SENP1 induced protein deSUMO modification increased the chemotherapy sensitivity of endometrial cancer side population cells / 中华肿瘤杂志

Objective: To inhibit the stemness maintenance potential of endometrial cancer and increase the sensitivity of endometrial cancer side population cells to chemotherapy drugs by inducing extensive deSUMOylation modification of proteins. Methods: Flow cytometry was used to sort and culture CD133(+) CD44(+) KLE endometrial cancer cell clone spheres. Protein expression level of small ubiquitin-related modifier 1 (SUMO1) and two stemness maintenance genes of tumor side population cells, octamer binding transcription factor-4 (Oct4) and sex determining region Y-box2 (Sox2), were detected by western blotting method. Lentivirus-mediated Sentrin/SUMO-specific proteases 1 (SENP1) gene was stably transfected into KLE side population cells. Western blotting was used to detect the protein expressions of SENP1, SUMO1, Oct4 and Sox2. The clone formation rate was compared between KLE side population cells with or without SENP1 overexpression. Flow cytometry was applied to detect cell cycle changes. 3-(4, 5-Dimethylthiazole-2)-2, 5-diphenyl-tetrazolium bromide (MTT) experiment and flow cytometry apoptosis method were used to detect the chemosensitivity of the side population of endometrial cancer cells to cisplatin. Tumor-bearing mouse models of endometrial cancer were established to detect the effect of SENP1 overexpression on the chemotherapy sensitivity of cisplatin. Results: Compared with CD133(-)CD44(-) KLE cells, CD133(+) CD44(+) KLE side population cells could form clonal spheres and express higher levels of SUMO1, Oct4 and Sox2 proteins (P<0.05). Compared with KLE side population cells that were not transfected with SENP1 gene, the expression level of SENP1 protein in KLE side population cells overexpressing SUMO1、Oct4 and Sox2 were lower. The clonal sphere formation rate was reduced from (25.67±5.44)% to (7.46±1.42)%, and cell cycle shifted from G(0)/G(1) phase to G(2) phase. IC(50) of cisplatin decreased from (55.46±6.14) μg/ml to (11.55±3.12) μg/ml, and cell apoptosis rate increased from (9.76±2.09)% to (16.79±3.44)%. Overexpression of SENP1 could reduce the tumorigenesis rate of KLE side population cells in vivo and increase their chemotherapy sensitivity to cisplatin (P<0.05). Conclusion: Overexpression of SENP1 can induce protein deSUMOylation modification, inhibit the stemness maintenance potential of endometrial cancer side population cells, and enhance their chemotherapy sensitivity, which provides a new reference for gene therapy of endometrial cancer.

A new view of treatment for bronchiolitis / 中国实用儿科杂志

Bronchiolitis is a common lower respiratory tract infection in infants and young children, mostly caused by respiratory syncytial virus. At present, the treatment mainly includes oxygen therapy, control of wheezing, maintenance of internal environment stability and so on. Most cases of bronchiolitis still lack specific antimicrobial agents. To explore a new treatment method for bronchiolitis is helpful to improve the symptoms, shorten hospitalization days,and improve the prognosis of children with moderate to severe bronchiolitis, especially those younger than 6 months with high risk factors.

Detection of neutralizing antibody to human adenovirus type 5 in marmosets / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a recombinant human adenovirus type 5 (Ad5) expressing luciferase and GFP reporter gene and detect neutralizing antibodies against adenovirus type 5 in common marmosets (Callithrix jacchus) to provide basic laboratory data for evaluating adenovirus vaccines.</p><p><b>METHODS</b>Luciferase and GFP reporter genes from plasmid pHAGE-CMV-GFP were inserted into pDC315 to construct the recombinant adenovirus shutter plasmid pDC315-Luc-GFP. The shutter plasmid was co-transduced with pBHGlox(delta)E1,3Cre in 293A cell line to package the recombinant adenovirus rAd5/Luc/GFP. Three rounds of plaque formation experiment were performed to select the monoclonal adenovirus followed by purification with cesium chloride density gradient centrifugation and virus titration with TCID50 method. Chemiluminescence assay and flow cytometry were employed to detect the neutralizing antibody levels in 14 common marmosets.</p><p><b>RESULTS</b>The shuttle plasmid pDC315-Luc-GFP was successfully constructed and the recombinant adenovirus rAd5/Luc/GFP was packaged with a the titer reaching 6.9×10(11.5) PFU/mL. In the 14 marmosets, chemiluminescence assay identified 4 (28.6%) marmosets that were positive for Ad5-neutralizing antibodies, including 2 with a antibody titer of 1/16 and another 2 with a titer of 1/32; flow cytomery detected Ad5-neutralizing antibodies in 3 marmosets at the titer of 1/16.</p><p><b>CONCLUSION</b>Chemiluminescence assay is a simple, sensitive, and accurate modality for detecting Ad5-neutralizing antibodies. Common marmosets have a very low positivity rate for Ad5-neutralizing antibodies and are therefore promising models for studying adenovirus-based vaccines and therapies.</p>

