0.05), but it increased writhing times of mice (P0.05), but HPPT was decreased(P

In order to construct recombinant adenovirus vector expressing Suppressor of cytokine signaling 3 (SOCS3) and obtain infectious adenoviral particles, SOCS3 gene was amplified from plasmid pcDNA3-SOCS3 and subcloned into the adenovirus shuttle plasmid pAdTrack-CMV. After sequence confirmation, the recombinant shuttle plasmid pAdTrack-CMV-SOCS3 was linearized by Pme I, and then transformed into BJ5183 competent cell, the recombinant plasmid pAd-SOCS3 was obtained by homologous recombination between pAdTrack-CMV-SOCS3 and the adenoviral backbone plasmid pAdEasy-1 in BJ5183. The pAd-SOCS3 was linearized by Pac I and transfected into HEK293 cells via liposome. The recombinant adenovirus was packaged and amplified in HEK293 cells. After purifying, virus titer was determined by tissue culture infectious dose 50 (TCID50). Using the recombinant adenoviruses to infect porcine primary adipocytes, the expression of green fluorescent protein (GFP) was observed by fluorescent microscopy, and SOCS3 gene was identified by RT-PCR and Western blotting. Restriction enzyme and PCR analysis demonstrated that the recombinant adenovirus vector was constructed correctly, and the virus titer reached 1.2x10(9) PFU/mL. The result of RT-PCR and Western blotting showed that SOCS3 mRNA and protein expression was remarkably increased in porcine primary adipocytes infected with recombinant adenovirus. In conclusion, this study successfully constructed the recombinant adenovirus containing SOCS3 gene, and can be helpful for further research on the function of SOCS3.
Adenoviridae ; genetics ; metabolism ; Adipocytes ; metabolism ; Animals ; Cloning, Molecular ; Green Fluorescent Proteins ; biosynthesis ; genetics ; HEK293 Cells ; Humans ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Suppressor of Cytokine Signaling 3 Protein ; Suppressor of Cytokine Signaling Proteins ; biosynthesis ; genetics ; Swine ; Transfection

OBJECTIVETo observe the inhibitory effect of dendritic cells (DCs) activated cytotoxicity T lymphocyte (CTL) combined with MAGE-1 nonapeptide on transplanted human hepatocyte carcinoma (HCC) in nude mice.

METHODSA model of HCC transplanted tumor was established by injecting BEL-7402 cell line HCC cells subcutaneously on the back of nude mice. Successful transplantation rate was 73%. Specific CTLs (1 x 10(6)), which were activated by DCs combined with MAGE-1 nonapeptide, were injected into the site of transplanted tumor (group A, n = 5). Another group of 17 mice were treated with same amounts of different kinds of cells, and they were divided into groups B, C, D, E, and F. The growth of tumor was observed, and pathological examination was also done.

RESULTS(1) The activated lymphocytes induced by DCs combined with MAGE-1 nonapeptide could suppress the growth of tumor and reduce the tumor size. In group A, 5/5 mice survived for at least two weeks, while the tumors grew rapidly and the majority of the mice died within two weeks in other groups (groups B, C, D, E, F) (P < 0.01). (2) Extensive necrosis and apoptosis were found in transplanted tumors in group A.

CONCLUSIONSThe DCs combined with MAGE-1 nonapeptide could not only inhibit the growth of HCC, but also result in produce death and apoptosis of HCC, hence preventing tumor metastasis and recurrence. The mechanism underlying tumor immunization resulted from DCs might be enhanced in apoptosis of tumor cells. MAGE-1 nonapeptide combined with DCs might be a potential novel tumor vaccine for the treatment of HCC.

