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BACKGROUND: By targeting inducing differentiation in vitro,mesenchymal.stem cells(MSCs) transform into osteoblasts,lipocytes,chondrocytes,muscular cells,neuronal cells,etc. Whether Chinese herbs act on induced differentiation of MSCs in rats or not?OBJECTIVE: To study the amplification of MSCs cultured in vitro in SD rats and efficacy of Chinese herbs on targeting inducing differentiation of neuron-like cells.DESIGN: Exploring study with repeated observation and measurement based on cells.SETTING: Department of pathophysiology in a medical college.MATERIALS: Experimental marrow collected from SD male tested-healthy rats.METHODS: By adhesion method,MSCs in rats were isolated for amplifying culture in vitro. Flow-type cell instrument was applied for the determination of its surface antigen expression. Various Chinese herbal components were used for the targeting inducing differentiation of MSCs into neuron-like cells. The cellular morphology was observed under optical microscope. and the specific antigen label of neuronal cells was determined with immunocyto-chemical method.MAIN OUTCOME MEASURES:①Results of MSCs isolation and amplification;②Results of identification of MSCs surface antigen and neuon-like cells.RESULTS: By adhesion method,MSCs in rats were isolated successfully and amplified in a large amount in vitro. It was indicated in the results determined by flow-type cell instrument that CD14,CD1 1α,CD34,CD38,CD45,CD80 and CD86 presented negative,and CD29,CD44,CD90,CD105 and CD166 presented positive. By induced with various kinds of Chinese herbs,like huangqi(Radix Astragali seu Hedysari),tianma(Rhizoma Gastrodiae),renshen (Radix Ginseng),danggui(Radix Angelicae Sinensis),naoxinshu,renshen fengwangjian for 1 to 3 hours,most MSCs transformed into neuron-like cells,presenting soma and neurite. With immunocyto-chemical staining,neuron-specific enolase(NSE) and nestin displayed positive and glial fibrillary acidic protein negative.CONCLUSION: MSCs in SD rats have the potential of multi-targeting differentiation,presenting a strong capacity of amplification and self-replacement. In a suitable inducing condition,MSCs may differentiate into neuron-like cells.
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Objective To investigate the main genotypes and the epidemic characteristics of TEM-type and SHV-type extended-spectrum beta-lactamases (ESBLs) of the gram-negative bacilli in Guangzhou.Methods The phenotype of ESBLs from 3 500 clinical isolates were primary screened and confirmed by the NCCLS confirmatory test according to guidelines of NCCLS(2001). PCR were performed on 3 500 clinical isolates using primers specific for blaTEM and blaSHV, respectively, the PCR product were purified and sequenced by ABI prism 377 DNA sequencer.Results Total of 3 500 un-replicated and consecutive Gram-negative bacilli were isolated from 13 hospitals in Guangzhou in the past two years, and the prevalence of ESBLs-producing clinical Gram-negative isolates was 31.0%(1 084/3 500). The positive rates of PCR results for blaTEM, blaSHV, and both of them in all the clinical Gram-negative isolates were 24.0%(840/3 500),10.8%(378/3 500),and 3.7%(128/3 500), respectively. All PCR products for blaTEM were further identified as TEM-1-type non-ESBLs gene, including TEM-1,TEM-1B,TEM-1D,and TEM-1F,by DNA sequencing analyses. However, almost all of the blaSHV gene were further identified as SHV-type ESBLs gene(the prevalence of SHV-1 is 7.2% only).The prevalence of SHV-12/5a accounted for highest(50.0%,152/304) in Guangzhou. Our test also showed that 53%(340/641) of blaTEM-producers [JP2] were Escherichia coli, and 57.9%(176/304) of blaSHV-producers were Klebsiella pneumoniae.Conclusions [JP]We could conclude from above results that SHV-type ESBLs, especial SHV-12/5a, was the prevalent genotype of ESBLs of clinical gram-negative bacilli in Guangzhou. TEM-type ESBLs did not exist in our city. In addition, SHV-12/5a-producing strains probably were main epidemic strains in Guangzhou. As for detection of ESBLs with regular phenotype methods, there was possibility of false negative and false positive.
