Establishment of a method for the evaluation of emergency granulopoiesis in mouse bone marrow with EdU / 中华微生物学和免疫学杂志

Objective To label granulocytes in a state of differentiation in mouse bone marrow (BM) with EdU (5-ethynyl-2′-deoxyuridine) for further understanding the changes in granulocyte produc-tion at different stages of differentiation during inflammation. Methods C57BL/6 mice were intraperitoneal-ly (i.p.) injected with EdU and heat-inactivated Escherichia coli(HI E.coli). BM cells were harvested at different time points after HI E.coli injection and then stained with fluorescent-conjugated antibodies(Abs). Myeloblasts,promyelocytes,myelocytes, metamyelocytes and band and segmented neutrophils were identi-fied by fluorescence-activated cell sorting(FACS). The percentage of EdU-positive cells in each population was recorded. Results The percentage of EdU-positive myeloblasts in mice increased by 10.0% at 24 h af-ter intraperitoneal injection with HI E.coli,but decreased by 75.0% and 23.0% at 48 h and 72 h,respec-tively. The percentage of EdU-positive promyelocytes declined by 23.0%,54.5%,64.3% and 77.8% at 24 h,48 h,72 h and 96 h,respectively. The percentage of EdU-positive myelocytes increased by 60.0% and 10.0% at 24 h and 48 h,but decreased by 80.0% and 90.0% at 72 h and 96 h. The percentage of EdU-positive metamyelocytes increased by 50.0% at 24 h,but decreased by 33.3%,61.5% and 66.7% at 48 h,72 h and 96 h. The percentage of EdU-positive band and segmented cells increased by 14.0% at 24 h,but decreased by 50.0%, 77.8% and 88.0% at 48 h, 72 h and 96 h. Conclusion Emergency granulopoiesis occurred 24 h after the establishment of HI E.coli-induced model of acute peritonitis, which meant that the proliferation of myeloid precursor cells,especially that of myelocytes and metamyelocytes,was accelerated and resulted in increasing number of mature neutrophils immigrating to sites of inflammation.

Vascular endothelial injury induced by anti-endothelial cell antibody in allogeneic hematopoietic stem cell transplantation / 中华血液学杂志

<p><b>OBJECTIVE</b>To clarify the role of endothelial cells (ECs) injury induced by anti-endothelial cell antibody (AECA) in allogeneic hematopoietic stem cell transplantation (allo-HSCT).</p><p><b>METHODS</b>Serum immunoglobulin (IgG) from allo-HSCT recipients were purified and incubated with human umbilical vein vascular endothelium (HUVEC) in vitro, then the functional changes and cell apoptosis were tested.</p><p><b>RESULTS</b>After incubation with AECA positive IgG, soluble adhesion molecules significantly elevated in culture supernatant. When concentration of IgG was 160, 320, and 640 μg/ml, concentrations of soluble intercellular adhesion molecule-1 in supernatant were statistically higher in AECA positive groups [(117.10 ± 12.82) vs (78.17 ± 4.90) pg/ml, (151.30 ± 15.35) vs (89.46 ± 6.02) pg/ml, (239.00 ± 32.53) vs (127.80 ± 13.86) pg/ml, P<0.01)]. When concentration of IgG was 40, 80, 160, 320, and 640 μg/ml, concentrations of soluble vascular cell adhesion molecule-1 in supernatant were also statistically higher in AECA positive groups [(38.51 ± 3.76) vs (24.78 ± 2.59) pg/ml, (61.34 ± 6.99) vs (38.20 ± 3.17) pg/ml, (135.60 ± 24.46) vs (63.73 ± 5.08) pg/ml, (221.30 ± 29.40) vs (112.80 ± 8.91) pg/ml, (420.90 ± 31.70) vs (224.40 ± 20.79) pg/ml, P<0.01]. Clotting activity factors also elevated in culture supernatant after incubation with AECA positive IgG. When concentration of IgG was 80, 160, 320, and 640 μg/ml, concentrations of von Willebrand factor were statistically higher in AECA positive groups [(19.51 ± 0.72) vs (17.17 ± 0.60) ng/ml, P=0.0193; (22.97 ± 1.18) vs (18.27 ± 0.61) ng/ml, (26.40 ± 1.54) vs (19.53 ± 0.70) ng/ml, (34.35 ± 1.60) vs (23.81 ± 0.92) ng/ml, P<0.01]. When concentration of IgG was 320 and 640 μg/ml, concentrations of thrombomodulin were statistically higher in AECA positive groups [(57.50 ± 4.50) vs (40.31 ± 4.39) pg/ml, P=0.0132; (59.18 ± 4.11) vs (38.84 ± 5.16) pg/ml, P<0.01]. However, inflammatory factors (IL-1β, IL-6, IL-8 and ANG2) were not statistically different in AECA positive and negative groups (P>0.05). Moreover, IgG from AECA positive samples did not change the proliferation or cell apoptosis.</p><p><b>CONCLUSION</b>AECA from allo-HSCT recipients dysregulates ECs' function in vitro, but do not induce apoptosis, which is valuable in the pathophysiology of graft-versus-host disease (GVHD) and other complications after allo-HSCT.</p>

