[Show abstract][Hide abstract] ABSTRACT: Objective
To observe enhancement of anti-tumor immunity by gene vaccine using nucleofection technology
The technique of nucleofection was used to transfer effectively plasmid DNA into immature dendritic cells (iDCs); we studied immune responses regulated by DNA vaccine using real-time quantitative polymerase chain reaction (PCR) and western-blotting to optimize the follow-up lymphocyte activation. The anti-tumor capacity of lymphocytes primed by DCs was analyzed using lactate dehydrogenase with a non-radioactive cytotoxicity assay
Human monocyte-derived dendritic cells (hMoDCs) were induced by interleukin (IL)-4 and granulocyte-macrophage colonystimulating factor (GM-CSF) in vitro from human monocytes for 5 or 6 days. DNA vaccine was transfected to iDCs with high transfection (35.73%) using nucleofection. Compared with the iDC group, the expression of Th1 cell cytokine IL-12, IL-18 and Th2 cell cytokine IL-4 increased after stimulation. CD86 and CD83 were upregulated compared with non-nucleofected groups 48 hours after nucleofection with DC-pVAX-PRA. The result of the cytotoxicity assay showed that DCs-pVAX-PRA primed non-adherent peripheral blood mononuclear cells (PBMCs) exhibit their highest cytotoxicity against target cells.
The results show that DNA vaccine was transfected to iDC with high transfection efficiency using nucleofection, priming autologous lymphocytes for anti-tumor immunity by upregulated expression of co-stimulatory molecules, adhesion molecules and cytokines. These results provided a basis to explore the molecular mechanism of DNA vaccine in vivo.
Preview · Article · Jun 2011 · Clinical Oncology and Cancer Research
[Show abstract][Hide abstract] ABSTRACT: To optimize a pretreatment method of urine proteomics in children with primary nephrotic syndrome.
Urine from children with primary nephrotic syndrome was treated in different pH and isolated by cold acetone precipitation for different durations. Then the amounts and kinds of proteins were compared by quantify, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE) in order to optimize a way to deal with urine protein.
Most proteins were obtained at pH 2.7. The amounts of protein precipitated by acetone for 0.5 hr was obviously less than those precipitated for 1 and 2 hrs (P<0.05), while there was no significant difference between the amount of protein precipitated for 1 and for 2 hrs. Protein precipitated by cold acetone for 1 hr at pH 2.7 was selected as the best pretreatment method. Satisfactory 2-DE maps can be acquired.
Urine protein can be best obtained at pH 2.7 and precipitated by cold acetone for 1 hr.
No preview · Article · Feb 2011 · Zhongguo dang dai er ke za zhi = Chinese journal of contemporary pediatrics
[Show abstract][Hide abstract] ABSTRACT: To explore the antineoplastic effect in vitro of earthworm coelomic fluid (ECF)on growth inhibition and its mechanism for the tumor cell lines Siha, SW480, Colo205 and PC12.
MTT colorimetric assay, flow cytometry and morphological analysis were used to test its antitumor activity on tumor cell lines and normal cell line Cos7 in vitro.
ECF can inhibit the cell growth of Siha, SW480, Colo205, PC12 and Cos7. But different tumor cell lines showed different sensitivity.
EFC can significantly inhibit the proliferation of tumor cells in vitro by inducing tumor cells apoptosis.
No preview · Article · Dec 2010 · Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology
[Show abstract][Hide abstract] ABSTRACT: To explore the antivirus function in vitro of earthworm coelomic fluid (ECF) by researching its effect on inhibiting respiratory syncytial virus (RSV).
By the method of Hep-2 cell culture and using ribavirin as a positive control, the anti-RSV effect of ECF was investigated by observing cytopathic effect (CPE) with MTT colorimetric assay.
In Hep-2 cells, the CC50 of ECF and ribavirin were 3.11 mg/ml and 1.35 mg/ml separately. In the experiment of ECF directly killing RSV, the IC50 of ECF was 184.1 microg/ml, SI was 16.87; In the experiment of ECF preventing RSV invasion, no antiviral function of ECF within the experimental concentration range was observed; In the experiment of ECF inhibiting RSV replication, the IC50 of ECF was 1555. 8 microg/ml, SI was 1.99.
ECF couldn't prevent virus from invading into host cell, but showed direct killing-virus function and inhibition of the virus replication.
No preview · Article · Apr 2010 · Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology
[Show abstract][Hide abstract] ABSTRACT: To explore the conditions for high expression of anti-HBsAg scFv A-15 in E. coli, increase the production of the scFv in the culture medium.
By changing induction occasion, concentration of inductor IPTG and induction time, influence of various conditions on expression of anti-HBsAg scFv A-15 was analyzed through ELISA. In addition, the effects of sucrose, glycine and Triton X-100 at different concentrations on the scFv excretion into culture medium was evaluation.
The optimal expression conditions were as follows: the induction was started after culturing for 4 h, the concentration of IPTG was 0.5 mmol/L, and the induction lasted for 8 h. The scFv affinity in culture medium with 0.3 mol/L sucrose, 2% glycine, 1% Triton X-100, 16.78-fold higher, respectively than that without the three chemicals. The final yield of anti-HBsAg scFv A-15 was estimated to be 7.4 mg/L.
The conditions for production of anti-HBsAg scFv A-15 were optimized, which provides a practical method for more efficient production of the scFv in E. coli for further studying structure and function.
No preview · Article · Feb 2009 · Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology
[Show abstract][Hide abstract] ABSTRACT: To purify a kind of deoxyribonuclease from earthworm Eisenia foetida (named earthworm DNase, EDNase) and study its characteristics.
Acetone precipitation, ion-exchange chromatography, high performance liquid chromatography, SDS-PAGE, Capillary electrophoresis isoelectric focusing and MALDI-TOP MS were used for the study.
This purified protocol improved 137-fold purification and 45.6% recovery of enzyme activity. The molecular mass of EDNase was estimated to be 63,000. Mg2+, Mn2+ and Ca2+ were strong inhibitors of EDNase, while Na+ slightly increased the enzyme activity. The enzyme was completely stable in the pH range from 4.4 to 5.2 and had a pH optimum of 4.8. The optimum temperature was 37 degree C and the enzyme was stable up to 40 degree C. The pI of the enzyme was 6.20. Km and Vmax for the enzyme were 1.52 g/L and 4.89 mg/(mL.min), respectively, with calf thymus DNA as substrate. The enzyme was able to degrade chromosomal DNA, linear lambda-bacteriophage DNA as well as supercoiled plasmid DNA, but didn't display any RNase activity.
This kind of deoxyribonuclease possesses unique characteristics, which is different from the deoxyribonucleases which we have known before.
No preview · Article · Nov 2008 · Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences