BACKGROUND:There have been no standard procedures regarding how to simply and efficiently separate and purify human peripheral blood monocytes and stimulate them into dendritic cells.OBJECTIVE:To investigate the efficacy of purification of human peripheral blood monocytes on gelatin-coated surfaces,analyze the phenotype of dendritic cells generated by these monocytes,and make a comparison with conventional plastic adhesion method.METHODS:Human peripheral blood mononuclear cells were harvested by Ficoll-Hypaque gradient centrifugation.Gelatin group and common plastic group were designated according to coating flasks with or without gelatin.Monocytes were harvested from each group and then were stimulated into dendritic cells.The number of monoctyes,CD14 positive rate of monocytes,contaminated T,B lymphocytes,the expression of CD1a,CD83 on immature and mature dendritic cells were determined.Cell viability was determined by trypan blue staining.Platelet contamination was compared between gelatin and common plastic groups.RESULTS AND CONCLUSION:The number of monocytes and CD14 positive rate were significantly higher in the gelatin group than in the common plastic group(P < 0.05).Contaminated T,B lymphocytes were significantly more in the common plastic group than in the gelatin group(P < 0.05).There were no significant differences in cell viability and dendritic cell phenotype between gelatin group and common plastic group(P > 0.05).Platelet contamination rate was significantly lower in the gelatin group than in the common plastic group.Gelatin-coated surfaces can effectively and simply isolate monocytes and successfully stimulate them into mature dendritic cells.

目的 研究高表达BCR/ABL融合蛋白对人鞘氨醇激酶(SPK)表达、活性及1-磷酸鞘氨醇(S1P)分泌的影响.方法 使用pLXSN和pLXSN-bcr/abl的重组逆转录病毒感染HL-60和ECV304细胞并筛选出pLXSN和pLXSN-bcr/abl的细胞.利用RT-PCR鉴定病毒转染.Western Blot检测SPK表达,γ-32P-ATP掺入法检测细胞SPK活性和培养上清的S1P含量.应用同样方法检测不同剂量Gleevec对K562细胞培养上清中S1P的影响.结果 RT-PCR结果显示bcr/abl基因转染成功. 高表达BCR/ABL融合蛋白的HL-60和ECV304均比转染空载体后的细胞SPK表达、活性和S1P分泌均显著提高.随着Gleevec浓度的增加,K562细胞培养上清中S1P的含量逐渐减少.结论 BCR/ABL融合蛋白可提高细胞SPK表达、活性和S1P分泌.

Objective:To purify the recombinant adenovirus by using chromatography.Methods:Source 15Q ion ex-change chromatography and Sepharose 4 Fast Flow size exclusion chromatography were used to purify Ad-GFP from the lysate of engineering cells HEK-293N3S infected by recombinant adenovirus and cultured in suspension.The purity of the product was determined by D260nm/D280nm and high performance liquid chromatography(HPLC).The infective titer was deter-mined by TCID50 assay.Reults:The D260nm/D280nm of the product was 1.21,and the purity determined by HPLC was 96.5%.The infective titer of the product was 1.0×1011 U /mL,the viral particle concentration was 1.76×1012/mL.The specific in-fectivity was 5.68%,which was higher than that required(3.3%)by the FDA,USA.Conclusion:We established the chromatography methods to purify recombinant adenovirus,and the purity and specific infectivity of the product meeting the standards established by State Food and Drug Administration(SFDA).

