Objective To investigate the difference of hypoxia-inducible factor 1a (HIF-1a) and vascular endothelial growth factor (VEGF) expression in endometrial cancer and para-carcinoma tissues, and to explore the clinical significance of hypoxia and the two proteins in the development and progression of endometrial cancer. Methods From Jan. 2011 to Dec. 2012, 128 patients with endometrial cancer underwent surgery in Tongji Hospital of Tongji University. The expression of HIF-1a and VEGF in cancer tissues and paired para-carcinoma tissues was detected using immunohistochemical method. The patients were followed up regularly, and the relationship between the expression of HIF-1a and VEGF and the prognosis of the patients was analyzed. The hypoxic cell model of human endometrial cancer was constructed to detect the expression of HIF-1a and VEGF proteins and observe the cell proliferation, invasion and apoptosis. Results The positive rates of HIF-1a and VEGF in cancer tissues were significantly higher than those in the para-carcinoma tissues (both P<0.05). The positive rate of HIF-1a was higher in the patients with lymph node metastasis, high histological grade, maximal tumor dimeter=4 cm or positive progesterone receptor (all P<0.05). The positive rate of VEGF was higher in the patients with lymph node metastasis, high histological grade, deep myometrial invasion, maximal tumor dimeter=4 cm, positive estrogen receptor, positive progesterone receptor or high pathological stage (all P<0.05). The 5-year overall survival rate of the patients with negative HIF-1a was significantly higher than that of the patients with positive HIF-1a (P<0.05), while there was no significant difference in the 5-year overall survival rate between the patients with negative and positive VEGF (P>0.05). In the hypoxic cell model of human endometrial cancer, the expression levels of HIF-1a and VEGF were significantly increased, cell proliferation and invasion were significantly increased, and the cell apoptosis was significantly reduced (all P<0.05). Conclusion HIF-1a and VEGF are related to the progress of endometrial cancer, and positive expression of HIF-1a indicates a poor prognosis.

A 2,4 -dichlorophenol degrading Pseudomonas strain GI241-1 was isolated from a soil sample. The dienelactone hydrolase gene, designated as dcpD which encodes dienelactone hydrolase involved in transforming cis-2-chloro-dienelactone into 2-chloromaleylacetic acid, was cloned from this bacterium strain. The gene cloning strategy was to construct genomic library after location of its neighbouring gene by Southem blot and to screen the aim transformant by dot blotting. Sequencing results showed that length of dcpD is 702bp. The sequence of dcpD and the deduced amino acid are different from the relative sequences registered in the GenBank.

2,4-Dichlorophenol is toxic and biorefratory organic pollutant. A 2,4-dichlorophenol degrading bacterial strain GT241-1, identified as Pseudomonas sp., was isolated from soil samples which was collected from drainage area of several 2,4-dichlorophenol producing factories. Strain GT241-1 had strong 2,4-dichlorophenol degrading ability, it could decompose 91% 2, 4-dichlorophenol of 90 mg/L within 48 hours at 25 - 30 degrees C, and could utilize 2,4-dichlorophenol, 2,4-dichlorophenoxyacetic acid (2,4-D), benzoate and catechol as sole carbon and energy source. Southern blot showed that 2,4-dichlorophenol hydroxylase gene (dcpA) of strain GT241-1 locates on the about 10kb EcoR I/Xba I fragment. This fragment was recovered, linked to the vecter pUC19 and transformed into the E. coli DH5alpha. A aim transformant, Z539, was obtained by dot blotting from about 1200 transformants. PCR and the sequencing results shew that the whole dcpA gene is contained within the 10kb EcoR I /Xba I fragment of pZ539. This fragment was shortened to about 2.4kb by HindmIII. The shorted fragment was subcloned to vecter pRSET-B to get a transformant BS1-12. The subcloned fragment was sequenced. Sequencing results showed that the whole length of the subcloned fragment containing dcpA is 2389bp and the nucleotide span of coding region is from number 276 to number 2072 (1797 bp), with ATG and TAA as start and stop codon respectively. The sequence analysis of dcpA and the deduced amino acid encoded by dcpA showed that they are different from the relative sequences registered in the GenBank. The subcloned fragment carry the promoter of dcpA, this can deduce from the fact that the upflow length of dcpA coding region is 275bp, and further confirmed by the 2,4-dichlorophenol hydroxylase activity measurement results. The 2,4-dichlorophenol hydroxylase activity of transformant Z539 and BS1-12 were detected, the results showed these transformants have 2,4-dichlorophenol hydroxylase activity. By comparison, the activity of these transformants were lower than that of the strain GT241-1.
Amino Acid Sequence ; Bacterial Proteins ; genetics ; metabolism ; Biodegradation, Environmental ; Chlorophenols ; metabolism ; Cloning, Molecular ; Environmental Pollutants ; metabolism ; Mixed Function Oxygenases ; genetics ; metabolism ; Molecular Sequence Data ; Pseudomonas ; enzymology ; genetics ; isolation & purification ; Soil Microbiology

