The human Interleukin-2 cDNA containing full-length of encoding region was cloned by RT-PCR from PBMNC and confirmed by DNA sequencing. The hIL-2 recombinant retroviral expressing vector (pLXSN-hIL2) was constructed by inserting hIL-2 cDNA into the BamHI cloning site of pLXSN retroviral vector. After packaged with CRIP packaging cell line, the hIL-2 retrovirions were produced at the titer of 7.6?105CFU/ml. Then a fibroblast cell clone(NIH3T3-hIL2) secreting 118.2U/ml hIL-2 was obtained by infecting the NIH3T3 fibroblast cells with hIL-2 retrovirions. The integration of hIL-2 provirus into the genome of NIH3T3-ML2 cells was confirmed by PCR analysis for NeoR gene. Our data showed that the hIL-2 retroviral vector was successfully constructed, which enables us to further the investigation of the hIL-2 gene therapy in clinical trial.

Objective To evaluate the effect of low-molecular weight heparin (LMWH) on biological behavior of a human cutaneous squamous cell carcinoma cell line A431.Methods To optimize the concentration of LMWH,A431 cells were treated with different concentrations (12.5,25,50,100 and 200 IU/ml) of LMWH for 24,48 and 72 hours followed by CCK-8 assay for the detection of cell viability.Then,A431 cells were cultured with or without the presence of LMWH at 200 IU/ml for 24,48 and 72 hours.Subsequently,flow cytometry was performed to assess cell cycle,real time quantitative PCR (RT-qPCR) and Western blot to quantify the expression of vascular endothelial growth factor (VEGF) mRNA and protein respectively,double-antibody sandwich enzyme linked immunosorbent assay (ELISA) to determine the expression level of VEGF protein in the supernatant of A431 cells,wound-healing assay,Transwell assay,and adhesion assay to observe the migration and adhesivity of A431 cells.Analysis of variance and t test were carried out for statistical analysis.Results The optimal concentration of LMWH was determined as 200 IU/ml according to the CCK-8 assay,and used in the following experiment.The LMWH of 200 IU/ml resulted in a decrease in cell viability,cell cycle arrest,an increase in cell percentage in G1 phase,and a reduction in cell percentage in S phase.The proliferation index was 23.41 ± 5.51 and 11.76 ± 5.13 respectively in A431 cells at 48 and 72 hours after treatment with LMWH of 200 IU/ml,significantly lower than that in untreated A431 cells (48.62 ± 4.50,t =6.14,P ＜ 0.05; 46.86 ± 3.51,t =9.78,P ＜ 0.05).A significant decrease was observed in LMWH-treated A431 cells at 48 and 72 hours compared with the untreated A431 cells in the expression level of VEGF mRNA (10.16 ±0.07 vs.18.77 ± 0.11,4.11 ± 0.01 vs.17.39 ±0.05,t=114.38,451.10,both P＜ 0.05),VEGF protein (0.16 ± 0.01 vs.0.20 ± 0.01,0.12 ± 0.01 vs.0.21 ± 0.01,t =4.90,11.02,both P ＜ 0.05),and in the supernatant level of VEGF protein ((67.17 ± 3.34) ng/L vs. ( 122.63 ± 23.17) ng/L, (28.14 ± 3.14) ng/L vs.(86.76 ± 1.18) ng/L,t =4.10,30.27,both P＜ 0.05).The percentage of adherent cells was 29.7％ ± 1.92％ and 17.5％± 0.79％ in LMWH-treated A431 cells at 48 and 72 hours,respectively,significantly lower than that in untreated A431 cells (36.9％ ± 0.35％,34.6％ ± 0.96％,respectively,both P＜ 0.05).The migration of A431 cells was also obviously inhibited by the treatment with LMWH for 24,48 and 72 hours.Conclusion LMWH may suppress the proliferation,migration and adhesion of A431 cells via downregulating cellular viability and VEGF expression.

