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BACKGROUND:Recently, the effects of human umbilical cord mesenchymal stem cel s (hUCMSCs) and placenta-derived mesenchymal stem cel s (PDMSCs) on treatment of acute graft versus host disease (aGVHD) have been confirmed in some in vitro studies or animal models. But there are stil no reports comparing the therapeutic effects of these two cel types. OBJECTIVE:To compare the immunosuppressive function of hUCMSCs and PDMSCs in vitro or in a mouse aGVHD model. METHODS:(1) In vitro experiment. Human peripheral blood mononuclear cel s (PBMCs) were isolated and divided into four groups:PBMCs cultured alone, PBMCs stimulated with phytohaemagglutinin (PHA), PHA stimulated-PBMCs cocultured with hUCMSCs, PHA stimulated-PBMCs cocultured with PDMSCs. After 5 days, PBMCs proliferation and interferon-γlevel in cel supernatant were measured. (2) In vivo experiment. Fifty-seven BABL/C(H-2d) mice exposed to 8.5 Gy irradiation were randomly divided into five groups:only saline injection group, syngeneic bone marrow transplantation group, al ogeneic bone marrow transplantation group, aGVHD group, hUCMSCs treatment group, PDMSCs treatment group. The clinical aGVHD score, histopathology of skin, liver, and smal intestine, and survival time were analyzed at days 11, 14, 21 after transplantation. RESULTS AND CONCLUSION:(1) In vitro test:compared with the hUCMSCs, PDMSCs had stronger anti-inflammatory function. (2) In vivo test:The clinical scores on acute graft versus host disease were significantly lower in the hUCMSCs and PDMSCs treatment groups than that in the aGVHD group (P<0.05). The survival rates of mice were significantly increased in the hUCMSCs and PDMSCs treatment groups compared to the aGVHD group (P<0.05). Evident skin lesions were not found in al groups. Although smal intestine mucosal lesions were found in al groups, the damage level seemed similar. Notably, significant difference was found in the liver that multifocal necrosis and a large number of inflammatory cel s were seen in the aGVHD group, but less necrosis and inflammatory cel s in the hUCMSC and PDMSC treatment groups. In conclusion, hUCMSC and PDMSC are comparably effective in the treatment of aGVHD in mice.
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BACKGROUND: Mesenchymal stem cells (MSCs) are an important component of the in vivo microenvironment and act on multiple biological behaviors of tumor cells. The potential clinical value of MSCs has become an issue of concern in recent years.OBJECTIVE: To investigate the gene expression profiles of acute promyelocytic leukemia (APL) cell line NB4 treated with umbilical cord-derived MSCs (UC-MSCs) using cDNA microarray.METHODS: In vitro co-culture system was constructed, and then cellular proliferation, apoptosis and differentiation status of NB4 cells treated with UC-MSCs were evaluated. Two cDNA probes were prepared through reverse transcription from mRNA of NB4 cells treated with or without UC-MSCs. The probes were labeled with fluorescence dyes individually, hybridized with cDNA microarray, and their fluorescent intensities were scanned. The genes were screened through the analysis of the difference in two gene expression profiles.RESULTS AND CONCLUSION: UC-MSCs promoted the proliferation and differentiation, while reduced the apoptosis of NB4 cells. The analysis of gene expression profiles indicated that after co-culture with UC-MSCs, 530 genes were up-regulated and 53 genes were down-regulated. Accordingly, specific gene function and pathway signaling related were also regulated to some extent. Overall, UC-MSCs influence can major biological behaviors of NB4 cells by changing expression of a large amount of genes, gene-related function and multiple intracellular signaling pathways.
