OBJECTIVETo isolate and culture human placenta derived adherent cells (hPDAC) and assay their hematopoietic growth factor expression.

METHODSBy enzyme digestion, hPDAC were isolated from human placenta tissue and cultured, and their biological characteristics were studied. The hematopoietic growth factor (HGF) mRNA expression of hPDAC was assayed by RT-PCR.

RESULTShPDAC was successfully isolated from human placenta tissue, which was further confirmed as mesenchymal stem cell-like cells. HGF including SCF, FL, G-CSF, GM-CSF, M-CSF and IL-6 were expressed in hPDAC.

CONCLUSIONhPDAC could be used as feeder layer for umbilical cord blood CD(34)(+) cells ex vivo expansion.

Cells, Cultured ; Female ; Gene Expression ; Granulocyte Colony-Stimulating Factor ; genetics ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Hematopoietic Cell Growth Factors ; genetics ; Humans ; Interleukin-6 ; genetics ; Macrophage Colony-Stimulating Factor ; genetics ; Membrane Proteins ; genetics ; Placenta ; cytology ; metabolism ; Pregnancy ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cell Factor ; genetics

Yeast is a widely used host for recombinant protein expression. However, glycoproteins derived from yeast contain N-glycan of high mannose type and are usually hyperglycosylated. alpha-1,6-mannosyltransferases gene (och1) encodes the enzyme that initiates the first step of out-chain elongation of high mannose type N-glycan in yeast, which is different from that in human. So, a high efficient method to knockout target gene by two-step recombination was established and was used to delete och1. In the first recombinant, a plasmid with och1::ADE1 and ura3 gene was linearized in the downstream of och1 and inserted to the och1 site of P. pastoris genome, where the upstream and downstream of och1 were duplicated. In the second recombinant, the duplicated fragments of och1 were exchanged and the och1 deletion strains were selected on the plates containing 5-FOA, but no adenine. Then the och1 deletion strain was applied to express an human serum albumin (HSA) granulocyte-macrophage colony-stimulating factor (GM-CSF) chimera. Different with the hyperglycosylated HSA/GM-CSF chimera expressed in wild type P. pastoris, the chimera expressed in the och1 deletion strain, contained smaller N-glycan. The results suggested that the och1 mutant yeast may be more suitable for production of recombinant glycoproteins. And the och 1 deletion strain could be used for further re-engineering to produce complex human glycoproteins.
Chimera ; Gene Deletion ; Gene Knockout Techniques ; Granulocyte-Macrophage Colony-Stimulating Factor ; biosynthesis ; genetics ; Mannosyltransferases ; genetics ; Pichia ; enzymology ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Serum Albumin ; biosynthesis ; genetics

OBJECTIVETo investigate the expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and GM-CSF/IL-3/IL-5 receptor common beta chain (beta c receptor) in an adult patient with idiopathic pulmonary alveolar proteinosis (PAP), so as to demonstrate the possible association of the GM-CSF and beta c receptor with the pathogenesis of human PAP.

METHODSThe GM-CSF levels were measured with a commercial ELISA kit (sensitivity 5 pg/ml) and the beta c receptor expression on the cell surface was detected by flow cytometry analysis. Reverse transcription polymerase chain reaction (RT-PCR) analysis was employed to detect the expression of the GM-CSF mRNA and the beta c receptor mRNA in peripheral blood mononuclear cells and alveolar macrophages. The entire coding regions of the GM-CSF cDNA and the beta c receptor cDNA were sequenced by the Sanger dideoxy-mediated chain termination method to detect possible mutations.

RESULTSThe patient with PAP failed to release the GM-CSF protein either from circulating mononuclear cells or from alveolar macrophages. The expression of the GM-CSF mRNA was normal after the stimulation of lipopolysaccharide, whereas a point mutation at position 382 of the GM-CSF cDNA from "T" to "C" was revealed by cDNA sequencing, which caused a change in amino acid 117 of the protein from isoleucine to threonine. The beta c receptor expression on the cell surface was normal, and the beta c receptor mRNA expression and the sequence of the entire coding region of the beta c receptor were also normal.

CONCLUSIONSThe decreased GM-CSF production is associated with the pathogenesis of human PAP. A point mutation of the GM-CSF cDNA may contribute to the decreased GM-CSF production in our adult PAP patient. The mutation of the beta c receptor in some of paediatric patients with PAP may not be a common problem in adult patients.