The objective of study was to explore the influence of high mobility group box 1 (HMGB1) on migration of cord blood CD34(+) cells and their mechanism of migration. The expressions of receptor for advanced glycation end products (RAGE), toll-like receptor-2 (TLR2) and TLR4 were detected by flow cytometry. The CD34(+) cells in umbilical cord blood (CB) were enriched by MiniMACS and were exposed to various concentration of HMGB1 (10, 50, 100, 1, 000 ng/ml), then the migration effect of HMGB1 on umbilical cord blood (UCB) CD34(+) cell count was determined by microscopy, the chemotactic index was calculated. The CD34(+) cells untreated with HMGB1 were used as control. The results indicated that the purity of the isolated CD34(+) cells was more than 98%. The HMGB1 could promote the migration of CD34(+) cells, and the migration effect of HMGB1 on CD34(+) cells in certain concentrations gradually increased along with raise of concentration, the strongest effect was observed in concentration of 100 ng/ml, there was significant difference as compared with control (p < 0.01). Anti-RAGE antibody partially inhibited the migration effect of HMGB1 on CD34(+) cells. It is concluded that the HMGB1 in certain concentration can enhance migration of CD34(+) cells, which may be mediated through RAGE.
Antigens, CD34 ; Cell Movement ; drug effects ; Cells, Cultured ; Female ; Fetal Blood ; cytology ; drug effects ; HMGB1 Protein ; pharmacology ; Humans ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; metabolism ; Signal Transduction

OBJECTIVETo evaluate the correlation of 64-multidetector-row CT (64MDCT) perfusion imaging with microvessel density(MVD) and vascular endothelial growth factor(VEGF) in colorectal carcinoma.

METHODS64MDCT perfusion imaging was performed in 33 patients with pathologically verified colorectal carcinoma. Time-density curves (TDC) were created from the region of interest (ROI) drawn over the tumor, target artery and vein by 64MDCT with perfusion functional software. The individual perfusion maps generated were for blood flow (BF), blood volume (BV), mean transit time (MTT) and permeability-surface area product (PS). MVD and VEGF expression of surgical specimens were examined by immunohistochemical staining with anti-CD34, anti-VEGF monoclonal antibody. MVD and VEGF were compared among the different types of TDC in colorectal carcinoma. The correlation of CT perfusion parameters with MVD and VEGF was also examined.

RESULTSTDC of colorectal carcinoma was divided into five types according to their shapes. MVD in the colorectal carcinoma was 22.61+/-9.01. VEGF staining was found in 25 of 29 tumors (86.2%). The score of VEGF expression was 4.15+/-1.09. No significant differences of MVD and VEGF expression among TDC types were found (F=2.59, 1.11, P>0.05). There were also no correlations of MVD and VEGF expression with any dynamic CT parameters (P>0.05).

CONCLUSION64MDCT perfusion imaging, MVD and VEGF may reflect angiogenic activity, but no significant correlations are found among them.

Adult ; Aged ; Colorectal Neoplasms ; blood supply ; diagnostic imaging ; Female ; Humans ; Male ; Microvessels ; Middle Aged ; Neovascularization, Pathologic ; Tomography, Spiral Computed ; methods ; Vascular Endothelial Growth Factor A ; metabolism ; Young Adult

OBJECTIVETo explore the impact of IL-2- and IL-15-activated donor natural killer (NK) cell infusion on graft-versus-host-disease (GVHD) and graft-versus-leukemia (GVL) effect post allogeneic hematopoietic stem cell transplantation (allo-HSCT).

METHODSThe C57BL/6 mice splenic NK cells were selected by microbeads, and then expanded in the media containing IL-2 and IL-15. The killing activity of NK cells was detected. In the leukemia mouse model, recipients (BALB/c) were intravenously inoculated with EL9611 leukemia cells 8 days before transplantation. Lethally irradiated BALB/c recipient mice were transplanted with 5 x 10(6) bone marrow cells (BMCs), or 5 x 10(6) BMCs plus 1 x 10(7) splenocytes with or without 1 x 10(7) activated NK cells. Additionally, NK cell infusion group mice were intraperitoneally injected with a mixture of IL-2 and IL-15 post transplant. Survival time, GVHD occurrence, lineage chimerism, TRBV spectra-typing were observed post transplant.