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BACKGROUND: Imatinib has a significant pro-apoptosis effect on chronic myelogenous leukemia (CML), but there are still some patients being resistant to it. Human umbilical cord mesenchymal stem cells (hUC-MSCs) affect the apoptosis of a variety of hematologic malignancies. However, the impacts of hUC-MSCs on the apoptosis of CML cells induced by imatinib remain unclear.OBJECTIVE: To investigate whether hUC-MSCs have an influence on the apoptosis of K562 cells induced by imatinib and to reveal the possible underlying mechanism.METHODS: K562 cells were cultured with hUC-MSCs or/and imatinib. Cellular apoptosis was measured with Annexin-V and PI staining by flow cytometry analysis. The protein expressions of Bax, Bcl-2, caspase-3, caspase-9 and cleaved-PARP in K562 cells were detected by western blot assay. Pan-caspase inhibitor Z-VAD-FMK was used to block apoptosis in each group, and during this process the effect of caspase apoptosis signaling pathway was detected.RESULTS AND CONCLUSION: The apoptosis of K562 cells was enhanced, when imatinib was combined with hUC-MSCs. Western blot analysis showed that the expression of pro-apoptotic protein Bax was enhenced and the expression of anti-apoptotic protein Bcl-2 was suppressed. Furthermore, the cleaved forms of caspase-9, caspase-3 and PARP in K562 cell were higher in the hUC-MSCs+imatinib group than in the imatinib group. The apoptosis of K562 cells induced by the hUC-MSCs combined with imatinib was significantly inhibited by Z-VAD-FMK. In conclusion, these findings indicate that hUC-MSCs can enhance imatinib-induced apoptosis of K562 cells by activating caspase apoptosis signaling pathway.
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BACKGROUND:There are various methodstoinduceadipogenic differentiation ofbone marrow mesenchymal stem cels, and the main componentfor adipogenic induction isindomethacin or rosiglitazone. However, there is a lack of comparative studyonthe induction efficiency and mechanism among these methods. OBJECTIVE:Tocompare the adipogenic responses ofhuman bone marrow mesenchymal stem celsto different induction methods, and to analyze the mechanismunderlyingdifferent induction efficiency. METHODS:After isolation and purification,the adipogenic abilitiesof human bone marrow mesenchymal stem cels in threedifferentculture systemswere comparedby oil red O staining and lipogenic geneassay. At 0, 1, 3 and 7 days of adipogenensis, mRNA expressionsof PPARγ, C/EBPα, Adiponectin and Leptin were detected.At7 daysofadipogenensis, protein expressionsof PPARγ and C/EBPβ were detectedby western blot assay,andeffects ofDIMIversusDIMRonphosphorylationofPPARγatSer273were compared. RESULTS AND CONCLUSION:Findings from oil red O staining andreal-time PCRshowedthat DIMR significantlyinducedadipogenicdifferentiation of bonemarrow mesenchymal stem cels compared with DIM and DIMI at 7 daysofinduction. Western blot showed thattheprotein expressionsof PPARγ and C/EBPβ in the DIMIgroupwere significantly higher than those in the DIMRand DIM at 7days ofinduction. In addition, the ratio ofPPARγphosphorylation atSer273was lowerin the DIMR group thantheDIMI group.To conclude,DIMR has the most potential to induce early adipogenesis ofhumanbone marrow mesenchymal stem cels by weakening the phosphorylationof PPARγ-Ser273.
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BACKGROUND:There are abundant cel populations in the placenta that attracts more and more attentions because of high content of CD34+cel s. It is expected to become a new source of hematopoietic stem cel s for the treatment of hematologic diseases and other malignant diseases. OBJECTIVE:To investigate the amount of cel s derived from placenta, their colony forming ability, and their chimerism analysis. METHODS:Five placentas obtained from five healthy ful-term cesarean women were treated with perfusion method and tissue digestion for the cel col ection. Flow cytometry was used to detect the proportion of CD34+cel s in the placenta and cord blood, fol owed by the culture of cel colonies as wel as regular observation of cel morphology and counting. PCR amplification with sequence-specific primers and sequence-specific oligonucleotide probes were used to examine HLA type of placenta, umbilical cord blood, and maternal peripheral blood;Short tandem repeat PCR was used for chimerism analysis. RESULTS AND CONCLUSION:There were more CD34+cel s in the placenta than in the umbilical cord blood. The placenta had good ability to form multiple colonies in vitro, and there were maternal source components in the placenta. It is concluded that the amount of cel s in the placenta and their biological functions exhibit the potential use of placenta as a new source of hematopoietic stem cel s.
