Objective To investigate effect to kill primary breast cancer cells by dendritic cell (DC)with self-freeze thawing antigens of primary breast cancer cells co-cultured with cytokine induced killer cell (CIK). Methods Self-neoplasm antigen by primary cells of breast cancer in log phase growth was prepared and DC and CIK cells was cultured from peripheral blood. The CIK of co-cultured DC loaded self-neoplasm antigen was compared to PBMC, CIK cells with self-neoplasm antigen and the single CIK cells. Results Killing efficiency of PBMC, CIK, Ag-CIK and Ag-DC-CIK were (34.35±3.28) %, (45.91±2.78) %, (50.88±3.22) %, (62.10±5.94) %. There were significant difference between Ag-DC-CIK group and CIK-Ag and CIK group to primary cells of breast cancer (P <0.01). Conclusion The CIK induced from cocultured DC loaded with self-neoplasm antigen show a special killing activity to self-primary tumor cells.

ObjectiveTo study the feasibility of ex vivo gene transfer to donor heart by delivering i ntracoronarily a reporter gene (Lac-Z gene) to donor heart at the time of trans plantation.MethodsThe model of heterotopic heart transplantation in murine was established and the method of perfusing intracoronarily was performed. Twelve male BALB/c mice were divided into 2 groups randomly: gene-transfer group (group 1) and control grou p (group 2). In group 1, plasmid vector (PSV-?-gal containing Lac-Z gene)/li posome (DOSPER) was perfused intracoronarily into each donor heart at the time o f transplantation. In group 2, normal saline was perfused. Donor hearts were har vested 3 days after transplantation. Freezing sections were made for detection o f the transfer and expression of Lac-Z gene by histochemical staining (X-gal). ResultsThe expression of Lac-Z gene was detected in the donor hearts of group 1. In tw o donor hearts, the expression of Lac-Z gene was detectable in the myocardial c ells in the mid-layer of ventriculus; In one donor heart the expression was fou nd in the myocardial cells under epicardium. But no expression of Lac-Z gene wa s detected in donor hearts of group 2.ConclusionEx vivo gene transfer intracoronarily to donor heart in the form of plasmid vector /liposome complex at the time of transplant ation is feasible.

Objective To analyze the superiority of implanting operation with autoskull stored at ultrahypothermia over low temperature's and frozen skull bone of oneself.Methods 42 case were observed and followed up after operation.Results Incision is healing by firct intention.there were many advantages,such as good biological activity,beautiful appearance,no repellent response,no immunoreaction and hard to infection,etc. After storing 1,3,6 and 12 months,the histological structure of frozen skull observed by electronscope was similar to those of fresh skull.Pestroyed skull bone cells were not found.All cases had no complication during the period of 3 to 12 months(mean 6.5 months) follow-up after operation.After 12 months,X ray and CT scan showed that skulls were healed.Conclusions Implanting operation with autoskull stored at ultra-hypothermia is one of the most effective technique for repairing skull defects.

Aim To examine the role and uderlying mechanisms of Lin28 /let-7d axis in the proliferation of lung fibrobalsts and fibroblasts-into-myfibroblasts tran-sition,and provide novel strategy for the treatment of idiopathic pulmonary fibrosis (IPF).Methods We induced experimental lung fibrosis in mice by intratra-cheally injection of bleomycin (BLM).Ang Ⅱ and TGF-β1 were used to induce fibrogenesis in cultured MRC-5 cells;qRT-PCR and Western blot were applied to determine the changes of Lin28B,collagen 1 α1 and collagen 3α1 ;MTT assay,Edu satining and immun-ofluoresence were used to examine the cell viability, proliferation and fibroblasts-into-myofibroblasts transi-tion in MRC-5 cells.Results Lin28B was increased in the lung of mice with experimental lung fibrosis and in MRC-5 cells treated with AngⅡ or TGF-β1 .Moreo-ver,Lin28B enhanced collagen deposition via inhibi-ting expression of let-7d,which maybe contribute to the progression of IPF.In addition,further studies showed that Lin28B promoted proliferation and fibro-blasts-into-myofibroblasts in MRC-5 cells.Conclusion Lin28B /let-7d axis contributes to fibrogenesis via promotes fibroblasts-into-myofibroblasts transition, which may provide novel approaches for lung fibrosis treatment.

