Objective To generate hemogenic endothelial cells(HECs)from human induced pluripotent stem cells (hiPSCs)in vitro in order to learn more about the mechanism by which the vascular niche affects HECs production and self -renewal.Methods hiPSCs with reporter gene runx1c were differentiated to hematopoietic cells by spinEB method.The CD34 positive cells were sorted by magnetic-activated cell sorting(MACS)at day 10 after hematopoietic differentiation. Afterwards,these CD34 positive cells were co-cultured with DLL4 overexpressed vascular niche cells VeraVec to further differentiate to HECs.The HECs derived from the hiPSCs were characterized by FACS.Results We first established an hiPSCs single cell culture method for spinEB differentiation.Single cell cultured hiPSCs with reporter gene runx 1c were differentiated to form embryonic bodies(EBs)by spinEB method.The HECs were enriched from the day 10.Meanwhile, we cultured the E4ORF1 transfected human umbilical vein endothelial cell(HUVEC)line(VeraVec)and examined the expression of NOTCH signaling pathway related genes.According to the results, VeraVec had a high expression level of NOTCH ligand DLL4 at both mRNA and protein levels.And the CD34 positive HECs were co-cultured with DLL4 overexpressed VeraVec cells,which promoted the expression of tdTomato during hematopoitic differentiation and increased HSCs production.Conclusion A method of inducing hiPSCs differentiation by spinEB has been established, which can enrich HECs.This model can be applied to study the mechanism by which the vascular niche promotes hematopoietic differentiation from hPSCs.The generated functional HSCs are of great social and military values for HSCs transplantation and battlefield radiation injury treatment.

OBJECTIVE Toprepareapolyclonalantibodyforspindlin1protein,anovelcancer related protein,and to provide the data for a better understanding of its functions and screening tu mor. METHODS Purifiedspindlin1proteinwasinjectedintorabbitstoproducethepolyclonalantiserumafter removing glutathione S-transferase (GST)from the fusion protein spindlin 1-GST that was expressed in Escherichia coli..The antiserum was purified through the Hitrap Protein A system,and the titer of spin-dlin 1 polyclonal antibody was detected by ELISA.The specificity of the polyclonal antibody was deter-minedbyWesternblottingandimmunohistochemistry.RESULTS Thetiterofspindlin1polyclonalanti-body was 1∶2000.Western blotting detection demonstrated that the spindlin 1 polyclonal antibody recog-nized myc-spindlin 1 reco mbinant fusion protein in HeLa cells transfected with pAdeasy-myc-spindlin 1 , which also corresponded with Myc.antibody.The HeLa cells were transfected with enhanced green fluo-rescence protein (EGFP)and spindlin 1 vector(pEGFP-C3-spindlin 1 ),which was confirmed by the in-dependent GFP fluorescence assay.The results of immunohistochemistry detection with the spindlin 1 polyclonal antibody suggested that spindlin 1 was mainly expressed in the nuclei of HeLa cells.More i m-portantly,in i mmunohistoche mical assays,the spindlin 1 antibody recognized nuclear spindlin 1 expres-sioninclinicalovariancancertissues.CONCLUSION Thespecificspindlin1polyclonalantibodyispre-pared,which may be used to detect cancer-related protein spindlin 1 in HeLa cells and ovarian cancer tissues.

Objective To investigate the relationship between the severity of insomnia and the curative effect in acute stage in patients with major depressive disorder (MDD). Methods The Insomnia Severity Index (ISI) was used to evaluate and group the severity of insomnia in the 57 patients with MDD. The Pittsburgh Sleep Quality Index (PSQI) was used to assess sleep quality. The 24－item Hamilton Depression Scale (HAMD24) was used to evaluate depressive symptoms, and the effect of acute stage (4～6 weeks) was evaluated with its reduction rate. The difference of curative effect was compared among patients with different insomnia levels. Results There was a significantly different recovery rate in acute stage in 3 groups of patients with mild, moderate and severe insomnia ( X2=22.34,P＜0.01). The severity of insomnia in patients with MDD (PSQI) was negatively correlated with the curative effect of acute stage (r=－0.44,P＜0.01). The total score, anxiety/somatization factor score, retardant factor score and despair factor score were significantly higher in severe insomnia group than in the moderate and mild insomnia groups after acute treatment (P＜0.01). Conclusion The severity of insomnia in patients with MDD can predict the curative effect in acute stage. The depressive patients with severe insomnia have residual anxiety/somatization, retardant, feelings of despair and other symptoms more obvious than mild and moderate insomnia patients after acute treatment.