DNA, Complementary ; chemistry ; Granulocyte-Macrophage Colony-Stimulating Factor ; analysis ; biosynthesis ; Humans ; Male ; Middle Aged ; Pulmonary Alveolar Proteinosis ; etiology ; metabolism ; RNA, Messenger ; analysis ; Receptors, Cytokine ; biosynthesis ; genetics

Paeonia lactiflora root (baishao in Chinese) is a commonly used herb in traditional Chinese medicines (TCM). Two isomers, paeoniflorin (PF) and albiflorin (AF), are isolated from P. lactiflora. The present study aimed to investigate the protective effects of PF and AF on myelosuppression induced by chemotherapy in mice and to explore the underlying mechanisms. The mouse myelosuppression model was established by intraperitoneal (i.p.) injection of cyclophosphamide (CP, 200 mg·kg(-1)). The blood cell counts were performed. The thymus index and spleen index were also determined and bone morrow histological examination was performed. The levels of tumor necrosis factor-α (TNF-α) in serum and colony-stimulating factor (G-CSF) in plasma were measured by Enzyme-Linked Immunosorbent Assays (ELISA) and the serum levels of interleukin-3 (IL-3), granulocyte-macrophagecolony-stimulatingfactor (GM-CSF), and interleukin-6 (IL-6) were measured by radioimmunoassay (RIA). The levels of mRNA expression protein of IL-3, GM-CSF and G-CSF in spleen and bone marrow cells were determined respectively. PF and AF significantly increased the white blood cell (WBC) counts and reversed the atrophy of thymus. They also increased the serum levels of GM-CSF and IL-3 and the plasma level of G-CSF and reduced the level of TNF-α in serum. PF enhanced the mRNA level of IL-3 and AF enhanced the mRNA levels of GM-CSF and G-CSF in the spleen. PF and AF both increased the protein levels of GM-CSF and G-CSF in bone marrow cells. In conclusion, our results demonstrated that PF and AF promoted the recovery of bone marrow hemopoietic function in the mouse myelosuppression model.
Animals ; Antineoplastic Agents ; adverse effects ; Bridged-Ring Compounds ; administration & dosage ; Cyclophosphamide ; adverse effects ; Drugs, Chinese Herbal ; administration & dosage ; Glucosides ; administration & dosage ; Granulocyte Colony-Stimulating Factor ; genetics ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; metabolism ; Hematologic Diseases ; etiology ; genetics ; metabolism ; prevention & control ; Humans ; Interleukin-3 ; genetics ; metabolism ; Interleukin-6 ; metabolism ; Male ; Mice ; Monoterpenes ; administration & dosage ; Paeonia ; chemistry ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

The objective of this study was to investigate the expressions of hematopoietic cytokines EPO, SCF and GM-CSF in bone marrow cells of patients with chronic aplastic anemia (CAA) and their significance. The mRNA expressions of EPO, SCF and GM-CSF from 35 CAA patients and 10 healthy individuals were detected by semi-quantitative RT-PCR. The results showed that the levels of EPO, SCF and GM-CSF in CAA patients were obviously lower than those in the healthy individuals (p < 0.01). It is concluded that the immunity disorder mediated by hematopoietic cytokines is one of the pathogenesis in the CAA patients, it provided theoretical basis for guiding clinical treatment.
Adolescent ; Adult ; Aged ; Anemia, Aplastic ; genetics ; metabolism ; pathology ; Bone Marrow ; metabolism ; pathology ; Child ; Chronic Disease ; Erythropoietin ; metabolism ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism ; Humans ; Male ; Middle Aged ; RNA, Messenger ; genetics ; Stem Cell Factor ; metabolism ; Young Adult

OBJECTIVETo study the feasibility of transfecting breast cancer BA46 gene into dendritic cells (DCs) using adeno-associated virus (AAV) to induce specific cellular immunity.

METHODSMononuclear cells (DC precursor) were isolated from the peripheral blood of healthy donors by density gradient centrifugation and infected with rAAV/BA46/Neo virus stock (transfection group) or pulsed with 293 cell lysate (control group). In both groups, maturation of the DC precursor was induced by granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor-alpha(TNF-alpha). On day 7, the DCs were collected and mixed with T cells at the ratio of 1 to 20 to induce cytotoxic T lymphocytes (CTL). The capacity of the DCs in stimulating T lymphocyte proliferation was assessed using (3)H-thymidine incorporation assay. The expressions of interferon-gamma (IFN-gamma), IL-4, CD4, CD8, CD25 and CD69 in the CTLs were analyzed with cytometry, and the cytotoxicity of the CTLs was evaluated with (51)Cr-release assay using BA46-positive breast cancer cell line Hs578T as the target.

RESULTSThe DCs transfected with BA46 gene exhibited potent capacity to stimulate T lymphocyte proliferation. The CTL population induced by the transfected DCs expressed high levels of CD8, CD69 and IFN-gamma, and showed strong cytotoxicity against BA46-positive breast cancer cell line Hs578T, which was BA46 antigen-specific and MHC-limited.

CONCLUSIONThe success in BA46 gene transfer in the DCs that induce specific cellular immunity provides the experimental basis for breast cancer immunotherapy using genetically modified cells.

