Objective　To obtain mammalian cell expression vector of human CD154 gene. Methods　A 820 bp cDNA fragment was amplified by RT-PCR method from total RNA of human peripheral blood mononuclear cell（PBMC） activated with 10 ng／mL PMA and 1 μg／mL PHA for 8 hours. The fragment was cloned into pcDNA3.1（＋） plasmids.The cloned insert was identified by double digestion of the recombinant plasmid with restriction enzymes BamH Ⅰ and EcoR Ⅰ and sequenced by Sangers-dideory-mediated chain termination. Results　This cDNA fragment included 820 bp entire coding region and a part of the 3 non-coding region. The recombinant mammalian cell expression vector of pcDNA3.1（＋）／hCD154 was constructed， the sequence of the insert was identical to the published sequence encoding human CD154 antigen. Conclusion　The recombinant mammalian cell expression vector of pcDNA3.1（＋）／hCD154 was successfully constructed.

BACKGROUND: At present, after transplantation of small diameter artificial blood vessel, long-term patency rate is low due to being lacking of endothelial cells for lining and anti-thrombus characters. In some studies,mature endothelial cells were tried to be seeded in the artificial vessel to boost up its anti-thrombus capability so as to improve the long-term patency rate, but we got unsatisfied effect due to the defects of seed cells and scaffolds. Therefore, in clinic, proper seed cells and vascular scaffolds have been searched for improving the long-term low pateney rate in transplantation of small diameter artificial blood vessel.OBJECTIVE: To investigate the feasibility that differentiation of bone marrow mononuclear cells induced in vitro into endothelial-progenitor cells (EPCs) and seed polyurethane small diameter artificial blood vessel so as to provide proper seed cells for endotheliazation of polyurethane small diameter artificial blood vessel.DESIGN: Observation experiment SETTING: Cardivascular Medical Department and Staff Room of Immunology, First Hospital Affiliated to Sun Yat-sen University MATERILAS: This experiment was carried out at the First Hospital Affiliated to Sun Yat-sen University from September 2004 to May 2005. About 10 mL of bone marrow from healthy adult volunteers (n=7) was used in this experiment.METHODS: Bone marrow mononuclear cells of healthy adult were collected and put in the fibronectin pre-coated DMEM culture medium, then induced by vascular endothelial growth factor and basic fibroblast growth factor. Induced cells were observed under fluorescence microscope and identified with immunohistochemical staining. The induced and proliferated EPCs were seeded onto the surface of polyurethane small diameter artificial blood vessel. Morphological change was observed under scanning electron microscope.MAIN OUTCOME MEASURES: ① Cellular morphological change.② Staining results of immunohistochemical VWF and CD 34 antibody . ③ Adhesive growth status of EPCs on the polyurethane small diameter artificial blood vessel RESULTS: ① In the vascular endothelial growth factor and basic fibroblast growth factor and other inducers , bone marrow mononuclear cells differentiated into EPCs , presenting typical "spindle-shaped" appearance under an inverted fluorescence microscope and became to form a monolayer that arrayed in "cobblestone-like" ② Immunohistochemical staining showed von willebrand factor(VWF) and CD34 antigen stained positive. ③ Under the scanning electron microscope, surface of polyurethane small diameter artificial blood vessel without seeded cells presented typical polyporous honeycomb-like structure , and the size of hole suited the crawling of EPCs. After seeding the cells, we observed the adhesion, crawling and spreading of the EPCs on the surface of polyurethane small diameter artificial blood vessel. Some EPCs grew into the honeycomb-like holes were seen occasionally.CONCLUSION: Bone marrow mononuclear cells can be induced and differentiated into EPCs, while induced and differentiated EPCs well grow adhesively in the polyurethane small diameter artificial vessels, suggesting that differentiation of bone marrow mononuclear cells induced in vitro into EPCS, which can be used as seed cells for endothelialization of polyurethane small diameter artificial blood vessels.

0.05).We did not find any difference of the expression of fibronectin,laminin and type IV collagen in them.Expression of ICAM and VCAM were(56.4?14.8)% and(55.6?12.2)%,respectively,obviously higher than those in control group(P