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ObjectiveToll-like receptors (TLRs) play important role in the progression and tumor immunity of some types of cancer,some research have demonstrated that agonist of TLR3 can trigger apoptosis of cancers.This study was proposed to investigate if Poly(I:C),the specific agonist of TLR3,could impact proliferation or apoptosis of progressive breast cancer cells MDA-MB-231,and to investigate the primary mechanism of the function.MethodsExpression of TLR1-10 mRNA was detected by quantitative real-time reverse transcription-polymerase chain reaction.Cell Counting Kit-8 was used to determine the inhibitory effect of Poly(I:C) on proliferation of MDA-MB-231 cells.Cell apoptosis was assayed by flow cytometry with V-FITC/PI staining.Results First,the toll-like receptors 1-10 were all expressed on MDA-MB-231 cells,while the expression level of TLR8 was lower than that of others.Second,according to the CCK-8,the proliferation of MDA-MB-231 cells was inhibited,but the apoptosis was not affected on the basis of Apoptosis Kit.At last,the mRNA expression of TNF-α、IFN-β and IFN-γ were elevated approximately 20 times after Poly(I:C) stimulation for 6 hours.ConclusionMDA-MB-231 cells express all toll-like receptors on mRNA level,and TLR8 was expressed lower than others.The stimulation of TLR3 with Poly(I:C) can inhibit the proliferation of MDA-MB-231,but had no effect on apoptosis.TNF-α、IFN-β and IFN-γ maybe participate in this process.
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Objective To investigate the influences of imatinib on Survivin gene expression in bcr-abl-transformed leukemia cells.Methods Firstly,PCR and Western blot were carried out to detected Survivin expression with imatinib treatment in 32Dcl3 and 32D-bcr-abl cell lines.Then the luciferase reporter plasmids containing human Survivin promoter as well as its deletion and site-directed mutation were constructed to identify the essential responsive elements for suppressing Survivin promoter activity by imatinib.Chromatin immunoprecipitation was performed to confirm the binding of c-myc to Survivin promoter.10058-F4,a small molecule c-myc inhibitor,was used to disrupt c-myc activity and evaluate its anti-leukemic effect combined with imatinib.Results Both of mRNA and protein level of Survivin in bcr-abl-transformed cells were downregulated upon imatinib treatment.The decrease of Survivin expression was controlled at the transcriptional level through a mechanism in which imatinib repressed survivin promoter activity by disturbing the interaction between c-myc and E-box elements.Interruption of c-myc activity by 10058-F4 exerted an anti-leukemia effect with enhancing the sensibility of K562/G01 cells to imatinib.Conclusion Imatinib down-regulates Survivin expression through c-myc-mediated transcription and interference with c-myc might be a potential utility for treatment of imatinib resistant leukemia.
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ObjectiveTo observe the effect of indomethacin on the migration of breast cancer cell line MCF-7 in vitro and investigate the mechanism involved.MethodsThe migration of MCF-7 cell line stimulated with or without indomethacin were tested using transwell plates consisting upper and lower chambers separated by Millipore polycarbonate membrance filters with 8 μm pore sizes; the levels of chemokine receptor 4(CXCR4),cyclooxygenase(COX-2),epidermal growth factor receptor(EGFR) and vascular endothelial growth factor(VEGF)expression in MCF-7 cell line were detected by flow cytometry,Real-time PCR and ELISA,respectively.Results Indomethacin decreased the migration ability of MCF-7 cell line significandy.CXCR4 membrane expression was significantly reduced in a time-dose dependent manner,and CXCR4,COX-2 and EGFR mRNA levels were significantly downregulated after indomethacin stimulation.However,exposure to indometahcin had no major effect on VEGF production of cells.ConclusionThe downregulation of CXCR4,COX-2 and EGFR expression might be the primary mechanism involved in the inhibitory effect of indomethacin on the migration of MCF-7 cell line.
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Objective Myocardial infarction and subsequent heart failure remain the most dominant health challenges worldwide.Therapeutic angiogenesis has emerged as a potential novel treatment for severe ischemic heart disease and there is increasing evidence that cell transplantation may improve the perfusion and contractility of myocardium in animal models.This study was designed to examine the endothelial growth potential and whether transplantation of human umbilical cord derived mesenchymal stem cells can improve local blood flow in a mouse ischemic hindlimb model.Methods The mesenchymal stem cells derived from human umbilical cord of passage 5 were differentiated in an endothelial differentiation medium containing vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in vitro.Samples were observed for 2 weeks.The human umbilical cord derived mesenchymal stem cells were transplanted into a hindlimb ischemia mouse model in vivo.Four weeks later,immunofluence was used to identify the migration and differentiation of the transplanted cells towards endothelial linage.Laser Doppler perfusion image was used to evaluate the local blood flow of the hindlimb.Results Results After incubation with VEGF and bFGF,the human umbilical cord derived mesenchymal stem cells started to form interconnected clusters and a network was formed.Four weeks after transplantation,the transplanted cells were sprouting f0rom the local injection and differentiated into endothelial cells,contributed to the recovery of local blood flow obviously as compared with control group.Conclusion Human umbilical cord derived mesenchymal stem cells have the ability to differentiate into endothelial cells,contribute to the local angiogenesis in a hindlimb ischemia mouse model and represent a new source for therapeutic angiogenesis for clinical applications.