Objective To observe the effect of short hairpin RNA (shRNA)-mediated vascular endothelial growth factor (VEGF) gene silencing on the growth of human skin squamous cell carcinoma(SCC) xenografts in nude mice.Methods Two eukaryotic expression plasmids targeting VEGF gene,including psilencer-VEGF1-shRNA (VEGF-s1) and psilencer-VEGF2-shRNA (VEGF-s2),as well as one negative control plasmid containing random target sequence (psilencer-Target-off-shRNA,T-off),were chemically synthesized,and transfected into a human skin SCC cell line A431 to develop stably transfected cell lines.Real time quantitative PCR (RT-qPCR) and double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) were carried out to measure the expression of VEGF mRNA and protein respectively in A431 cells.Twelve nude mice were divided into 4 goups to be subcutaneously inoculated in the axillary region with untransfected A431 cells as well as A431 cells transfected with VEGF-s1,VEGF-s2 and T-off,respectively.The tumor growth was observed in nude mice every 5 days.Twenty days after the inoculation,the mice were sacrificed,and transplanted tumors were obtained from the mice and subjected to an immunohistochemical study for the measurement of VEGF,proliferating cell nuclear antigen (PCNA) and CD34 expression.Data were statistically analyzed by using the Stata 7.0 software,and t test was conducted to compare the differences between groups.Results The mRNA and protein expression levels of VEGF were significantly lower in A431 cells transfected with VEGF-s1 and VEGF-s2 than in untransfected A431 cells (27.85 ± 3.95 and 24.69 ± 2.83 vs.54.06 ± 6.38,t =6.05,7.29,both P＜ 0.01; 32.67 ± 2.52 and 29.27 ± 1.10 vs.52.85 ± 2.23,t =8.04 and 11.53,both P＜0.01 ).Twenty davs after the inoculation,the volume and weight of xenografted tumors in mice inoculated with VEGF-sl- and VEGF-s2-transfected A431 cells were significantly lower than those in mice with untransfected A431 cells ( ( 192.50 ± 10.90) mm3 and (203.67 ± 3.21 )mm3 vs.(272.00 ± 21.07) mm3,t =5.80 and 5.55,both P＜ 0.01; (0.05 ± 0.03) g and (0.13 ± 0.04) g vs.(0.25 ± 0.02) g,t =9.60 and 4.64,both P＜ 0.01 ).Decreased expression rate of VEGF,PCNA and number of CD34-positive vessels were observed in the xenografted tumor tissue from mice inoculated with VEGF-sl- and VEGF-s2-transfected A431 cells compared with that from mice with untransfected A431 cells (52.00％ ± 2.00％ and 56.67％ ± 3.06％ vs.70.00％ ± 2.00％,both P ＜ 0.01;37.01％ ± 2.41％ and 33.94％ ± 3.25％ vs.72.11％ ± 3.02％,both P＜ 0.01; 2.05 ± 0.07 and 1.72 ± 0.10 vs.4.01± 1.27,both P ＜ 0.01).No significant differences were observed in the above parameters between cells transfected with VEGF-s1- and VEGF-s2-transfected A431 cells,between untransfected and T-off-transfected A431 cells,between tumor xenografts derived from VEGF-sl- and VEGF-s2-transfected A431 cells,or between tumor xenografts derived from untransfected and T-off-transfected A431 cells (all P ＞ 0.05).Conclusions The shRNA targeting VEGF gene can significantly inhibit the expression of VEGF in A431 cells and A431-derived tumor xenografts in nude mice,in turn suppress the growth and attenuate the malignant phenotype of tumor.

Objective To evaluate the effect of try psin on the proliferation,migration and adhesion of a skin squamous cell carcinoma cell line A431.Methods Cultured A431 cells were divided into several experimental groups treated with trypsin at concentrations of 0.1,1,10 and 100 nmol/L for 24,48 and 72 hours respectively,and a control group treated with DMEM complete medium only.Cell counting kit-8 (CCK8) assay was conducted to evaluate cellular proliferative activity to select the optimal concentration of trypsin.Then,some A431 cells treated with trypsin at the selected concentration for 24,48 and 72 hours respectively (or 48 hours only) served as the experimental groups (or group),and other A431 cells treated with DMEM complete medium served as the control group.Flow cytometry was performed to assess cell cycle distribution and proliferation index,fibronectin-based adhesion assay to estimate cell adhesive capacity,and wound healing assay and Transwell assay were conducted to evaluate the migratory capacity of cells in two-and three-dimensional space.Statistical analysis was carried out by using analysis of variance,paired samples t test and chi-square test.Results The proliferative activity of A431 cells increased along with the increase of trypsin concentrations,with the strongest increasing effect observed at 100 nmol/L.After treatment with 100 nmol/L trypsin,the experimental group showed a decrease in the percentage of G1-phase cells,but an increase in the percentage of S-phase cells,proliferation index,migratory and adhesive capacity compared with the control group (all P ＜ 0.05).Conclusion Trypsin can promote the proliferation,migration and adhesion of A431 cells.