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AIM: To investigate the differentiation from adult rat and human bone marrow mesenchymal stem cells (BMMSCs) into neuron with musk polypeptide (Mu-P).METHODS: Adult rat and human BMMSCs were induced with Mu-P.Neuron-specific enolase (NSE),neurofilament (NF),Nestin,glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry.RESULTS: Simple methods with Mu-P induced adult rat and human BMMSCs exhibiting a neuronal phenotype,expressing Nestin at 3 hours to 5 hours,and expressing NE and NF at 5 hours to 7 days.But the neuron-like cells didn't express the glial astrocyte marker GFAP.CONCLUSION: Adult rat and human BMMSCs can be induced to differentiate into neurons with Mu-P.
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AIM:To study the differentiation of rat marrow-derived mesenchymal stem cells(MSCs) into myoid cells in vitro . METHODS:The MSCs of SD rat were cultured?passaged?induced and differentiated in vitro used by routine culture technique, and evaluated by FACScan flow cytometer,detected by immunohistochemistry and analyzed by Hitachi H-600 transmission electron microscope(TEM). RESULTS:FACScan shows that cells expressed the antigens of CD29 and CD44, not those of CD11b and CD45; cells show positive response of staining with desmin and myoglobin after processing by two compounds of 5-azacytidine and amphotericin B. There were stripform zone of myofilament without any organells beside the edge of membrane in myoid cell of induction and differentiation checked by TEM. CONCLUSION: The passaged cells were MSCs and the MSCs may have specific gene structures that can differentiate into myoid cells. The demethylation or hypomethylation may conduct by compounds of 5-azacytidine and amphotericin B, which could be involved in activating phenotype-specific genes to differentiate MSCs into myoid cells. There are good outlook on clinical treatment of illness of myatrophy using by MSCs.
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AIM: To study the effects of cotransplantation of donor-derived bone marrow mesenchymal stem cells on graft versus host disease in a rat allogeneic bone marrow transplantation model. METHODS: Fisher 344 rat bone marrow MSCs were isolated and cultured to the fifth passage (P5) in vitro . The recipient Wistar rats were conditioned with lethal total body irradiation and transplanted with F344 rat bone marrow cells and spleen cells in the presence or absence of (P5) MSCs. The onset time of graft versus host disease (GVHD), incidence of GVHD and survival time were monitored. RESULTS: Cotransplantation of MSCs deferred the onset time of GVHD[(19.1?1.7) d vs (15.6?1.5) d, P
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AIM: To investigate the differentiation potential of human bone marrow mesenchymal stem cells (hBMMSCs) into hematopoietic cells in vivo. METHODS: hBMMSCs prepared from the bone marrow-aspirate sample obtained from healthy human donors were culture-expanded in vitro with 5-8 passages. hBMMSCs(P5-8, 4.8?10 5 cells/mouse) were injected into the severe combined immuodeficiency (SCID) mice treated by cyclophosphamide(CPA) and various tissues were analyzed at 35 days post-transplant for the presence of differentiated human cells. RESULTS: hBMMSCs(P5-8) viability, which was determined by typan blue staining at the end of the harvest and before infusion, was greater than 95% in every infusate at both time points. Cells characterized by flow cytometry using human MSC-specific monoclonal antibodies were uniformly positive for CD29, CD44, CD90, CD105, CD106, CD166 and negative for CD11a, CD14, CD34, CD38, CD45, CD80, CD86 which are common on cells of the hematopoietic lineages. Analysis of PB demonstrated that 5 of 6 hBMMSCs transplanted SCID mice had low level of circulating human CD45 +/ H-2D d- cells(range from 0.17% to 0.36%)and CD34 +/ H-2D d- cells(range from 0.10% to 0.50%). Analysis of BM for the presence of hematopoietic chimerism demonstrated human CD45 +/ H-2D d- cells and CD34 +/ H-2D d- cells in the marrow of 4 out of 6 hBMMSCs transplanted SCID mice (0.10%-0.19% and 0.03%-0.52%, respectively). Human hematopoietic cells with these same phenotype were also detected in the spleen 4 of the hBMMSCs transplanted SCID mice (range from 0.19% to 1.65% ,from 0.20% to 0.26%, respectively). No human hematopoietic cell was seen either in the PB, BM or spleen of all control animals. CONCLUSION: hBMMSCs have the ability to differentiate into blood cells of multiple lineages, including CD34 + hematopoietic stem cells/progenitor cells (HSC/HPC).
