Female ; Heart Defects, Congenital ; surgery ; Heart Septal Defects, Ventricular ; surgery ; Humans ; Male ; Pulmonary Artery ; abnormalities ; Pulmonary Atresia ; surgery

The sequence of Neurospora crassa(XP_322380)and Gibberella zeae PH-1(EAA76971)ribosomal protein gene were subjected to local tBlastn searching against the Chaetomium globosum ESTs datebase.The 765 bp full length cDNA encoding 60S ribosomal protein L10a gene was obtained.The open reading frame was 654 bp and encoded 217 amino acids.The protein molecular mass was 23.9 kD.The BlastP analysis revealed that amino acids sequence of ribosomal protein L10a gene from C.globosum shared 89% high similarity with N.crassa and 78% low similarty with Ustilago maydis.The cDNA and deduced amino acid sequence of 60S ribosomal protein L10a gene were accepted by GenBank(accession numbers: AY669070,AAT74578).

With the rapidly development of the biotechnology industry,large quantities of recombinant proteins are needed for specific therapeutic and diagnostic applications.Bacterial cells are most often used for the production of recombinant proteins.However,recombinant proteins expressed in the cytoplasm of bacteria are often misfolded as insoluble inclusion bodies and therefore inactive.To circumvent this problem,several eukaryotic expression systems have also been developed over the years,ranging from yeast to mammalian cell-based technologies.For many mammalian proteins,especially those secreted and modified posttranslationally,a more compatible expression system is highly desirable because proper folding or modification can only be provided with closely related cells,i.e.,mammalian cells.Large scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins.Transient expression by means of extrachromosomal replication in COS cells is frequently used to check the functional integrity of genes/plasmids and to produce small quantities of cell supernatants containing the protein of interest.As it is allowed for easy and efficient purification,many recombinant proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted.The use of a proper signal peptide is one of the major determinants for the efficient secretion of heterologous proteins from mammalian cells.The noncatalytic C-terminal hemopexin-like domain of MMP-2,PEX,can block angiogenesis and tumor growth in vivo.Large quantities of biochemically active recombinant PEX are required for the study of their functions and biochemical properties,as well as for their industrial applications.For this purpose,the rat growth hormone,mouse IgG? chain and MMP-9 signal peptides were used for expression of PEX in COS7 cells,and their secretion efficiencies were compared by Western blotting and ELISA.Western-blotting of PEX protein from culture media,resulted in detection of proteins with the predicted molecular mass,which indicate that all of the signal sequences could direct PEX secretion successfully.The MMP-9 signal peptide seems to be superior to the signal peptides from IgG and rGH both in terms of extracellular yield and in terms of secretion efficiency.Thus,expression of pM9PEX construct resulted in higher yields of extracellular PEX and the majority of the produced PEX was secreted and not trapped intracellularly.To examine whether the observed difference in secretion yields is promoted at the transcriptional level,a RT-PCR analysis was performed at 6 h after transfection.The presence of mRNA transcripts of PEX was observed in all the DNA constructs.Moreover,semiquantitative reverse transcription(RT-PCR)results show that there were no significant differences in the expression levels of PEX among the constructs at 6 h after transfection.Though there was no difference in the expression levels of PEX at an early time point after transfection,the presence of an ER-targeting signal peptide sequence in the expression vector affected the trafficking of expressed proteins in the cells.Hence,the described difference in exported yields is probably promoted at the secretion level,rather than at the transcriptional level.Chick chorioallantoic membrane(CAM)bioassay show that the PEX protein purified from cell culture had biological activity to inhibit the angiogenesis.The MMP-9's signal peptide is used for the first time as leader sequence for secretion of foreign proteins.The results revealed that higher amounts of secreted PEX were obtained when vectors containing MMP-9 signal peptide were used and it is also indicated that MMP-9 signal sequence could be effective on promoting the secretion of other heterologous proteins in eukaryotic cells.