RESULTSThe purity of isolated splenic NK cells was 95.7% - 97.1%. The killing activity of NK cells after activation was increased by 3 times. GVHD did not occurred in allogeneic BMCs infusion group, whereas did from 1 week after transplant in allogeneic BMCs + splenocytes infusion group. The severity of GVHD in total body irradiation (TBI) experimental group was significantly lower than in splenocytes infusion group (P < 0.05). The survival time was 9.5 - 14.0 d in TBI alone conditioning group. In leukemia mouse model, 100 day survival rate was 10% the rest of them were died of leukemia while in experimental group, the more than 100 days survival rate was 80% (P < 0.01). PB NK cells at 2 week post-transplant were 4.8% in experimental group and 2.8% in control group. NK cells recovery in experimental group was earlier than that in control group (P < 0.05). TRBV reconstitution was faster in experimental group than in control group, moreover, the number of TRBV family expression was more in experimental group than in control group which mainly expressed monoclone or oligo-clone.

CONCLUSIONSDonor alloreactive NK cells can be efficiently expanded and activated with IL-2 and IL-15. Donor activated NK cell infusion and IL-2, IL-15 treatment can promote immune reconstitution, mitigate GVHD and reduce leukemia relapse.

Animals ; Cells, Cultured ; Graft vs Host Disease ; prevention & control ; Graft vs Leukemia Effect ; Hematopoietic Stem Cell Transplantation ; Interleukin-15 ; immunology ; pharmacology ; Interleukin-2 ; immunology ; pharmacology ; Killer Cells, Natural ; cytology ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL

The study was aimed to explore the influence of co-culture ex vivo of CD34+ cells from two units of cord blood (CB) on the homing-related adherent molecule expression of each other. Mesenchymal stem cells (MSCs) were obtained from human bone marrow. Two units of CB CD34+ cells were co-cultured on 12 Gy gamma-ray irradiated MSC layer. Their adherent molecule expressions were assessed by flow cytometry. The results showed that the purity of the isolated CD34+ cells was (98.25+/-0.93)%. After co-culture on MSC layer for 6 days, the proportion of CD34+ cells of each unit was dropped to (60.4+/-6.32)% and (60.2+/-5.12)% respectively, but there was no significant difference from the control groups. The expressions of CD44, CD62L, CD184 and CD26 on CD34+ cells of each unit remained unaffected. The expression of CD162 was downregulated and CD54 was first increased but then dropped to the level before co-culture. But there was no significant difference between the experimental and control groups. In conclusion, co-culture of CD34+ cells from two units of CB may have no effects on the adherent molecule expressions of each other.
Antigens, CD34 ; metabolism ; Bone Marrow Cells ; cytology ; Cell Adhesion Molecules ; metabolism ; Coculture Techniques ; Fetal Blood ; cytology ; metabolism ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Mesenchymal Stromal Cells ; cytology

This study was aimed to investigate the therapeutic efficiency and complications after allo-hematopoietic stem cell transplantation (allo-HSCT) with reduced-intensity conditioning regimens in hematologic malignancies. 10 patients (6 CML patients, 2 AML patients, 1 ALL patient and 1 NHL patient) underwent related allogeneic hematopoietic stem cell transplantation with reduced-intensity conditioning regimens. The conditioning regimens consisted of "FLU + CY + TBI" basically and was appropriately improved in accordance with status of patients. Cyclosporin A (CsA) and mycophenolate mofetil (MMF) were used to prevent the graft-versus-host disease (GVHD). Detection of bone marrow cells, chromosomes, fused gene, ABO blood group and STR-PCR were used to observe engraftment, relapse, GVHD, transplantation- related complications (TRC) after transplantation and to evaluate patients quality of life. The results showed that the 10 patients successfully accepted the transplantation and their primary diseases were cured. In one patient, severe pulmonary infection happened, and in another one CMV infection occurred. Grade IV of acute GVHD occurred in one case and grade I of acute GVHD in 2 cases, the no chronic GVHD appeared. 5 patients relapsed after transplantation at various time points, the donor lymphocytes infusion (DLI) or drugs rescued these 5 patients. During median follow-up of 5 - 35 months, 2 out of which died, 8 survived, the overall survival rate was 80%, and the survivors live in a high-quality life. In conclusion, the hematopoietic stem cell transplantation with reduced intensity conditioning regimens was feasible with relatively low toxicity for recipients. GVHD and TRC were low, and life quality of patients after transplantation was high. DLI could cure the primary diseases even relapsed after transplantation.
Adult ; Cyclosporine ; administration & dosage ; Female ; Graft vs Host Disease ; prevention & control ; Hematologic Neoplasms ; therapy ; Hematopoietic Stem Cell Transplantation ; methods ; Humans ; Lymphocyte Transfusion ; Male ; Middle Aged ; Mycophenolic Acid ; administration & dosage ; analogs & derivatives ; Transplantation Conditioning ; methods

OBJECTIVETo study both the release of HMGB1 from irradiation-treated mesenchymal stem cells (MSCs) and the effects of HMGB1 on human cord blood CD34(+) hematopoietic progenitor cell proliferation and differentiation.