Preparation and characterization of monoclonal antibodies against BP26 protein of Brucella melitensis M5-90 / 中国地方病学杂志

ObjectiveTo prepare high specific monoclonal antibodies(mAbs) against BP26 of Brucella(B.)melitensis.Methods A recombinant plasmid pET-28a-BP26 was constructed and transformed into competent Escherichia coli BL21 (DE3),and then the bacteria were induced by 1 mmol/L isopropylthio-β-D-galactoside (IPTG).After induction,the recombinant BP26 protein (rBP26) was purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PGAE) and nickel ion affinity chromatography(Ni-NTA).Mice were inoculated with rBP26 antigens for three times at 2-week intervals.The first subcutaneous injection contained 100 μg rBP26 with 0.1 ml complete Freund adjuvant.The second subcutaneous injection was 50 μg rBP26 with 0.1 ml incomplete Freund adjuvant.The antibody titers to rBP26 were determined 2 weeks after each reimmunization.Three days before cell fusion,the mice with the highest titer were intraperitoneally injected with 50 μg rBP26 in 0.1 ml PBS.Pre- and post-immunization sera were collected and used as negative or positive controls for screening mAbs.Mice with the highest titer were sacrificed and spleen cells were isolated.The spleen cells of rBP26 immunized mice were fused with SP2/0 myeloma cells in a ratio of 5 ∶ 1 by polyethylene glycol(PEG) 1450.Antibody-producing hybridomas were primarily screened by an indirect enzyme-linked immunosorbnent assay(ELISA) with rBP26.Reactive hybridomas were subcloned for 3 times,then the strains of hybridoma cells secreting antibodies against BP26 were obtained.Supernatant of cloned hybridoma cultures was collected for mAb analyses.These mAbs were named by the hybridoma clone number and tested their reactivity to membrane proteins extracted(NMP) from B.melitensis vaccine strain(M5-90) by Western blotting and Dot-ELISA.mAbs isotyping and kappa(κ) or lambda(λ) light chain was identified by Mouse Monoclonal Antibody Isotyping Kit.Results A total of two mAbs reactive to rBP26 of B.melitensis were selected from antibody screening hybridomas by indirect-ELISA.The two mAbs were named 3C3 and 5A5,and identified as IgG1 (κ) and IgG2(κ),respectively.They could react with NMP from M5-90.Conclusions Results of identification show that two mAbs against rBP26 can be produced.The two mAbs can recognize natural BP26 protein,giving the experimental materials for further research on identification of its epitopes.

A comparative analysis of the methods for titering adenoviruses / 南方医科大学学报

<p><b>OBJECTIVE</b>To compare different methods commonly used for titering adenovirus and analyze the advantages and limitations of each method.</p><p><b>METHODS</b>Four recombined adenoviruses (Ad-G-AT2R-EGFP, Ad-CMV-EGFP, Ad-mif-shRNA-EGFP and Ad-CBA-GFP) were amplified and purified, and each was titered by optical absorbance, real-time PCR, green fluorescent protein (GFP)-labeled method, immunoassay, and cytopathic effect (CPE). The results were then comparatively analyzed.</p><p><b>RESULTS</b>No significant difference was found in the titer amounts derived from GFP-labeled method, immunoassay, and cytopathic effect method (P>0.1). A positive correlation was noted in the titer amounts determined by real-time PCR and immunoassay (r=0.965), even though the value (vg/ml) obtained by real-time PCR was 10 times higher than that by immunoassay (ifu/ml).</p><p><b>CONCLUSION</b>GFP-labeled method and immunoassay allow rapid determination of the adenoviral titer. Real-time PCR can not directly determine the real infectious titer of the adenovirus, but the result is well correlated to that of immunoassay and reflects, though indirectly, the actual infectious titer of adenovirus. Considering the procedural convenience and shorter time consumption, real-time PCR is still a practical method for adenoviral titration.</p>

Construction of an expression vector of GPC-3 CTL epitope / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct the expression vectors of GPC-3 CTL epitope.</p><p><b>METHODS</b>The HBsAg gene with three different EYILSLEEL (EYI) sites was named EYI1, and another with one EYI replacing CTL epitope FLG or SIL of pcHBsAg were named EYI2 and EYI3, respectively. All the three DNAs were amplified by SEOing PCR from pcHBsAg plasmid and linked into pBSSK+ vector to construct Pbssk/EYI1, pBSSK/EYI2, and pBSSK/EYI3. The three plasmid were identified by PCR, double digestion and sequencing, and the fragments with EYI1-3 were obtained by double digestion and then inserted into pcDNA3.1+ vector.</p><p><b>RESULTS AND CONCLUSION</b>PCR, enzyme digestion and sequence analysis confirmed successful construction of the eukaryotic expression vectors pcDNA-EYI1/HBsAg, pcDNA-EYI2/HBsAg, pcDNA-EYI3/HBsAg, which facilitate further studies of the GPC3-HBsAg multiple peptides vaccine for HBV infection.</p>