplets in the bone marrow mesenchymal stem cells from systemic lupus erythematosus model mice was significantly less than the control group, and PCR detection also showed significantly decreased expression of adipogenic genes, including PPARγ2 and lipoprotein lipase. These findings suggest that the bone marrow mesenchymal stem cells from systemic lupus erythematosus model mice exhibit lower osteogenic and adipogenic capacities than those from normal C57BL mice, and also have the impaired multi-differentiation ability.
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BACKGROUND:Metabolic syndrome is based on sugar, fat and other metabolic disorders and central obesity, hypertension as features in a series of syndrome. The traditional treatment is not yet possible to fundamental y improve and cure metabolic syndrome. OBJECTIVE:To provide an overview of the research progress of stem celltransplantation in the treatment of metabolic syndrome. METHODS:The first author retrieved PubMed database and CNKI database for articles regarding basic research on progress of stem celltransplantation in the treatment of metabolic syndrome, insulin resistance, diabetes, hyperlipidemia and hypertension published from 2002 to 2012. The key words were“stem cells, metabolic syndrome, insulin resistance, diabetes, hyperlipidemia, hypertension, stem cells transplantation”in English and Chinese, respectively. Outdated and repetitive studies were excluded, and 43 literatures were included for summarization. RESULTS AND CONCLUSION:Stem cells are the origin of the body cells, and have self-replicating, highly value-added and differentiation capacity. Stem celltherapy can promote a variety of damage repair and renew aging or death of cells, so as to improve the structure and function of tissues and organs, and to promote the utilization and excretion of metabolites. Studies have shown that stem cells can treat lipid metabolism, insulin resistance, hypertension, hyperglycemia, atherosclerosis and other hazards of metabolic syndrome disorders through a variety of mechanisms. There are many problems to be solved in the treatment of metabolic syndrome with stem celltransplantation. But the existing research data have been confirmed, and stem celltransplantation in the treatment of the metabolic syndrome is a promising new approach.
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BACKGROUND: The inoculation of hybridoma cell strain onto mouse abdominal cavity may obtain ascites containing mass antibody. Previous method to purify monoclonal antibody in ascites is complex and difficult to operate.OBJECTIVE: To prepare, purify and label anti-human leukocyte antigen (HLA)-I class molecule light chain monoclonal antibody, and to detect the expression of tumor cell surface HLA-I class molecules. METHODS: Hybridoma cells were inoculated onto the mouse abdominal cavity. Ascites containing anti-human light chain beta2-microglobulin antibody were obtained and purified with the modified caprylic acid-ammonium sulfate method. The purified monoclonal antibody was labeled with fluorescein isothiocyanate to detect peripheral blood mononuclear cells, T2 cells expressing blank HLA-A2 molecule and K562 cells surface HLA-I class molecules. The expression of HLA-I class molecules was determined by using flow cytometry and fluorescent microscopy. RESULTS AND CONCLUSION: The purified anti-human light chain beta2-microglobulin-fluorescein isothiocyanate monoclonal antibodies accounted for 96% purity. Flow cytometry results showed that, the HLA-I class molecules were highly expressed in peripheral blood mononuclear cells surface, lowly expressed in T2 cells, and not expressed in K562 cells surface. It is a simple and convenient method to purify ascites with the modified caprylic acid-ammonium sulfate method, and according prepare anti-human light chain beta2-microglobulin-fluorescein isothiocyanate. This method is effective to distinguish the levels of HLA-I class molecules expressed in various cells.
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BACKGROUND: Antiviral drugs can reduce the incidence of early-onset cytomegalovirus(CMV)disease,but are associated with strong toxicity and the development of late-onset CMV disease.In order to prevent CMV disease better,cytotoxic T lymphocytes(CTL)may play a critical role in controlling CMV reactivation.Fluorescent HLA-peptide tetramers are used to monitor the recovery of CMV CTL in recipients of allogeneic transplants.OBJECTIVE: To explore the effect of HLA-peptide tetramers and adoptive immunotherapy in treating CMV disease.METHODS: A computer-based online search of Pubmed and Wanfang databases was performed for articles related to CTL detection,application of antiviral drugs and HLA-peptide tetramers,and adoptive immunotherapy with key words"HLA-peptide tetramers,cytomegalovirus,specific CTL,adoptive immunotherapy"in English and Chinese.Repetitive articles were excluded and 29 articles were included.RESULTS AND CONCLUSION: Adoptive immunotherapy with CMVs cytotoxic T cells as preemptive therapy is a very elegant strategy; however,generation of these cells is costly and time-consuming,and therefore the therapy is not available at every transplantation center.Magnetic selection of CMVs CD8+T cells from peripheral blood of CMV-seropositive donors by using HLA-peptide tetramers may be very hopeful,which simplifies adoptive immunotherapy.