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Objective To investigate the infect capability of recombinant adenovirus mediated PDGF to human umbilical cord derived mesenchymal stem cells (MSCs). Methods The PDGF cDNA sequence was amplified with RT-PCR and constructed recombinant adenovirus vector AdPDGF. The human umbilical cord derived MSCs were isolated and cultured. In vitro AdPDGF infected MSCs with various MOI,and determine the efficiency using FACS and fluorescence microscope. Trypan blue and MTT assay was used to detect cell viability and proliferation. ELISA was used to quantify PDGF secretion. Results Recombinant adenovirus AdPDGF was successfully and constructed,in which infection efficiency to MSCs was dose dependent. The highest efficiency was around 87.36 % when MOI =50,at which cell viability and proliferation wasn' t impaired. Cell viability of uninfected,AdPDGF infected and control virus infected MSCs was (97.8 ±2.3) %,(91.9±4.0) % and (92.8±4.0) %,separately. The cell proliferation was (100±16.8) %,(95.9±12.0) % and (87.5±9.7) %,separately. There was no statistic significance among AdPDGF infected,control virus infected and uninfected MSCs. 48 hrs post infection,PDGF secretion can be measured from infectious MSCs' supernatant. The secretion level was (1.53±0.37),(3.03±0.68) and (5.25±0.92) ng/ml,separately,when MOI= 10,30,50. No PDGF can be detected from MSC-GFP and control MSC group. Conclusion Recombinant adenovirus AdPDGF was constructed and can infect human umbilical cord derived MSC at high efficiency.
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BACKGROUND:An effective freezing-thawing technique is crucial for the clinical application of human umbilical cord mesenchymal stem cells(UC-MSCs).OBJECTIVE:To investigate biological characteristics of UC-MSCs after cryopreservation.METHODS:UC-MSCs were isolated from human umbilical cord and frozen in liquid nitrogen.The survival rate and the suppressive effect of γ-interferon(IFN-γ)of cryopreserved-thawed and fresh human UC-MSCs were compared.Furthermore,the multiple potentials and phenotype of UC-MSCs were estimated after cryopreservation.RESULTS AND CONCLUSION:There was no significant difference between cryopreserved-thawed and fresh human UC-MSCs on the survival rate and the suppressive effect of IFN-γ of peripheral blood mononuclear cells(PBMCs).After cryopreservation,human UC-MSCs had the potential differentiation and the phenotype of mesenchymal stem cells.
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Objective To explore the regularity of hematopoietic materials during the clonal evolution of aplastic anemia(AA), paroxysmal nocturnal hemoglobinuria (PNH) , myelodysplastic syndrome (MDS) and acute leukemia(AL). Methods Patients diagnosed as AA, PNH, MDS and AL were recruited as cases and health volunteers were recruited as controls. Serum folic acid, vitamin B12 and ferritin levels were measured by radioimmunoassay and competitive enzyme immunoassay,before and after-treatment. Results Before treatment,the level of serum folic acid in PNH group were significantly lower than that in the control group(P<0. 05). Vitamin B12 and ferritin levels of MDS patients were higher than the control group (P < 0. 05); Serum Folic acid level in AL patients was significantly lower than the control group (P < 0. 05). In contrast, vitamin B12 and ferritin levels were higher than the control group(P <0. 05). Compared to pre-treatments AA patients, vitamin B12 and ferritin levels of MDS patients were significantly higher(P <0. 01) ;Serum folic acid level in AL patients was significantly lower(P < 0.05). However,vitamin B12 and ferritin levels were higher. Compared to pre-treatments MDS patients, serum folic acid level in AL patients was significantly lower(P < 0. 05) , whereas vitamin B12 and ferritin levels were higher(P < 0. 05). The comparison of hematopoietic materials between pre-and post-treatments among the groups showed that there was no significant difference for AA patients between pre- and post-treatments in the levels of serum folic acid and vitamin Bl2 (P > 0. 05), whereas ferritin was significantly higher after treatment caused by transfusion in AA patients(P<0. 05) ;ln PNH patients,serum folic acid was significantly higher after treatment(P<0. 05) ,and there was no significant difference in the levels of vitamin B12 and ferritin between pre-and post-treatments (P >0. 05). In MDS patients, there was no significant difference in the level of ferum folic acid between pre-and post-treatments (P > 0. 05) , whereas vitamin B12 and ferritin levels were significantly lower after treatment (P < 0. 05); In AL patients,serum folic acid was significantly higher after treatment(P <0. 05) .whereas the levels of vitamin B12 and ferritin were significantly lower after treatment (P <0. 05). Conclusions There are significant difference in serum folic acid,vitamin B12 and ferritin among the patitents of AA,PNH,MDS and AL and would be helpful in discovering the interrelationship among the four diseases pertinent to the clonal evolution,prognosis,treatment and prognosis.