METHODSMSCs were obtained from human bone marrow. HMGB1 released by the MSCs after treatment with 12 Gy gamma-ray irradiation was determined by enzyme linked immunosorbent assay (ELISA). CD34(+) cells were positively selected with a MACS CD34 isolation kit. The freshly isolated CD34(+) cells were cultured in the presence of HMGB1 for 6 days. Phenotype of cultured cells surface molecules (CD13, CD14, CD11c, CD41 and CD71) were analyzed by flow cytometry. The proliferation and differentiation capacities of cord blood HSCs were assayed by colony forming cell assay. The receptors of HMGB1 (RAGE, TLR2 and TLR4) on cord blood CD34(+) cells were detected by flow cytometry.

RESULTSHMGB1 level in the supernatant \[(4.3 +/- 0.9) ng/ml\] of the irradiated MSC was significantly higher than that in control \[(0.4 +/- 0.2) ng/ml\] (P < 0.01). Human cord blood CD34(+) cells expressed the HMGB1 receptors RAGE, TLR2 and TLR4. The HMGB1-treated CD34(+) cells contained higher proportions of CD13(+) \[(32.6 +/- 5.9)% vs (18.4 +/- 3.8)%\], CD14(+)\[(25.4 +/- 4.4)% vs (12.6 +/- 2.7)%\], CD11c(+) \[(20.3 +/- 3.9)% vs (9.8 +/- 2.1)%\], CD71(+) \[(47.1 +/- 7.4)% vs (26.6 +/- 4.6)%\] cells compared with control group did. But HMGB1 did not induce the generation of CD41(+) cells \[(1.3 +/- 0.5)% vs (1.1 +/- 0.4)%\]. Furthermore, HMGB1 profoundly induced the growth of BFU-E, CFU-GM and total CFU in a dose-dependent manner, and this effect was partially inhibited by TLR2 and TLR4 antibodies.

CONCLUSIONHuman MSC treated with gamma-ray irradiation can release HMGB1, which can induce the proliferation and differentiation of human cord CD34(+) cells.

Antigens, CD34 ; metabolism ; Cell Differentiation ; Cells, Cultured ; Fetal Blood ; cytology ; HMGB1 Protein ; Hematopoietic Stem Cells ; cytology ; Humans

OBJECTIVETo observe the effect of curcumin on the expression of PI3K (phosphatidylinositol-3-kinase, PI3K) and p-P3 K (phosphated phosphatidylinositol-3-kinase, p-PI3K) in the hippocampus of Alzheimer's disease (AD) model (APP/PS1 double transgenic) mice.

METHODA total of 60 three-month-old APP/PS1 double transgenic mice were randomly divided into model group, rosiglitazone group(10 mg . kg-1 . d-1) and curcumin large(400 mg . kg-1 . d-1), medium(200 mg- kg-1 . d-1) and small(100 mg . kg-1 . d-1) dose group. Twelve C57BL/6J mice in the same age and genetic background as APP/PS1 double transgenic mice were used as normal control group. All the 6 groups of mice were intragastrically administered for 3 months. After 3 months, the expression of PI3K and p-PI3K were detected by immunohistochemistry and Western blot.

RESULTThe expression of PI3K and p-PI3K positive cells in hippocampus CA1 region significantly decreased in model group compared with normal control group (P < 0. 05) , while compared with model group, PI3K and p-PI3K positive cells of all the curcumin intervention groups increased to varying degrees in hippocampus CA1 region,especially the middle dose group(P <0. 01). Besides,Western blot results of the curcumin high dose group were also increased obviously (P <0. 05).

CONCLUSIONCurcumin can recover the decreased PI3K and p-PI3K and improve the insulin-signaling transmission in the hippocampus of APP/PS1 double transgenic mice. The mechanism of curcumin maybe by regulating the insulin signal transduction to treat AD.

Alzheimer Disease ; drug therapy ; genetics ; metabolism ; Animals ; Curcumin ; pharmacology ; therapeutic use ; Disease Models, Animal ; Hippocampus ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Phosphatidylinositol 3-Kinases ; genetics ; metabolism ; Thiazolidinediones ; pharmacology ; therapeutic use