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AIM: To explore the potential of human bone marrow mesenchymal stem cells (hMSCs) differentiating into hematopoietic cells with murine fetal liver stromal cell-conditioned medium (FLSC-CM) in vitro. METHODS: 12.5-14.5 days post coitus (dpc ) of KM mice were used for the preparation of fetal liver stromal cell-conditioned medium (FLSC-CM) and embryonic fibroblast feeder layer (FD). Culture-expanded hMSCs were directly contacted with FLSC-CM, FD, and the combination of human interleukin-6 (IL-6) or stem cell factor (SCF), respectively. Seven days later, the non-adherent cells were collected and characterized by morphology, immunophenotypes, and colony forming unit-granulocyte/macrophage culture assay. RESULTS: The number of nonadherent cells derived from hMSCs cultured with FLSC-CM was increased remarkably than those with either FD or cytokines. The non-adhered cells with the morphology of monocyte-or small lymphocyte-like cells were positive for human CD34, CD45 and had the capacity to form the hematopoietic progenitor colonies in methylcellulose cultures containing recombined human granulocyte/macrophage-colony stimulating factor (rhGM-CSF). CONCLUSION: hMSCs were successfully induced toward their differentiation into CD34+CD45+ hematopoietic progenitors after being cultivated with FLSC-CM. This study suggests that hMSCs have the hematopoietic differentiation potential in vitro. [
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AIM: To investigate the basic biological characteristics of adult rat bone marrow mesenchymal stem cells(rBMMSCs), and compare to that of human BMMSCs (hBMMSCs). METHODS: rBMMSC and hBMMSCs were separated from bone marrow with the difference of adherence and Ficoll-Paque reagent, and expanded in culture medium in vitro, respectively. The proliferation and growth characteristics of the primary and different passage culture of rBMMSCs and hBMMSCs were analysed. The neural differentiation capacity of rBMMSCs with passages were observed. To detect the surface antigens of rBMMSCs, the labeled cells were analysed on a FACScan flow cytometer. The karyotype of rBMMSCs were detected by blocking cellular fission with colchicines. RESULTS: rBMMSCs and hBMMSCs have a strong self-renewal capacity. Approximately (4-8)?10~(12) and (3-4)?10~(12) cells were obtained after passage 15 in vitro, respectively. The ability of proliferation, CFU-Fs, and neural differentiation of rBMMSCs and hBMMSCs were decreased gradually with passages, but the ability of proliferation and CFU-Fs of rBMMSCs were higher than that of hBMMSCs at different passage. FACScan result showed rBMMSCs were uniformly positive for CD29 and CD44, and negative for CD11b, CD45, CD61, CD71, CD80, CD86,MHC Ⅰ and MHC Ⅱ. rBMMSCs had an normal karyotype, which had an average of 37.0?4.0 to 40.5?2.5 chromosomes. CONCLUSION: Adult rBMMSCs have strong self-renewal and neural differentiation capacity, and have an normal karyotype. So rBMMSCs can be used as the seed cells for tissue engineering.