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AIM: To prepare a soluble hemangiopoietin(HAPO) protein and to construct pET22b(+) expression vector, to obtain pure recombinant HAPO protein and to measure its bioactivity. METHODS: HAPO cDNA was amplified using RT-PCR method from a commercial human fetal liver cDNA library. The resulting product was cloned into pET22b(+) vector and transformed into E.coli BL21(DE3). The recombinant protein was isolated and purified by Ni~(2+)-NTA chelating resin and the chromatographies of SP Sepharose FF. The adhesion of human umbilical vein endothelial cells (HUVEC) were measured by adhesion assay. RESULTS: HAPO gene with a reading frame of 897 bp was successfully cloned from human fetal liver cDNA library, the expressed pET22b(+)-HAPO fused protein existed in a soluble form, with the yield above 10% total bacterial protein and its purity achieved above 80%. The activity assay showed that the treatment of HAPO enhanced total adherence of HUVEC in a concentration-dependent manner. CONCLUSION: HAPO protein can be expressed in a soluble form. HAPO may facilitate the homing of hematopoietic stem/progenitor cells in vitro.
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BACKGROUND:It is not certain that whether human adipose-derived mesanchymal stem cells express telomerase reverse transcriptase (TERT).OBJECTIVE:To investigate whether human adipose-derived mesenchymal stem cells express telomerase reverse transcriptase or not.DESIGN,TIME AND SETTING:The cytology in vitro experiments were performed at TEDA Life and Technology Research Center,Institute of Hematology and Blood Diseases Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College in August 2009.MATERIALS:Adult adipose tissue was obtained from 5 healthy donors undergoing liposuction at the Tianjin Yili Medical Cosmetology Plastics Outpatient.Hela cells were supplied by the Cell Center of Basic Medical Sciences,Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences.METHODS:Human adipose-derived mesenchymal stem cells were isolated from 20 mL human edipose tissue of each healthy donor by collagenase digestion.Cells were digested by trypsin when 80% confluency.Hela cells served as controls,and treated with 1640 medium containing 10% fetal bovine serum,and then digested using trypsin when 80% confluency.MAIN OUTCOME MEASURES:Morphology,phenotype and differentiation of human adipose-derived mesenchymal stem cells.TERT of human adipose-derived mesenchymal stem calls were detected by reverse transcription-polymerase chain reaction (RT-PCR).Telomerase activity was measured by telomeric repeat amplification protocol assay ELISA (TRAP-ELISA).RESULTS:Human adipose-derived mesenchymal stem cells adhered to the flask,presented spindle shape,and whirlpool-shape when cell density was large.Human adipose-derived mesenchymal stem cells were labeled positively for CD73,CD90 and CD105,but negatively for CD19,CD34,CD11b,CD45,and HLA-DR.Following adipogenic induction,oil red O staining showed significant red oil drop.Following ostesgenic induction,Von Kossa staining showed black particles,which indicated calcium deposition.Human adipose-derived mesenchymal stem cells at P1,P4 and P7 did not express telomerase reverse transcriptase gane.Human adipose-derived mesenchymal stem cells from 5 different donors did not expression telomerase reverse transcriptase gane.Hela cells had telomerase activation,but human adipose-derived mesanchymal stem cells did not express telomerase activation.CONCLUSION:Human adipose-derived mesanchymal stem cells do not express mRNA of hTERT,nor the activity of telomerase reverse transcriptase.
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Objective To study the dietary nourishment of adult patients with leukemia and compare acute leukemic patients with chronic leukemic patients. Methods Adopting dietary review of 24 hours and seven consecutive days of dietary records method to obtain the food category and quantity of 122 patients with acute leukemia and 10 patients with chronic leukemia. Using statistic software SPSS11.0 to calculate the patients'intake of various kinds of nutfiments. and the difiences between acute and chronic leukemic patients were analyzed. Results The rate of most ontrients of patients'intake reaches RNI/AI is lower,especially vitamin A,vitamin C and caleium.There's a tendency that intake diet,energy and nourishments of acute leukemic patients is lower than that of those chronic leukemic patients. Conclusion There is a tendency of unbalanced dietary intakes in leukemic patients.including the low intakes.There is the tendency that nutritional status of acute leukemic patients iS poorer than that of chronic leukemic patients.
