AIM: To investigate inhibition of K562 cell growth by antisense drug targeted VEGF mRNA. METHODS: X7, 20-mer antisense sequences were selected, synthesized and modified with phosphorothioate. The drug was transfected into K562 cells in the present of lipofection. Cell growth was assayed by trypan blue dye exclusion assay and MTT. The level of VEGF protein in the media was determined by ELISA. The morphology of apoptotic cells were observed by Giemsa staining, and the propotion of apoptotic cells was detected by flow cytometry. RESULTS: The antisense drug inhibited growth of K562 and downregulated expression of VEGF protein significantly, compared with Scrambed control group and showed dose-dependent relation. Signs of apoptosis of K562 cells were not observed. CONCLUSION: Inhibition of K562 cell proliferation, but not cells apoptosis induction is the mechanism of inhibing growth of K562 cells by antisense drug targeted VEGF mRNA. At same time, VEGF has function of promoting K562 cell proliferation, and VEGF mRNA may be a new target attached by drugs. [

Aim To explore the mechanisms of lovastatin protecting EPCs.Methods EPCs were preincubated with lovastatin or LOX-1 mAb for 24 h and then exposed to oxLDL for 48 h.The abilities of migration,adhesion,and tube structure formation of EPCs were examined.To explore the mechanisms,the level of NO,the expression of eNOS and LOX-1 protein and mRNA were assayed.Results Incubation of EPCs with oxLDL resulted in the impairment of migration,adhesion and tube structure formation.Furthermore,oxLDL caused the decrease of NO generation,the down-regulation of eNOS mRNA and protein expression,the up-regulation of LOX-1 mRNA and protein expression.However,the detrimental effects of oxLDL on EPCs function were attenuated by lovastatin and LOX-1 mAb.Moreover,the effects of oxLDL on NO generation,eNOS and LOX-1 expression were reversed by lovastatin and LOX-1 mAb.Conclusion Lovastatin protects EPCs by the regulation of eNOS and LOX-1 expression.

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.

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.

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.

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.

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.

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.

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.

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.