AIM: To induce lymphoid stem cells and/or T-cell precursors to diffe rentiate into functional mature T lymphocyte, and to increase the surface marker of T lymphocytes such as CD + 3, while embryonic stem(ES) cells differentiate d into hematopoietic stem/progenitor cells(HSPCs) in vitro . When they were i njec ted into lethally irradiated mice, these differentiated cells had the advantage in immune reconstitution. METHODS: Embryonic stem cells formed e mbryoid bodies(EBs) in the medium containing methycellulose, hematopoietic growt h factors(HGFs) was added to the culture system on the 6th day, thymopeptide was added at the same time. Flow cytometry were performed to detect the surface mar ker CD 34 and CD 3 of the differentiated cells. Finally the differentiated cells were injected into lethally irradiated mice, 60 days later, the incidence rate of graft versus host disease(GVHD) was taken as the mark of cell mediated immunity, PCR was performed to detect the sex determining region of the Y-chromo some(Sry) in bone marrow cells and spleen cells of the survival host female mice . RESULTS: The percentage of CD + 3 T lymphocytes was 10.52% a nd the incidence rate of GVHD was 0% on the 13th day, but they respectively rose up to 22.93% an d 100% if thymopeptide was added in the procedure of inducing ES cells to differ entiate into HSPC in vitro . CONCLUSION: The quantity of CD + 3 T lymphocytes increased in medium containing thymopeptide when ES cells differe ntiated into CD 34 + HSPC.

In this study, the peripheral blood mononuclear cells of healthy adult were acquired and inducted by vascular endothelial growth factor, et cetera. The differentiated endothelial cells were observed and identified as EPCs by the double positive staining of fluorescent labeled acetylated-LDL and lectin, seeded on the polyurethane small-diameter artificial vessels, treated by shear stress of 15 dyn/cm2, and observed by scanning electronic microscopy. As a result, the peripheral blood mononuclear cells differentiated into EPCs. They were positively stained by labeled acetylated-LDL and lectin. Under observation of scanning electronic microscope, the unseeded polyurethane small-diameter artificial vessel being suited for the growth and spreading of the cells; the cell lineage on surface of artificial vessels of stationary group being arrayed in chaos, and that of shear stress group being arrayed in direction. Therefore, the peripheral cells can differentiate into EPCs, and EPCs can be used as novel source cells for the accelerated endothelialization of small diameter artificial vessel. Shear stress contributes to the mechanic moulding of cell lineage on the surface of artificial vessel.
Bioartificial Organs ; Biocompatible Materials ; Blood Vessel Prosthesis ; Cell Adhesion ; Cell Differentiation ; Cells, Cultured ; Endothelial Cells ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Polyurethanes ; chemistry ; Prosthesis Design ; Shear Strength ; Stem Cells ; cytology ; Stress, Mechanical

This study was designed to investigate the changes of prostaglandin I2 (PGI2) and nitric oxide (NO) secreted by endothelialized polyurethane small diameter artificial blood vessel. The peripheral blood mononuclear cells of healthy adult were separated and induced into endothelial progenitor cells (EPCs), which were identified by the methods of discrepancy microphage and fluorescent immunology labeling. After the induced cells being seeded on the polyurethane small-diameter artificial vessels, the endothelialized polyurethane small diameter artificial blood vessels were divided into four different experimental groups, including stationary group, low-flow shear stress group (5 dynes/cm2), medium-flow shearstress group (15 dynes/cm2), and high-flow shear stress group (25 dynes/cm2). Then, the levels of 6-ketoprostaglandin F1alpha (6-keto-PGF1alpha) and NO of different time were measured by enzyme-linked immunosorbent assay and nitrate reductase methods. The peripheral blood mononuclear cells differentiated into EPCs. They presented typical "spindie-shaped" appearance, and they were positively labeled by fluorescent acetylated-LDL, lectin, FLK-1 and vWF. Shear stress enhanced the production of NO and 6-keto-PGF1alpha by EPCs in a dose-dependent manner. Therefore, shear stress increases the secretion of NO and PGI2 by EPC, which suggests that shear stress can improve the antithrombogenic potentials of endothelialized polyurethane small diameter artificial blood vessel.
Bioartificial Organs ; Biocompatible Materials ; chemistry ; Blood Vessel Prosthesis ; Cell Adhesion ; Cell Differentiation ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Epoprostenol ; metabolism ; Fibrinolytic Agents ; metabolism ; Humans ; In Vitro Techniques ; Leukocytes, Mononuclear ; cytology ; Nitric Oxide ; metabolism ; Polyurethanes ; chemistry ; Stress, Mechanical

AIM: To clone and express mouse telomerase reverse transcriptase(mTERT) cDNA and obtain eukaryotic expression vector.METHODS: The cDNA of mTERT was amplified by RT-PCR and PCR.After purification,the gene fragment was cloned into a vector PUC-19.The sequence of inserted mTERT gene fragment was also detected.RESULTS: The recombinant plasmid PUC-19-mTERT was constructed.Positive clones were screened and identified by PCR and digestion with restriction enzyme.The size of gene fragment was(3 369) bp and in accordance with the expected one.CONCLUSION: The mTERT cDNA was obtained and the recombinant eukaryotic expression vector was successfully constructed.The study lays foundation for DCs vaccine modified by mTERT gene for the treatment of tumor.