Antigens, Surface ; genetics ; metabolism ; Breast Neoplasms ; genetics ; immunology ; Cells, Cultured ; Dendritic Cells ; immunology ; metabolism ; Dependovirus ; genetics ; metabolism ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Immunity, Cellular ; Immunotherapy ; Interleukin-4 ; pharmacology ; Milk Proteins ; genetics ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection

A new method for the preparation of oligonucleotide microarray for gene expression detection was found. The key techniques and standards of quality controlling for preparation of oligonucleotide microarray was explored using gene of human 23 kD highly protein and Luciferase and mouse cytokine-associated genes. By the using of a software system MProbe, oligonucleotide probes were designed and BLAST. All the probes have a very high specificity, i.e. except target sequence, the similarity between the probe and non-target sequences is less than 70% and the hairpin structure are not exist in all probes. All the probes have the same length 40. GC contents in all probes are in a narrow scope (from 45% to 55%). All the probes are modified with amino at 5' or 3' terminal. The satisfied images with good sensitivity and very high specificity were obtained by the using of the methods above and also using of positive and negative controls and some internal controls(house keeping gene) to quantitate and balance expression of genes. High specificity, good sensitivity and stability have been verified by three continuous experiments using the oligonucleotide microarray to study gene expression profile of normal mouse breast grand tissue. The oligonucleotide microarray for expression detection prepared using our method have high specificity, good sensitivity and stability et al. It may be a more advanced method for analysis of gene expression profile.
Animals ; Cytokines ; genetics ; DNA, Complementary ; genetics ; Gene Expression Profiling ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Interleukin-10 ; genetics ; Mammary Glands, Animal ; metabolism ; Mice ; Nerve Growth Factor ; genetics ; Oligonucleotide Array Sequence Analysis ; methods ; Polymerase Chain Reaction

To investigate the effect of hSCGF-alpha on human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs), we obtained hSCGF-alpha using genetic engineering, hSCGF-alpha gene was amplified from hUCMSCs cDNA using two-step PCR and was inserted into pET-28a(+) plasmid vector. Induced by IPTG at 20 degrees Celsius for 24 h, the fusion protein expressed in E. coli BL21 (DE3) was mainly existing in soluble form. The recombinant hSCGF-a was purified using NI-NTA affinity chromatography and the purity was up to 90%. The colony forming test revealed that combined use hSCGF-alpha and rmGM-CSF (recombinant murine GM-colony stimulating factor, rmGM-CSF) had granulocyte/macrophage (GM) promoting effects on murine bone marrow GM progenitor. In addition, the results indicated that hSCGF-alpha and rhGM-CSF had stimulatory effect on hUCMSCs and their synergetic effect was the strongest.
Cell Proliferation ; drug effects ; Cells, Cultured ; Cloning, Molecular ; Drug Synergism ; Escherichia coli ; genetics ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Stem Cell Factor ; biosynthesis ; genetics ; Umbilical Cord ; cytology

In order to evaluate the biological activity in vitro and the antitumor effects of 131I-conditionally replicating oncolytic adenovirus KH901 on HepG2 human hepatoma xenografts, the leves of GM-CSF expression were determined by ELISA method. A panel of tumor and normal cells was infected with recombinant adenovirus KH901 at MOI of 10 PPC. The medium was harvested to determine the bioactivity of GM-CSF after 24 hours. Nude mice bearing HepG2 human hepatoma xenografts were given 131-KH901. Antitumor effects were assessed using endpoints of tumor growth delay. The data showed that after 24 hours 131-KH901 replicated hugely in tumor cells and produced significant amount of GM-CSF 183.27 +/- 6.90 pg/ml, while producing very small amount of GM-CSF 20.44 +/- 0.77 pg/ml in normal cells. In the treatment of tumor, 131I-KH901 showed higher restraint rate (71.3%) compared to 131I (22.7%) or KH901 (52.7%). Therefore, 131-KH901 can inhibit the growth of human hepatoma cell in nude mice and it may be a potential drug for treating liver cancer.
Adenoviridae ; genetics ; metabolism ; Animals ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; metabolism ; Hep G2 Cells ; Humans ; Iodine Radioisotopes ; Liver Neoplasms, Experimental ; diagnostic imaging ; pathology ; virology ; Male ; Mice ; Mice, Nude ; Oncolytic Virotherapy ; Oncolytic Viruses ; genetics ; metabolism ; Radionuclide Imaging

To develop the stable transformants of the silkworm (Bombyx mori) BmN cells that could continuously express the exogenous gene based on a non-transposon vector, an expression cassette containing human granucyto-macrophage colony-stimulating factor (hGM-CSF) gene driven by ie-1 promoter from B. mori nucleopolyhedrovirus was inserted into pIZT-V5-His to form a recombinant vector pIZT-IE-hGM-CSF, followed by transfecting the constructant into BmN cells, the stable ie-hGM-CSF cell lines were obtained after being selected with Zeocin. PCR result using the genomic DNA of the transformed BmN cells as template illustrated a specific fragment of ie-hGM-CSF, and Western blotting analysis using an antibody against hGM-CSF demonstrated a specific band with a molecular weight of 22 kDa in the transformed cells, meanwhile, the expression level of hGM-CSF determined by ELISA was about 2 814.7 pg in 10(6) transformed BmN cells.
Animals ; Animals, Genetically Modified ; Bombyx ; cytology ; genetics ; metabolism ; Cell Line ; Fibroins ; genetics ; Genetic Vectors ; genetics ; Granulocyte-Macrophage Colony-Stimulating Factor ; biosynthesis ; genetics ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transformation, Genetic