BACKGROUND:Shear stress can directly mediate the expression of endothelial cells, especially some cytokine genes whose codes are related to angiogenesis. Otherwise, flow shear stress of blood plays an importantly biological role in regulating vascular structure and function.OBJECTIVE: To observe the effects of laminar flow shear stress on the proliferation of vascular endothelial cells and the expression of protooncogene c-myc in human cerebral arteriovenous malformation.DESIGN: Randomized controlled study.SETTING: Department of Neurosurgery, Shenyang General Hospital of Military Area Command of Chinese PLA.MATERIALS: The experiment was carried out in the Neuromedical Institute, General Hospital of Shenyang Military Area Command of Chinese PLA from November 2006 to February 2007. Fresh samples of human cerebral arteriovenous malformation were derived from 20 patients who were of grade Spetzler Ⅱ -Ⅲ and received resection of human cerebral arteriovenous malformation in the Department of Neurosurgery, General Hospital of Shenyang Military Area Command of Chinese PLA in 2006. All cases were diagnosed with whole-brain angiography before operation. The main reagents and equipments were detailed as follows: M199 culture media (Gilbco BRL), quality fetal bovine serum (HyClone), endothelial cell growth supplement (ECGS; Sigma, USA), CO2 incubator (Forma Scientific, USA), flow cytometry analysis of cell cycle kit (BD Company), flow cytometer (FACS Calibur, BD Company), rat-anti-human c-myc monoclonal antibody (Santa Cruz Company, USA), and reverse transcription polymerase chain reaction (RT-PCR) kit (Promega).METHODS: Tissue explants adherent method was used to culture vascular endothelial cells of human cerebral arteriovenous malformation, and then the cells were classified into 4 groups based on degree of shear stress, including control group, low shear stress group, moderate shear stress group and high shear stress group. Cultured endothelial cells of human cerebral arteriovenous malformation were put in a parallel plate flow chamber. In addition, cells in the low,moderate and high shear stress groups were stressed by low, moderate and high shear stress for 8 hours, respectively.However, shear stress in the control group was 0 Pa. Flow cytometry was used to measure proliferation index, and the expression of c-myc protein and c-myc mRNA were determined by immnocytochemistry and RT-PCR analysis respectively.MAIN OUTCOME MEASURES: Expressions of c-myc protein and c-myc mRNA and proliferation index in endothelial cells under various degrees of shear stress.RESULTS: ① Proliferation index: Proliferation index was higher in the moderate and high shear stress groups than that in the control group (P ＜ 0.05, 0.01). ② Expression of c-myc protein: Immuneposjtjve expression of c-myc protein was gradually increased with the increase of shear stress and there were significant differences in the three shear stress groups as compared with control group (P ＜ 0.05-0.01). ③ Expression of c-myc mRNA: Proliferation index of endothelial cells was higher in the low and moderate shear stress groups than that in the control group (P ＜ 0.05).CONCLUSION: Flow shear stress can induce expression of c-myc and activate expression of c-myc gene based on gene transcription so as to promote the proliferation of vascular endothelial cells in human cerebral arteriovenous malformation