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<p><b>BACKGROUND</b>To investigate the effects of cisplatin on proliferation, telomerase activity, cell cycle, p53, bcl-2 and proliferating cell nuclear antigen (PCNA) expressions of human lung adenocarcinoma SLC-89 cells induced by cisplatin and to find out the possible mechanisms.</p><p><b>METHODS</b>SLC-89 cells were treated with cisplatin of different concentrations for 72 h. Then, the proliferation of the cells was measured by MTT method, telomerase activity was measured by telomeric repeat amplification protocol with ELISA (TRAP-ELISA), and cell cycle, p53, bcl-2 and PCNA expressions of the cells were detected by flow cytometry (FCM) respectively.</p><p><b>RESULTS</b>Cisplatin could obviously inhibit the proliferation of the cells, and IC₅₀ value for cisplatin treatment was 18.47 mg/L. Cisplatin could obviously down-regulate telomerase activity, decrease S phase cells, increase G₀/G₁ phase cells, decline the expressions of bcl-2 and PCNA proteins and induce the expression of p53 protein of SLC-89 cells in a concentration-dependent fashion.</p><p><b>CONCLUSIONS</b>Cisplatin can obviously inhibit the proliferation of SLC-89, change the distribution of cell cycle, decline telomerase activity and expressions of bcl-2 and PCNA proteins, and induce expression of p53 protein, which may be the important mechanisms of cisplatin's anticancer action.</p>
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<p><b>OBJECTIVE</b>To investigate the effect of luteolin on the proliferation and collagen expression of hepatic stellate cells.</p><p><b>METHODS</b>The effect of luteolin on proliferation and collagen synthesis of hepatic stellate cells isolated from the liver of Wistar rats were determined by (3)H-TdR and (3)H-Pro, and procollagen gene expression was also detected by DIG-labeled gene probe and in situ hybridization.</p><p><b>RESULTS</b>The proliferation and collagen synthesis were significantly and dose-dependently inhibited by luteolin when the concentrations reached 10 micromol/L and 20 micromol/L respectively (t=2.542, P<0.05; t=3.650, P<0.01). The type I, III procollagen mRNA expression was decreased by 25 micromol/L luteolin, in which the type I procollagen mRNA was reduced with statistical significance (x(2)=6.850, P<0.01).</p><p><b>CONCLUSIONS</b>Luteolin inhibits the proliferation and collagen expression of hepatic stellate cells in vitro. It may have a preventive or therapeutic role in liver fibrosis.</p>
Subject(s)
Animals , Rats , Cell Division , Cells, Cultured , Collagen , Expectorants , Pharmacology , Flavonoids , Pharmacology , Liver , Cell Biology , Luteolin , Protein Synthesis Inhibitors , Pharmacology , Rats, WistarABSTRACT
Objective To investigate effect on telomerase activity of Hep-2 cells treated by anticancer drugs(hydroxycamptothecine, cisplatin and cytoxan).Methods By MTT method,we measured the 50% inhibitory concentration(IC 50 ) at 72h,and compared to untreated control cells. Telomerase activity of Hep-2 cells treated by the drugs in different concentration based on IC 50 for different time was observed by Telomeric Repeat Amplification Protocol with ELISA(TRAP-ELISA).Results Hydroxycamptothecine and cytoxan could inhibit proliferation of Hep-2 and down-regulate telomerase activity of Hep-2 cell. However, cisplatin promoted proliferation of Hep-2 and up-regulated telomerase activity of Hep-2 cell.Conclusions Hydroxycamptothecine and cytoxan could down-regulate telomerase activity of Hep-2 cell by direct or indirect pattern, which may correlate with drug concentration and time-dependent pattern.Cisplatin could up-regulate telomerase activity of Hep-2 cell, which mechanism is not clear.
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Objective To investigate effect on telomerase activity, apoptosis and p53/bcl-2 gene expression of MCF-7 cells line induced by paclitaxel.Methods By techniques of cell culture in vitro, telomeric repeat amplification protocol with ELISA(TRAP-ELISA) and flow-cytometry (FCM), MCF-7 cell line was treated by paclitaxel in various concentration for 72h.Results Paclitaxel down-regulated telomerase activity of MCF-7 cell, induced apoptosis of the cell in a concentration-dependent manner, significantly decreased expression of bcl-2 gene protein and increased expression of p53 gene protein. There was a positive correlation between telomerase activity and apoptosis and the expression of p53/bcl-2 gene protein.ConclusionPaclitaxel could down-regulate telomerase activity,induce apoptosis, decrease expression of bcl-2 gene protein and increase expression of p53 gene protein, which may be one of important mechanisms of Paclitaxel′s anticancer action.
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Objective To study the alteration of telomerase activity of MCF 7 cell line of breast cancer in the presence of different anticancer drugs. Methods The hyperplasia and viability of MCF 7 cell were detected by cell counting and trypan blue exclusion, and the telomerase activity was measured by TRAP.The alteration of MCF 7 cell and its related factors of telomerase activity were observed on cell growing in different condition. Results In abscence of drug, there was a positive correlation between hyperplasia and telomerase activity of the cell(r= 0.901 ). Adriamycin, paclitaxel and cisplatin could obviously inhibit the growth and reduce the telomerase activity of the cell in a dose-dependent and time-dependent fashion, and this reduction in telomerase activity closely correlated with the reduction in the number of viable cells. Conclusions Adriamycin, paclitaxel and cisplatin can inhibit the growth of MCF 7 cell, which may be correlated with the reduction in telomerase activity and cell viability.