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BACKGROUND: At present, hemopoietic stem cells have been proved to differentiate into nerves in rodents animals. As for the human, this topic is in debate.OBJECTIVE: To investigate the neural differentiation potential of human umbilical cord blood-derived AC133+ cells. DESIGN, TIME AND SETTING: Control experiments by grouping were performed in the Hematology Institute of Tianjin Hematology Hospital and Central Laboratory of Neurosurgery in Yuquan Hospital of Tsinghua University from August 2005 to December 2007.MATERIALS: Human umbilical cord blood was sampled from full-term newborn infant. Fetal brain-derived trophic support cells were harvested from aborted fetus of 22 weeks old.MAIN OUTCOME MEASURES: After the induction, human cord blood cells were collected at weeks 1, 2 and 4. RT-PCR was used to detect the expression of nestin, bone morphogenetic protein-2 and neural cell adhesion molecule. Immunocytochemistry method was applied to detect the cytotype-specific antigen. RESULTS: In the culture medium containing epidermal growth factor and basic fibroblast growth factor, human cord blood AC133+ cells could express nestin and bone morphogenetic protein-2, which were down-regulated even closed up in suboptimal condition. In the DMEM/F12 medium supplemented with epidermal growth factor, basic fibroblast growth factor and brain-derived neurotrophic factor, the gene expression of bone morphogenetic protein-2 and nestin continued in optimal condition at 2 weeks. Moreover neural cell adhesion molecule, another gene of neural cells, also expressed in this condition. AC133+ cells co-cultured with fetal brain-derived trophic support cells exhibited similar expressions. In the optimal non-cell-cell contact co-culture system, glial fibrillary acidic protein-positive cells were found by immuocytochemistry, while neuronal marker β-tubulin Ⅲwas expressed in the cell-cell direct contact system. These outcomes indicated that human cord blood isolated AC133+ cells may have an effect through gene rearrangement on inducing stem cells to express nerve cell development factors.CONCLUSION: The human umbilical cord blood-derived AC133+ cells contain some multipotential stem cells with differentiation potential, neural differentiation-related antigen when exposed to a suitable microenvironment.
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BACKGROUND:Human umbilical cord blood (CB)-derived CD133+ cells are a minority population of primitive cells with extensive proliferation and differentiation potentials,which are considered to have ability of neural differentiation.OBJECTIVE:We hypothesized a possible application of CB CD133+ cells in the cognitive and survival function of mice with dementia,the present study observed the changes of the cognitive function and survival of amyloid precursor protein(APP)transgenic mice after CB CD 133+ cells transplantation to verify the above assumption.DESIGN,TIME AND SETTING:A completely randomized block design of animal experiments was performed in the Hematology Institute of Tianjin Hematology Hospital from September 2005 to December 2007.MATERIALS:Forty-eight eight-month-old male APP 695 transgenic C57BL/6 (BDF1/KM) mice were selected in this experiments All mice were divided randomly into three groups:control group (n=8),CD133+ transplantation group (n=20) and CD133 transplantation group (n=20).METHODS:Mice in control groups received an intraventricular injection of 10 μL phosphate buffered saline (PBS).The transgenic mice that received an intraventricular injection of 10 μL CD133+ (5×104/μL) and CD133 CB cells (5×104/μL) respectively.MAIN OUTCOME MEASURES:Radial ann water maze (RAWM) was used to evaluate cognitive function of the mice and the survival days of mice in different groups were recorded,lmmunohistochemical assessments and Dil Fluorescence labeled way was used to detect the differentiation phenotype of transplanted cells.RESULTS:The cognitive function of the mice in CD133+ transplantation group was significantly improved compared with the mice in CD 133- transplantation and control groups both 30 and 180 days after transplantation (P＜0.05).The mean survival time of the mice in CD133+ transplantation group was significantly increased compared with CD133 transplantin group and control group (P＜0.05).It was observed that the transplantation CB CD133+ cells labeled with Dil migrated into several brain regions at day 30 post-transplantation.These cells were stained for human βⅢ-tubulin,neuralfilement(NF),neuron specific enolase (NSE),and glial fibriliary acidic protein(GFAP).However,in the brain of mice that received CD133 cells transplantation,CB cells were distributed mainly in and around the lateral ventricle at day 30 and 180 post-transplantation and GFAP-,βⅢ-tubulin- and NSE-positive cells were rarely detected.After intraventricular transplantation of CB CD133+ cells,the percentage of transplanted Dil-labeled CB cells expressing βⅢ-tubulin was significant higher at day 30 than at day 180,and the percentage of CB cells expressing NSE was significant lower at day 30 than that at day 180 (both P＜0.01).The percentage of CB cells expressing GFAP was relatively constant between the days 30 and 180 after transplantation (P＞0.05).CONCLUSION:The result of this experiment suggested that the cognitive and survival function improvement achieved by transplantation of CB CD133+ cells is mainly due to a replacement of dysfunctional cells or augmentation of neural circuit by CB CD133+ cells transplantation.