Humans ; Traumatology

When mature adipocytes are subjected to an in vitro dedifferentiation strategy referred to as ceiling culture, these mature adipocytes can revert to dedifferentiated fat (DFAT) cells. DFAT cells have many advantages compared with adipose-derived stem cells (ASCs) and bone marrow mesenchymal stem cells (BMSCs). For example, DFAT cells are homogeneous and could be obtained from donors regardless of their age. Furthermore, DFAT cells also have the same multi-lineage potentials and low immunogenicity as ASCs. As an excellent source of seed cells for tissue engineering and stem cell transplantation, DFAT cells have better prospects in the treatment of many clinical diseases, such as bone defects, neurological diseases, ischemic heart disease and kidney disease. It is necessary to make more intensive studies of DFAT cells. This article summarizes progresses in the immunological characteristics, differentiation ability and potential clinical applications of DFAT cells.
Adipocytes ; cytology ; Cell Dedifferentiation ; Cell Differentiation ; Cells, Cultured ; Humans ; Stem Cell Transplantation ; Tissue Engineering

Genetic Therapy ; Humans ; Liver Neoplasms

Objective To investigate the effect of TanshinoneⅡA (TSN) on the cell hypertrophy induced by angiotensinⅡ(AngⅡ) in the primary culture of neonatal rat cardiomyocytes. Method The effect of TSN on cardiomyocyte was evaluated by the 3-[4, 5-dimethylthiazol-2-yl] -3, 5-diphenylformazan (MTF) assay. As the index of eardiomyocyte hypertrophy, protein synthesis rate was measured by H-Leucine incorporation and the cell size was determined by phase contrast microscope. The proto-oncogene c-los mRNA and c-jun mRNA expression was assessed using reverse transcription polymerase chain reaction (RT-PCR). Results Exposure of the myocytes to TSN (5～80 mmol/L) for 24hours produced no cytotoxicity. Protein synthesis rate and proto-oneogene c-fos and c-Jun mRNA expression of eardinmyoeytes increased significantly after AngⅡtreatment, and TSN inhibited these effect of AngⅡ.Conclusions TSN can prevent the hypertrophy of eardiomyocytes induced by AngⅡ, which be attributable relate to the decreased expression of proto-oncogene c-los and c-jun mRNA by TSN.

Objective To investigate the method of directed differentiation dendritic cells from embryonic stem cells(ESC) and to amplify high purity DCS in vitro for immunity therapy.Methods E14 ESC line were generated ESC-derived dendritic cells(ES-DC) in complete medium further supplemented with granulocyte-macrophage colony-stimulating factor(GM-CSF) and interleukin-3(IL-3).ES-DCs was used flow cytometry to determine CD11c,CD80,CD86,MHC-Ⅱ cell surface phenotype. Lipopolysaccharide (LPS) were added to induce the ES-DCs matured. The matured ES-DCs was harvested 24 hours later to be identified with morphology, transmission electron microscopy, analyzed by flow cytometry and compared with the immatured ES-DCs phenotype. The antigen presenting was evaluated by mixed lymphocyte responses.Results The ES-DC had obviously dendritic processes under scanning electron microscope . The immature DCs express low level of CD11c(4.33±0.23)%,CD80 (7.62±0.19) %, CD86 (4.77±1.22) % and MHC-Ⅱ (9.68±0.15) %, but the mature DCs express higher lerve of CD11c(47.36±2.68)%,CD80 (74.4±1.47) %, CD86 (29.77±2.00) % and MHC-Ⅱ (87.56±2.75) %. MLR showed that ES-DCs could effectively stimulate lymphocyte to proliferate.Conclusion These results provide evidence that DCs can be generated from E14 ESC with GM-CSF and IL-3, express high level of CD11c,CD80, CD86, MHC-Ⅱ and can effectively stimulate lymphocyte to proliferate. ES cells may become new origin for DCs which provided the immunotherapy.

Adolescent ; Adult ; Dermatitis, Occupational ; etiology ; immunology ; Female ; Humans ; Male ; Middle Aged ; T-Lymphocyte Subsets ; drug effects ; immunology ; Trichloroethylene ; adverse effects ; Young Adult

Animals ; Animals, Newborn ; Hyperoxia ; physiopathology ; Lung ; drug effects ; pathology ; Oxygen ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Tretinoin ; pharmacology ; therapeutic use