OBJECTIVETo probe factors of influencing therapeutic effects of acupuncture in the patient of insomnia.

METHODSAccording to scores of degrees of anxiety and depression, 52 cases of insomnia were divided into 3 groups, group I (mild or less degree) and group II (moderate degree) and group II (serious degree). The Pittsburgh sleep quality index (PSQI) were compared before and after treatment in the 3 groups, and between two groups after treatment. Results There were significant differences in the therapeutic effect as the groups I, II compared with the group III (P < 0.01). The total sleep quality in the group I was better than that in the group II (P < 0.05).

CONCLUSIONThe degree of anxiety and depression in the patient of insomnia is one of important factors influencing therapeutic effect of acupuncture on insomnia.

Acupuncture Therapy ; Anxiety ; Depression ; Depressive Disorder ; Humans ; Sleep Initiation and Maintenance Disorders

Humans ; Minimally Invasive Surgical Procedures ; Spine ; surgery

Animals ; Fatty Liver ; drug therapy ; metabolism ; prevention & control ; Liver ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism ; Taurine ; therapeutic use

OBJECTIVETo study the cytotoxic T lymphocyte (CTL) response induced by dendritic cells (DC) transduced with recombinant adenovirus vector bearing hepatitis B virus surface antigen (HBsAg) gene in hepatocellular carcinoma HepG2. 2. 15 cells in vitro.

METHODSFull length HBsAg cDNAs were subcloned into pIND vector, followed by being cloned into pShuttle vector. The HBsAg gene fragments resulted from the pShuttle-S digested with PI-Sce and I-Ceu were linked to the linear adeno-X virus DNA. After packaged with HEK293 cells, the adenovirus expression vector was obtained. Then the recombinant adenovirus expression plasmid AdVHBsAg was transfected into human monocyte-derived dendritic cells, to construct AdVHBsAg hepatocarcinoma tumor vaccine. The effectiveness of transfection was detected by Western blot. Surface molecules of AdVHBsAg-DC were detected by FACS. Autologous T cell proliferation stimulated by AdVHBsAg-DC was detected by 3H-TdR assay. Cytotoxic CTL activity induced by AdVHBsAg-DC in vitro was detected by LDH assay.

RESULTSHBsAg gene in the inserted DNA of AdVHBsAg was confirmed by PCR, and predictive fragments proved by restriction enzyme digestion analysis were exhibited. Cell pathological changes appear after 10 days HEK293 cells transfected AdVHBsAg. Western blot analysis showed that HBV surface antigen gene was expressed in transfected DC, indicating that the transfection was effective. AdVHBsAg-DC was able to upregulate CD1a, CD11c, CD80, CD86 and HLA-DR. Autologus T cell proliferation induced by AdVHBsAg-DCs was significantly higher than that in DC control group and LacZ-DC group (P < 0.05). AdVHBsAg-DC activated CTL presented the specific killer ability to the hepatocellular carcinoma cells expressing HBsAg.

CONCLUSIONDC transduced with recombinant adenovirus HBsAg can express HBV-related hepatocellular carcinoma antigen (HBsAg), and AdVHBsAg-DC can induce potent immune response against HBsAg-positive hepatocellular carcinoma cells in vitro.

Adenoviridae ; genetics ; Antigens, CD1 ; metabolism ; CD11c Antigen ; metabolism ; Cancer Vaccines ; immunology ; Carcinoma, Hepatocellular ; immunology ; pathology ; virology ; Cell Line, Tumor ; Cell Proliferation ; Cytotoxicity, Immunologic ; immunology ; Dendritic Cells ; cytology ; immunology ; metabolism ; Genetic Vectors ; Hepatitis B Surface Antigens ; genetics ; metabolism ; Humans ; Liver Neoplasms ; immunology ; pathology ; virology ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Transfection

AIMTo discuss the effect of Pitavastatin on angiogenesis in vivo and its mechanism in Klotho heterozygous deficient mice.