Adenovirus vectors are one of the most promising gene transfer systems. They are of great value for gene therapy because these vectors achieve temporal high-level transgene expression and high gene transfer efficiency. To meet increasing needs of adenovirus vectors for gene therapy programs, parallel development of efficient, scalable and reproducible production processes is required. Perfusion cultivation of 293 cells is one of the most commonly used methods to produce adenovirus vectors and it is suitable for industrialized production specially. Experimental studies had been carried out to produce recombinant adenovirus containing the green fluorescent protein gene (Ad-GFP) by perfusion cultivation of HEK-293 N3S cells in a 5L stirring bioreactors. Perfusion rate was 1-2 volume/day. To infect the 293 N3S cells with Ad-GFP at the density of (2-4) x 10(6) cells/ ml. The time of collecting cells was 48 hours post infection. After three rounds of freeze/thaw and centrifugation, the crude viral lysates were stored at--80 degrees C until use. Then to get the Ad-GFP products by 2 x CsCl-gradient purification. The purity of the products was determined by the A260/A280 ratio and a high performance liquid chromatography (HPLC) assay. The infective titer was determined by a TCID50 assay. The culture term was 10-12 days. The infectious titer, the number of virus particle and the ratio of infectious titer to virus particle for the product were 1.0 x 10(11) IU/mL, 1.68 x 10(12) VP/mL and 6.0% IU/VP respectively. The A260/A280 ratio was 1.33, and the purity determined by HPLC was 99.2%. The cell specific productivity was around 1000 IU/cell. By perfusion cultivation of 293 N3S cells in a 5L stirring bioreactors, we established the production process for Ad-GFP, which paves a way to produce other recombinant adenovirus for gene therapy.

近十年来,腺病毒载体已经成为了基因治疗的有效载体,各种重组腺病毒在抗肿瘤和治疗遗传病等方面发挥了重要作用。本文介绍了腺病毒载体系统的特点,腺病毒的感染动力学及包装细胞代谢的变化和定量计数方法,腺病毒生产和纯化方法及其产品的质量控制,对腺病毒生产的发展趋势和存在问题进行了阐述。

Objective: To investigate the role of replication-defective adenovirus vector encoding human extracellular domain of vascular endothelial growth factor receptor-2 (hVEGFR-2 or hKDR) in induction of immunity against murine hepatocellular carcinomas and in breaking the immune tolerance. Methods: Ad hKDRE were constructed and were used to immunize C57BL/6 mice. Seven days after immunization mice splenocytes were collected as effectors and Hepa 1-6/mKDR cells were used as target cells for lactate dehydrogenase (LDH) release assay to analyze the cytotoxic activity of antigen-specific cytotoxic T lymphocytes (CTL). In addition, immunized mice were transplanted with Hepa 1-6 cells and the survival of tumor-bearing mice was observed. Results: Seven days after the immunization, 6 h cytotoxic activity of CTL elicited by the Ad hKDRE were (81.5±5.6) %, (68.4± 5.5) %, and (39.6±3.9) % at the ratio of effector∶target (E∶T) of 100∶1, 50∶1, and 25∶1, respectively. In addition, no tumor formed 2 months after implantation of 2×106 hepatoma cells in Ad hKDRE-immunized mice; after implantation of 5×106 hepatoma cells, 60% of the mice survived without bearing tumor. The above CTL activity and protection against!tumor disappeared when CD8+ and CD4+ T lymphocytes were depleted from mice. Conclusion: Adenovirus vector-mediated xenogeneic extracellular domain of KDR can effectively break the immune tolerance to murine hepatocellular carcinomas in mice and induce a strong antigen-specific T cell response, which is dependent on CD8+ and CD4+ T cells.

Objective:To explore the differences between the influences of hepatocyte growth factor (HGF) on the fibroblasts of normal skin and that of hypertrophic scar tissue with the aid of transfection of the recombinant adenovirus carrying HGF gene (Ad-HGF). Methods:Primary cultured fibroblasts from normal skin and hypertrophic scar were transfected with the recombinant adenovirus carrying green fluorescence protein gene (Ad-GFP) at different multiplicity of infection (MOI), and the transfection efficiencies were evaluated by flow cytometry.Levels of HGF, matrix metalloprotease-1 (MMP-1) and TGF-β1 in cell culture medium were determined by enzyme linked immunoadsorbent assay (ELISA) after the fibroblasts were transfected with Ad-HGF at 100 MOI. Immunohistochemistry was used to determine the expression of MMP-1 and TGF-β1 in normal skin and hypertrophic scar.Results:There was no significant difference from the transfection efficiencies and the expressions of HGF between the two fibroblasts. HGF could obviously promote the expression of MMP-1 in HFB, and inhibit the secretion of TGF-β1 in HFB only. The results of immunohistochemistry revealed that the difference in the expression level of MMP-1 and TGF-β1 between normal skin and hypertrophic scar was distinct.Conclusion:The mechanism underlying the efficacy of HGF in treatment of hypertrophic scar may be associated with the ability of HGF to inhibit TGF-β1 expression and promote MMP-1 secretion in hypertrophic scar fibroblasts.