Objective To study the curative effects and adverse effects of the thalidomide combined with COMP chemotherapy in treating multiple myeloma(MM).Methods 42 patients were initially diagnosed as MM and 27 patients were refractory and relapsed MM.The small dose of thalidomide combined with COMP management and COMP management alone were used.The effective rate and adverse effects were analyzed.Changes of M-protein in serum,percentage of plasma cells in bone marrow and the level of hemoglobin were also analyzed in both pre-treatment and post-treatment periods.Results In 42 patients who were initially diagnosed as MM,the effective rate was 40.9% for 22 patients treated by chemotherapy alone and 70.0% for 20 patients treated by the thalidomide combined with chemotherapy.Statistic difference was observed between those two group.As to the 27 patients who were refractory and relapsed MM,the effective rate was 42.9% for 13 patients treated by chemotherapy alone and 84.6% for 14 patients treated by the thalidomide combined with chemotherapy.Statistic significance was present between them.Adverse effects were less and tolerated.Conclusion Treatment of small dose of thalidomide combined with COMP chemotherapy could significantly improve the effective rate with less adverse effects.

Yttrium vanadate:europium nanoprobes (YVO4∶Eu NPs) with good fluorescence properties and water solubility were synthesized by solvent thermal method.Due to the overlapping of the excitation spectrum of YVO4∶Eu NPs and the absorption spectrum of tryptophan, fluorescent internal filter effect (IFE) occurred, in which YVO4∶Eu NPs were the fluorophore and tryptophan was the absorber, leading the fluorescence of YVO4∶Eu NPs was quenched.Therefore, a new method for the determination of tryptophan was established by using fluorescent YVO4∶Eu as nanoprobes based on IFE.Some experimental parameters, such as the adding amount of YVO4∶Eu NPs, pH value of the reacting solution, and reacting time, were investigated.Under the optimum reaction conditions, the linear range of the method was 4.0×10-6-4.0×104 mol/L and the detection limit was 1.0×10-6 mol/L (3σ).The content of tryptophan in the soy sauce was determined with the recovery of 95.2% and 97.3%.This method is simple, rapid, sensitive and accurate.

Objective To establish a rat model of superior mesenteric vein thrombosis by vein ligation and to simulate the pathological process of the disease, and to provide the basis for studies of its pathogenesis and treatment.Methods Ninety-six SPF male SD rats were randomly divided into three groups: Group A (sham operation group), group B (strangulation group) and group C (simple group), 32 rats in each group.Rats in group A were only opened the abdominal cavity but not blocked the blood supply.The rats were sacrificed at 8, 24, 48 and 72 h after operation.The rats in groups B and C were subjected to establish the strangulation and simple models by superior mesenteric vein thrombosis, respectively, and were sacrificed at 8, 24, 48 and 72 h after modeling.Histological changes (H&E staining) in the rat intestinal tissues were evaluated by a pathological scoring system.The levels of intestinal fatty acid binding protein (IFABP) and α-glutathione S-transferase (α-GST) were detected by ELISA.Results The rat model of mesenteric vein thrombosis was successfully established, with a success rate of 100% (96/96).The pathological analysis revealed that compared with the group A, different degrees of blood stasis and injuries were observed in the intestinal tissues of groups B and C, and the injury were gradually increased in the group B, while gradually reduced in the group C.The degrees of blood stasis and injury were positively correlated with the scope of ligation.The result of ELISA showed that the serum levels of IFABP and α-GST of the rats in groups B and C were significantly higher than those in group A (P < 0.05), and the degree of elevation was positively correlated with the scope of ligation.Conclusions In this study, the rat model of superior mesenteric vein thrombosis is successfully established by vein ligation.This model is simple and easy to operate with a high success rate, and can be used in related research.