Animals ; Antigens, Neoplasm ; Apoptosis ; Cancer Vaccines ; immunology ; Dendritic Cells ; immunology ; Humans ; Liver Neoplasms, Experimental ; immunology ; pathology ; therapy ; Melanoma-Specific Antigens ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Proteins ; administration & dosage ; genetics ; Neoplasm Transplantation ; T-Lymphocytes, Cytotoxic ; immunology ; Transplantation, Heterologous

BACKGROUND: The area of myocardial infarction is the determinative factor of acute myocardial infarction prognosis. Amelioration of blood transportation and replacement therapy can reduce infarction area. Bone marrow mesenchymal stem cells can differentiate into cardiovascular tissue and are easy to obtain. After cultured and expanded in vitro, they can become the ideal cells for cardiovascular replacement therapy.OBJECTIVE: To evaluate the therapeutic effect of intracoronary transplantation of bone marrow mesenchymal stem cells in the treatment of myocardial infarction. DESIGN: Self-control observation taking the patients as subjects.SETTING: Department of Cardiology, Department of Nuclear Medicine,Echocardiogram Room, Nanjing First Hospital Affiliated to Nanjing Medical University.PARTICIPANTS: Totally 20 patients with acute myocardial infarction who received the therapy of bone marrow mesenchymal stem cells transplantation in the Department of Cardiology, Nanjing First Hospital Affiliated to Nanjing Medical University during March 2003 to March 2004 were recurited. Informed consents were obtained from the patients, and the complete postoperative follow up was over 3 months. The patients include 15 male and 5 female, and they were aged (64±10) years.METHODS: All the patients underwent percutaneous coronary intervention (PCI) to treat infarction-related blood vessel. Autologous bone marrow was taken from the patients, then stem cells were extracted to be performed in vitro induction, differentiation and proliferation, and transplanted infarction-related blood vessel through coronary artery at the mean number of (21.7±30.14)× 107 within 2 weeks. Before and 3 months after transplantation of stem cells, patients underwent gated dual-isotopic myocardial perfusion/metabolic imaging (18-fluoro-2-deoxy-glucose, 18F-FDG) examination. Survived and necrotic myocardia were predicted and infarction area was obtained. At the same time, wall motion and heart function index were evaluated with ultrasound cardiography (UCG)examination, and they were re-checked 3 months after operation to evaluate the amelioration of wall motion and heart function index. A 5-point scale was used in the evaluation of gated dual-isotopic myocardial perfusion/metabolic imaging (18F-FDG) examination: point 0: normal, 1: sparse, 2:obviously sparse, 3: defected. Evaluative standard of UCG: point 1: normal,2: reduced, 3: obviously reduced, 4: no ventricular wall motion or paradoxical motion; Wall motion with 2 points or more than 2 points suggests it is improved.MAIN OUTCOME MEASURES: ① Results of gated dual-isotopic myocardial perfusion/ metabolic imaging (18F-FDG-SPECT); ②Infarctionrelated myocardial segment score and heart function index before and after stem cell transplantation of patients in ECG follow-up observation.RESULTS: All the 20 patients participated in the result analysis.Results of gated dual-isotopic myocardial perrusion/metabolic imaging (18F-FDG-SPECT): The myocardial perfusion defect area of 20 patients was significantly reduced after therapy than before therapy [(33±15)%,-(44±18)% ,P ＜ 0.05]; Metabolie defect area was significantly reduced after therapy than before therapy [(33±17)%, (43±21)% ,P ＜ 0.05];Before therapy, there were 199 segments, in which blood flow reperfusion was matched to glycometabolism defect, and they were determined as necrotic myocardium. After therapy, blood flow perfusion metabolism was improved in 79 segments, but blood flow perfusion and glycometabolism were not improved significantly in 120 segments (P ＜ 0.05). Results of UCG: ejection fraction of patients was significantly larger after therapy than before therapy [(53±8)%, (42±7)% ,P ＜ 0.05].CONCLUSION: Intracoronary transplantation of human bone marrow mesenchymal stem cells for treating myocardial infarction is simple to operate. After therapy, the infarction area is obviously reduced, myocardial blood flow perfusion and metabolism of necrotic area improve, myocardial segments without survival determined before operation reduce sigrificantly and the heart function of patients improve.