Objective:To observe in vivo anti-tumor effect of adenovirus-mediated human wild-type p53,GM-CSF and B7-1 genes,their synergistic effects with chemotherapy or radiotherapy on Lewis lung cancer in mouse.Methods:Lewis lung cancer model in C57 mouse was established and the effective dose was determined by intratumorally injecting different dosages of Ad-p53,GM-CSF and B7-1 agent.The synergistic effects of recombinant adenovirus with chemotherapy or radiotherapy were observed at the same time.Results:It was shown that 5×10~8pfu of recombinant adenovirus had significant therapeutic effect on Lewis lung cancer.In addition,when combined with chemotherapy or radiotherapy,the agent remarkably increased chemotherapy or radiotherapy sensitivity of cancer cells.Conclusion:Ad-p53,GM-CSF and B7-1 agent had satisfactorily therapeutic effect on Lewis lung cancer in C57 mouse.And the recombinant adenovirus significantly increased sensitivity of chemotherapy or radiotherapy by Lewis lung cancer cells.

The purpose is to construct a recombinant adenovirus carrying human hepatocyte growth factor(HGF) gene (Ad-HGF). Then it was multipiled and purified and its quality was evaluated. To construct a shuttle recombinant plasmid carrying HGF gene (pXCJL1-CMV/HGF/pA), the plasmid was subjected to co-transfection with another plasmid GT4050 containing replication-defective Ad5 lacking E1 and E3~( )domains in 293 cells. This recombinant replication-defective adenovirus (Ad-HGF) was recovered by homogenous recombination. The presence of HGF cDNA in the viral genome was verified by PCR. Ad-HGF was multiplied to a large quantity in 293 cells growing in DMEM containing 5% FBS. Then Ad-HGF was purified by cesium chloride gradient centrifugation and desalted using Dialysis Cassettes. The particle number and the purity of Ad-HGF were determined by absorption at A_(260()nm) and the ratio of A_(260()nm)/A_(280()nm) using a DU-640 spectrophotometer. The final plaque-forming units (PFU) were determined by titration on 293 cells under an agar overlay (Low Gelling Temperature, Sigma). The presence of replication-competent adenovirus was detected. A recombinant replication-defective adenovirus carrying HGF gene was successfully constructed. The adenovirus has good purity and high viral titer, and the replication-competent adenovirus was not detected. The Ad-HGF constructed and prepared here has a potential application value.

To detect the effect of different proportions of supercoiled plasmid on transfer efficiency and expression level in target cells. A plasmid pcDNA3-HGF carrying human hepatocyte growth factorHGF gene and a plasmid pcDNA3-LacZ carrying LacZ reporter gene at first were constructed then expanded and purified supercoiled plasmids of different proportions with DNA purification kit. The proportion of supercoiled plasmids was determined by a Gel image analysis system. Subsequently transfection of the plasmids to fibroblast NIH3T3 cells was carried out in vitro by LipofectAMINE reagent. The transfer efficiency was observed by X-Gal staining and the expression level was quantitated by ELISA. The transfer efficiency of supercoiled plasmid pcDNA3-LacZ 85.45% and 48.44% proportions to NIH3T3 cells was 23.4%±3.8% and 9.3%±2.5% respectively at 48 h after transfection. The former is 2.5 times than the later. The amount of expressed HGF of supercoiled plasmid pcDNA3-HGF with 93.28% and 40.53% proportions in 1×106 NIH3T3 cells was 46.5±6.3 ng and 25.6±4.2 ng respectively insupernatant at 48 h after transfection. The former is 1.8 times than the later. These results demonstrate that proportion of supercoiled plasmid may affect its transfer efficiency and expression level in target cells. Higher proportion of supercoiled plasmid may lead to higher transfer efficiency and expression level.