BACKGROUND:Antisense gene therapy offers immense promise for the management of human cerebral arteriovenous malformation through inhibiting expression of vascular endothelial growth factor and angiogenesis in endothelial cells.OBJECTIVE: To observe the inhibitory effect of vascular endothelial growth factor-antisense oligonucleotide (VEGF-ASODN) on the proliferation of vascular endothelial cells in human cerebral arteriovenous malformation.DESIGN: Observational contrast study.SETTING: Department of Neurosurgery, General Hospital of Shenyang Military Area Command of Chinese PLA.MATERIALS: The experiment was carried out in the Neuromedical Institute, General Hospital of Shenyang Military Area Command of Chinese PLA from August to December 2006. A total of 18 patients with human cerebral arteriovenous malformation were selected from Department of Neurosurgery, Shenyang General Hospital of Military Area Command of Chinese PLA. There were 12 males and 6 females and their mean age was 40 years. Cerebral arteriovenous malformation was classified based on Spetzler grade: grade Ⅱ (n =10) and grade Ⅲ (n=8). All cases were diagnosed with whole cerebral angiography before operation and they provided the confirmed consent. Main reagents were detailed as follows: endothelial cell growth supplements (ECGS, Sigma, USA), 391 DNA automatic synthetic device (Shanghai Shenggong Liyong Company, PE, USA), anaerobic incubator (DY-1, Zhejiang), human vascular endothelial growth factor enzyme-linked kit (TBD Company, Beijing), 96E enzyme-labeling device (ERMA, INC), cell cycle analytical reagent kit (BD Company), and flow cytometer (FACS Calibur, BD Company).METHODS: ①Experimental procedure: Tissue explants adherent method was used to culture vascular endothelial cells from human cerebral arteriovenous malformation. The third generated cells were used and randomly divided into antisense group, sense group and control group with four bottles of cells in each group. Sense and antisense phosphorothioate oligodeoxynucleotides of artificial vascular endothelial growth factor selected from the antisense group and the sense group were covered with positive liposomes, and then they were used to transfected vascular endothelial cells cultured from human cerebral arteriovenous malformation; however, cells in the control group were not dealt with any treatments. Cells in the three groups were incubated in anaerobic incubator (including 0.95 volume fraction of N2 and 0.05 volume fraction of CO2) at 37 ℃ for 2, 4 and 8 hours, respectively. ② Experimental evaluation: Cell cycles were measured, protein content of vascular endothelial growth factor was measured, and mRNA expression of vascular endothelial growth factor was also detected.MAIN OUTCOME MEASURES: Expression of mRNA and protein of vascular endothelial growth factor and proliferation exponent at different times of hypoxia.RESULTS: ①mRNA expression of vascular endothelial growth factor: At 2, 4 and 8 hours after hypoxia, mRNA expression of vascular endothelial growth factor was higher than that before hypoxia in the control group (P ＜ 0.05);however, mRNA expression was lower in the antisense group than that in the control group (P ＜ 0.05). ② Protein content of vascular endothelial growth factor: At 2, 4 and 8 hours after hypoxia, protein content of vascular endothelial growth factor was higher than that before hypoxia in the control group (P ＜ 0.05); however, protein content was lower in the antisense group than that in the control group (P ＜ 0.05). ③ Proliferation exponent: At 4 and 8 hours after hypoxia,proliferation exponent of endothelial cells cultured from human cerebral arteriovenous malformation was higher than that before hypoxia in the control group (P ＜ 0.05); however, proliferation exponent was lower in the antisense group than that in the control group (P ＜ 0.05).CONCLUSION: Hypoxia may induce gene expression of vascular endothelial growth factor in endothelial cells at the transcriptional level. Antisense vascular endothelial growth factor can obviously inhibit gene expression of vascular endothelial growth factor cultured from human cerebral arteriovenous malformation and proliferation under hypoxic conditions.