METHODSThe heterozygous deficient Klotho mice (kl +/-) and wild mice (kl +/+) from the same litter were used to establish the animal model of hind-limb ischemia and grouped into control and Pitavastatin group, respectively. Hind-limb blood flow was evaluated using Laser Doppler perfusion imager (LDPI) before treatment and after operation of hind-limbs. The capillaries in muscle of limbs were counted by means of CD-31 labeled immuno-fluorescence. The phosphorylation of Akt (Protein kinase B) in cells was measured by direct immunohistochemical technique. The expression of vascular endothelial growth factors (VEGFs) in muscle of limbs was assessed using Western blotting.

RESULTSAfter treatment of Pitavastatin, the blood flow in ischemic limbs of the Kl +/- and wild mice improved obviously, the ratio of blood flow area in ischemic limb to that in non-ischemic limb increased and the density of capillaries increased in ischemic limbs of the Kl +/- and wild mice. Pitavastatin enhanced the phosphorylation of Akt and the expression of VEGF in ischemic limbs of the Kl +/- and wild mice.

CONCLUSIONPitavastatin has the pro-angiogenesis effect in vivo and the VEGF-p-Akt-NO pathway may be involved in the mechanism of the effect of Pitavastatin.

Angiogenesis Inducing Agents ; pharmacology ; Animals ; Heterozygote ; Ischemia ; Male ; Mice ; Mice, Knockout ; Quinolines ; pharmacology ; Vascular Endothelial Growth Factor A ; metabolism

Objective To investigate the clinical application of detecting serum PPAR-γmRNA,MMP-9mRNA in the diagnosis of ruptured intracranial aneurysm.Methods The expression of serum PPAR-γmRNA,MMP-9mRNA were detected for 87 cases of patients with intracranial aneurysm,including ruptured group and non-ruptured group,respectively,with 57 cases and 30 cases of patients.The control group should be established to compare the changes of the above indicators.Results The expression of serum PPAR-γmRNA in the ruptured group,the non-ruptured and the controls group were 0.23±0.03,0.59±0.11 and 0.87±0.15,which of MMP-9mRNA were 0.93±0.17,0.63±0.13 and 0.25±0.05.Compared with those in the controls group,the expression of serum PPAR-γmRNA in the ruptured group significantly lowered (t=23.79,P＜0.01),which of MMP-9mRNA raised (t=25.63,P＜0.01).There were statistically significant differences.The expression of PPAR γmRNA in the ruptured group were lower than those in the unruptured group,which of MMP-9 mRNA were higher (t=15.32,16.27,P＜0.01).To establishing the receiver-operating characteristic curve (ROC curve) in evaluating the clinical significances of the two markers to use the rupture group and non-ruptured group as the dependent variable,the AUC of the expression of serum PPAR-ymRNA,MMP-9mRNA were 0.858 (95 ％ CI:0.775 ～ 0.940,P =0.000),0.842 (95 ％CI:0.756～0.929,P=0.000).As the dependent variable in the control group and unruptured group,the AUC of the expression of serum PPAR-γmRNA,MMP-9mRNA were 0.827 (95％CI:0.734～0.920,P=0.000);0.818 (95％CI:0.722 ～0.914,P=0.000).Conclusion Detection of serum PPAR-γ mRNA,MMP-9 mRNA can be applied in assessment of occurrence and progression for the intracranial aneurysm,and to provide evidences for the early detection of ruptured intracranial aneurysm.

Objective To investigate the effect of microRNA-223-3p (miR-223-3p) on megakaryocytic differentiation and maturation, and explore the potential mechanism .Methods The endogenous expression of miR-223-3p during megakaryocyte ( MK) differentiation was detected by real-time PCR.Flow cytometry further indicated that alteration of miR-223-3p in human cell lines exerted effects on MK differentiation and maturation .By performing integrative bioinformatic analysis, the potential miR-223-3p target gene, MYH10,was identified.Real-time PCR, luciferase reporter assay and flow cytometry revealed that MYH10 was a direct target of miR-223-3p.Results Endogenous expression of miR-223-3p was in-creased with the differentiation and maturation of MK .The expression of megakaryocytic surface markers CD41 and CD61 and the ploidy were significantly increased in K562 and Meg-01 cells after transfection with miR-223-3p mimics.The expression of MYH10 decreased with the increase in miR-223-3p.Using a luciferase reporter assay ,we demonstrated that MYH10 was a direct target of MiR-223-3p.Furthermore, direct downregulation of MYH10 promoted MK polyploidization . Conclusion MiR-223-3p might regulate the polyploidization of MK by targeting MYH10.