Adenovirus harboring E. coli. cytosine deaminase gene (AdCD) and adenovirus encoding with lymphotactin gene (AdLtn) were used for gene therapy in vivo. BALB/c mice were inoculated subcutaneously with CT26 colon adeno-carcinoma cells and 3 days later received combined injection of AdCD and/or AdLtn followed by continuous injection with 5-fluorocytosine(5-FC) 300mg/kg. The results demonstrated that mice received combined therapy developed tumors most slowly and survived longest when compared with mice treated with AdCD/5-FC, AdLtn, AdlacZ/5-FC or PBS. To further explain the immunological mechanism of the antitumor effects by the combined therapy, we found that combined treatment with suicide gene and Ltn gene therapy achieved maximal cytotoxic effects of nature killer cells and specific cy-totoxic T lymphocytes. FACS analysis of the tumor mass demonstrated that AdCD/5-FC in combination with AdLtn therapy increased the expression of H-2K~d and B7-1 expression on tumor cells. The CD4~+ and CD8~+ cells infiltrated in the tumor mass after combined therapy were significantly increased when measured by FACS analysis. Our results demonstrated that combined transfer of suicide gene and lymphotactin gene induce nonspecific and specific antitumor immunity of the host and elicit more potent antitumor effect.

Objective To observe clinical curative effect of the FLAG regimen combined donor lymphocyte infusion after granulocyte colony stimulating factor(G‐CSF) mobilization(G‐DLI) ,for the acute myeloid leukemia (AML) of allogeneic Peripheral blood hematopoietic stem cell trans‐plantation (allo‐HSCT) after recurrence of hematology .Methods For the patients with recur‐rence after allo‐HSCT ,giving the FLAG regimen chemotherapy when the WBC dropped to the lowest point ,followed by giving G‐DLI that infusion peripheral blood stem cell from the original donors ,to observe curative effect and survival situation .And searched the literature review through the PubMed etc .Results Through FLAG regimen combined G‐DLI ,3 cases of relapse after transplan‐tation again obtained complete remission (CR) .Case 1 :disease‐free survival (DFS) was 13 month and overall survival(OS) was 23 months after G‐DLI .The patient has been the central recurrence and remission in bone marrow ,he was dead after 23 months due to multipleorgan function failure .He occurred Ⅱ acute GVHD in Skin and Ⅰ acute GVHD in liver after G‐DLI and obtained effective control ,not chronic GVHD .Case 2 :DFS and OS were 12 months and 13 months ,as bone marrow relapse again and giving up treat‐ment ,so died a month later .Respectively ,he has limitations chronic GVHD in skin after G‐DLI .Case 3:DFS was 16 months after G‐DLI since the disease‐free survival ,had limitations GVHD in skin that was control for given small dose of immunosuppressive drugs .Conclusion Joint FLAG scheme and G‐DLI may be one of the effective treatment of postoperative recurrence of allo‐HSCT .

Objective To study the clinic effect and safety of MEAD chemotherapy regimen for adult patients with relapsed or refractory acute lymphocyte leukemia. Methods Between July 2006 and July 2009,twenty-two adult patients with relapsed or refractory acute lymphocyte leukemia received MEAD regimen (mitoxantrone 6 mg/d dl-3 iv drip,cytarabine 100 mg/d dl-5 iv drip,etoposide 100 mg/d dl-5 iv drip,dexmethasone 10 mg/d dl-8 iv drip). Results The complete remission (CR) rate of adult patients with relapsed or refractory acute lymphocyte leukemia was 31.8 %,the partial remission(PR) rate was 22.7 % and the overall response (OR) rate 54.5 %. The cumulitive CR rate was 50.0 %,and the PR rate 40.9 % after two times MEAD chemotherapy regimen. The main adverse effect was different level of myelosuppression,and other toxicity of vital organ was mild. Conclusion MEAD regimen is effective and can be tolerated for adult patients with relapsed or refractory acute lymphocyte leukemia,and its side effect is mild.