Objective To investigate the accumulation and maturation status of pulmonary conventional dendritic cells (cDCs) in the early phase of acute lung injury (ALI),and to explore the way of the inflammatory responses and lung injury modulated by cDCs in vivo.MethodsMale C57BL/6 mice were randomly ( random number) divided into the normal control group,6 h-ALI group and 24 h-ALI group.Murine model of ALI was made by intra-tracheal administration of lipopolysaccharide (LPS) and lung specimens were taken 6 h or 24 h later.The accumulation and maturation status of pulmonary cDCs were assessed by flow cytometry.IL-6 and TNF-α were quantified to evaluate the lung inflammation.Transcription factors T-bet/GATA-3 mRNA ratio was determined to estimate the balance between Th1/Th2 responses.IFN-γand IL-4 were quantified to evaluate Thl-specific and Th2-specific cytokine production respectively.Lung injury was estimated by lung wet weight/body weight ratio (LWW/BW) and histopathological assessment.Comparison between groups was performed using one -way ANOVA.ResultsCompared with normal control group,LPS challenge resulted in higher level of IL-6 and TNF-α,increased LWW/BW ratio and significant histopathological changes (P ＜0.01 ).The accumulation and maturation of pulmonary cDCs in 6 h-ALI group were significantly increased after LPS challenge (P ＜0.01 ),while the accumulation and maturation of pulmonary cDCs in 24 h-ALI group were significantly lower than that in 6 h-ALI group ( P ＜0.01 ).Compared with normal control group,the expression of T-bet mRNA in 24 h-ALI group was markedly enhanced ( P ＜ 0.01 ) and the production of IFN-γ was increased as well ( P ＜ 0.01 ).ConclusionsThe accumulation and maturation of pulmonary cDCs peaked within 24 h after LPS challenge,pulmonary cDCs may initiate and amplify acute lung inflammation of ALI by enhancing the Th1 immune response and ensuing cytokine production.

Objective To assess the diagnostic capability of spiral CT for giant pure seminoma in intraabdominal undescended testis.Methods Spiral CT of 6 cases with pure seminoma of inabdominal testes as proved by surgery and pathology were analyzed.All patients were male,and the age ranged from 31 to 45 years old with the mean of 35.2 years old.Results All tumors were located along the path of testicular descent on CT images.The arterial-supply of tumors all came from the testicular artery ipsilaterally.The draining vein could be seen between the mass and inferior vena cava or left renal vein in 5 cases.Isolaterally spermatic cord was absent in the inguinal region.Isolateral kidney was shifted upward.CT scans typically demonstrated a unilateral,mixed solid and cystic mass,with areas of solid located at the lateral aspect and areas of necrosis at the medial aspect.Contrast-enhanced CT scan showed mild enhancement of solid areas and band-like septal enhancement in areas of necrosis.There is no evidence of calcification or fat within the mass.Conclusion Spiral CT proves to be a very useful preoperative imaging modality for the giant intraabdominal seminoma.

Objective: To observe the effects of electroacupuncture on withdrawal anxiety in heroin-dependent patients. Methods: Sixty heroin-withdrawal subjects with negative urine morphine were digitally randomized into two groups: acupuncture group in which 30 cases were treated by electroacupuncture and control group in which 30 cases were given no treatment. The patients were assessed by Hamilton anxiety scale before treatment and one week, two weeks and three weeks after treatment. Results: After treatment, the anxiety symptoms of heroin-dependent patients were improved more significantly in the acupuncture group than in the control group (P<0.01). Conclusion: Electroacupuncture has good effects on anxiety symptoms and emotions of heroin-dependent patients.

Objective To explore the effect of hepatitis B virus (HBV) X protein (HBx) on autotaxin (ATX) expression and its significance. Methods The recombinant eukaryotic expression vector of HBx ,pcD-NA3.1(+)-HBx,and the recombinant luciferase reporter gene vector of ATX promoter,pGL3-ATX,were con-structed and used to co-transfect HepG2 cells to examine the effect of HBx on the activity of ATX promoter. The sta-ble cell expressing HBx,HepG2.HBx,was constructed,and Western blot(WB)was used to detect the effect of HBx on ATX expression. Results The luciferase activity of pcDNA3.1(+)-HBx and pGL3-ATX group was 1.47 times as that of the empty vector cDNA3.1(+)and pGL3-ATX group(P<0.000). WB detection showed that the expression of ATX protein was increased in HepG2.HBx cells,and 1.75 times as that of HepG2 cells(P<0.05). Conclusion HBx can activate ATX promoter and up-regulate ATX expression ,thus suggests that HBV infection might enhance ATX/LPA signaling.