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Aim:To investigate the effect of Hemangiopoietin (HAPO) on the hematopoiesis reconstitution in sub-lethally irradiated Balb/c mice.Methods: Balb/c mice were underwent total body irradiation at 700 cGy 137Cs ? radiation and were treated with HAPO or recombinant human granulocyte colony stimulating factor (rhG-CSF) after irradiation. The hematopoiesis reconstitution of mice were detected. Cells from bone marrow of Balb/c mice were cultured with HAPO or rhG-CSF for 24 hours or 72 hours before or after the cells were irradiated. The viability of cells were assessed and the ability of in vitro hematopoiesis reconstitution were also detected. Result: rhG-CSF and HAPO treated mice both showed increased survival rate and increased colony forming units. The peripheral WBC number increased greatly. The HAPO group was most quickest compared with rhG-CSF group and PBS control group. The number of bone marrow cells at day 14 of rhG-CSF group was higher than that in HAPO group, but the number of bone marrow cells at day 32 of rhG-CSF was lower than that in HAPO group. The number of bone marrow cells at day 42 of rhG-CSF was below normal. The number of bone marrow cells at day 42 of HAPO group was nearly normal. The number of CFU-GEMM in HAPO group was most compared with that in rhG-CSF group and PBS control group at day 7, 14 and 21 after radiation. The survival rate of cells after radiation in HAPO group was markedly higher than that in PBS control group, but the survival rate of cells after radiation in rhG-CSF group was no notable difference compared with that in PBS control group. In MTT assay, both HAPO and rhG-CSF incubation stimulated proliferation of bone marrow cell at 72 hours after radiation. Bone marrow cells formed Hematopoietic islands in HAPO group after radiation and were positive for sca-1 and CD31. CD31 positive endothelial cells increased around the Hematopoietic islands. There was no Hematopoietic islands formation, few CD31 positive endothelial cells and no sca-1 positive cells in PBS control group. Conclusion: HAPO can promote hematopoiesis reconstitution in sub-lethally irradiated Balb/c mice. It can increase the survival rate of mice and stimulate the proliferation of hematopoietic stem cells.
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BACKGROUND:Granulocyte colony-stimulating factor (G-CSF) can strongly mobilize bone marrow hematopoietic stem cells (HSCs). It has been proved that G-CSF has the ability to mobilize both HSCs and mesenchymal stem cells (MSCs).OBJECTIVE:To investigate the therapeutic effect of G-CSF in mobilizing autologous bone marrow stem cells entering cerebral infarction zone on ischemic cerebral infarction in rats.DESIGN:A randomized grouping design, animal experiment.SETYING: Center for Stem Cell Biology and Tissue Engineering of Sun Yat-sen University.MATERIALS: This experiment was carried out at the Animal Experimental Department of Sun Yat-sen University (North District) and Center for Stem Cell Biology and Tissue Engineering of Sun Yat-sen University from September 2004 to January 2005. Totally 200 male Wistar rats were chosen and randomly divided into autologous bone marrow stem cells transplantation group and control group, with 100 rats in each group.METHODS:Rats of two groups were made cerebral infarction models by line occlusion. Transplantation group introduced intraperitoneal injection of 60 μg/kg G-CSF one hour after operation. The control group introduced intraperitoneal injection of saline of the same dosage at the same time. ①All rats were weighed before operation and 24 hours, 48 hours, one week after operation to evaluate body mass loss rate. They were also given neurological grading. Grading criteria: Grade 0 is normal. Grade Ⅰ is that the right forelimb bends. Grade Ⅱ is that the right forelimb grasped weakly when the tail is lifted. Grade Ⅲ is that the rat has no directivity in automatic action and circumrotates to right when the tail is lifted. Grade Ⅳ is that the rat circumrotates to right in automatic action. ②15 rats in each group were selected. 24 hours, 48 hours, one week after operation, we opened the skulls, took out the brain and used 2,3,5-Triphenyltetrazoluim Chloride (TTC) staining to measure infarction volume, hematoxylin-eosin(HE) staining to observe the pathological change , and immunohistochemistry to detect the infiltration of CD34+ cells.MAIN OUTCOME MEASURES:Body mass loss rate, neurological grade,infarction volume, pathological change and infiltration of CD34+ cells.RESULTS: Totally 180 of 200 rats were successfully made cerebral infarction model. 48 rats died in seven days after operation. As a result, 132 rat models were alive and 120 rats were randomly selected for data analysis. ①Measurement of body mass and neurological grading: There was no significant difference in body mass loss rate between two groups 24 hours and 48 hours after operation (P ＜ 0.05);one week after operation, body mass loss rate was significantly lower in transplantation group [(10.5±8.2)%]than in control group [(17.8±7.1)%] (P ＜ 0.05). There was no significant difference in neurology grade between two groups. ②Infarction volume:Infarction volume and the percent of infarction volume in the whole brain in control group were all higher than those in the transplantation group,with significant difference [ (251.69±52.77) mm3 vs(145.72±28.05)mm3,(17.00±2.69)% vs (9.90±1.62)% ,P ＜ 0.01]. ③Pathological change: 24 hours after operation, the brain tissue of two groups got classical pathological change of cerebral ischemia infarction. There were some mono-nucleus cells infiltrating in transplantation group while none in control group. 48 hours after operation, most nerve cells disappeared and the glial cells were degenerated. There were many mono-nucleus cells infiltrating in transplantation group while a few in control group. One week after operation, tissues in the infarction zone were liquescent with many monocaryons and lymphocytes infiltrating around them in control group. In transplantation group, part of the infarction zone was plerosised through proliferation of newly born capillaries and glial cells and inflammatory cells were not evident. ④Immunohistochemistry: CD34+ mono-nucleus cells were detected in the ischemic territory in transplantation group 24 hours after operation while none in the brain of other side and control group. There were CD34+ mono-nucleus cells and pyramidate cells with mutations in transplantation group 48 hours after operation while none in the brain of other side and control group.CONCLUSION:The stem cell transplantation in situ therapy, which employs self-marrow stem cells mobilized by G-CSF can relieve the ischemic degree and reduce the infarction volume.
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<p><b>OBJECTIVE</b>To investigate the association between the plasminogen activator inhibitor-1 (PAI-1) 4G/5G gene polymorphism and the occurrence of myocardial and cerebrovascular infarctions in individuals from Tianjin, China.</p><p><b>METHODS</b>The PAI-1 genotype was determined using allele-specific polymerase chain reaction (AS-PCR) in 56 myocardial infarction (MI) patients, 54 cerebrovascular infarction (CI) patients and 83 unrelated healthy controls. All subjects' clinical features and plasma PAI-1 activity levels were determined.</p><p><b>RESULTS</b>The PAI-1 genotype distribution frequency of the single guanine deletion/insertion 4G/5G polymorphism (located -675 bp upstream from the start of transcription) significantly differed between the patients and healthy controls. In the MI group, the 4G/4G-genotype frequency was increased, but the 4G/5G-genotype is decreased when compared to the control group. In the CI group, both the 4G/4G- and 4G/5G -genotypes occurred at a lower frequency than those in the control group (P < 0.001). The plasma PAI-1 activity level in the MI group was lowered as the presence of the 4G allele decreases. In the CI group, the frequency of 5G/5G was much higher than that of the control group (P < 0.001). The plasma PAI-1 activity level in the CI group was elevated as the presence of the 5G allele increased. Furthermore, positive correlation between triglyceride, glucose levels and PAI-1 activity were found in all three groups (P < 0.001).</p><p><b>CONCLUSIONS</b>The PAI-1 4G/5G gene polymorphism is associated with a higher risk of MI and CI in individuals in Tianjin, China. The deletion/insertion polymorphism is probably an important hereditary risk factor for heart diseases. Moreover, triglyceride and glucose levels of plasma have functional importance in regulating PAI-1 activity.</p>