Screening and identification of a novel small-molecule TNFβinhibitor / 天津医药

Objective To explore a novel and highly specific small-molecule TNFβinhibitor by using computer-aid?ed virtual screening and cell-based assays in vitro. Methods Computer-aided drug design and virtual screening were used to design and identify chemical compounds that targeted TNFβbased on the crystal structure of the TNFβ-TNFR1 com?plex. The effect of the small-molecule compound against TNFβ-induced cytotoxicity of L929 cells was detected by MTT as?say, and the efficacy of the compound to inhibit TNFβ-induced apoptosis of L929 cells was determined by flow cytometry as?say. The impact of the compound on L929 cell cycle was examined by Propidium Iodide (PI) staining and flow cytometry, and the influence of the compound on TNFβ-triggered signal pathway was analyzed by Western blot assay and Ultra VIEW VOX 3D Live Cell Imaging System. Results No.35 compound (named as C35 thereafter) could effectively inhibit TNFβ-induced cell death in a dose dependent manner, and the half-maximum inhibition concentration (IC50) was 8.19μmol/L. Furthermore, C35 had lower cytotoxicity and minimal effect on L929 proliferation. Here we further revealed that C35 could affect TNFβ-induced apoptotic pathway by blocking the activation of Caspase 3, and markedly reduce L929 cell apoptosis induced by TNFβ. Conclusion A novel TNFβsmall-molecule inhibitor was identified by combining computer-aided virtual screening with functional assays, and which could block TNFβ-triggered apoptotic pathway and efficiently inhibit the cell death in?duced by TNFβ.

Construction and expression of disulphide stabilized anti-CD3/anti-Pgp diabody / 生物工程学报

We constructed and expressed an anti-CD3/anti-Pgp (P-glycoprotein) diabody previously. However, the two chains of diabody are associated non-covalently, resulting in being capable of dissociating. The aim of this study is to enhance the stability of the diabody. We introduced cysteine residues into the CD3 or Pgp V-domain to covalently lock the two chains together. The disulphide crosslinked diabody were expressed by Escherichia coli (E. coli) 16C9 and purified by a cation exchange column and an anti-Etag affinity chromatography. The purified proteins were verified through SDS-PAGE. Flow cytometry (FCM) was used to analyse the binding properties, competitive binding capacity and stability in vitro. The dsPpg-diabody failed to form disulphide bond properly. The designed disulphide bridge between the different chains of dsCD3-diabody was formed correctly. FCM demonstrated the dsCD3-diabody has specific antigen binding activity, the same binding activity and competitive binding activity as its parent diabody. The dsCD3-diabody retained the full activity even after 72 h incubation at 37 degrees C in human serum, in contrast, the parent diabody began to lose activity after only 1 h and lose all its activity 24 hours later. The induced disulphide bond in the CD3 V-domain effectively enhanced the stability of anti-CD3/anti-Pgp diabody. The method of stabilizing a diabody by introducing a disulphide bond into is practical.

Soluble-expression, purification and activity analysis of extracellular domain III of flt1 / 生物工程学报

To prepare a soluble human extracellular III domain of Flt1 and analyze its biological activity. The gene encoding extracellular domain III of Flt-1 was cloned into the expression vector pAZY by RT-PCR from human umbilical vein endothelial cell (HUVEC), and induced to express in Escherichia coli by low phosphoric medium, the product was purified by E-tag affinity chromatography. SDS-PAGE and Western blotting analysis showed that Flt-1 gene domain III gene was expressed in E. coli and the yield of the soluble fusion protein was about 1.10 mg/L. Enzyme-Linked ImmunoSorbent Assay (ELISA) revealed that the Flt-1 domain III was able to bind to VEGF165 dose-dependently. Monolayer denudation assay and Transwell assay showed that the fusion protein could inhibit HUVECs migration induced by conditional medium with 50 ng/mL VEGF165 and 100 ng/mL bFGF. In conclusion, Flt-1 gene domain III gene has been successfully cloned and expressed in E. coli, which will be useful in both the research on the function of Flt-1 gene domain III and preparation of anti-Flt-1 monoclonal antibody in the future.

Construction,expression and in vitro specific killing activity of a recombinant fusion protein:anti-CD20Fab-LDM / 中国药理学通报

Aim To construct and express anti-CD20Fab-LDP,generate anti-CD20Fab-LDM and identify its biological activity.Methods PCR and overlapping PCR were used to construct anti-CD20Fab-LDP.DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide.The product was purified by affinity chromatography and analyzed by Western blot and its antigen-binding activity was examined by FACS.Specific killing activity in vitro of anti-CD20Fab-LDM was analyzed by MTT.Results The data of DNA sequence showed that anti-CD20Fab-LDP was correct.The fusion protein was recovered in high yield(up to 4 mg·L~(-1))after proteinG purification.The fusion protein could bind to Raji cells(CD20+),and similar affinity data were obtained with anti-CD20Fab.Anti-CD20Fab-LDP showed potent cytotoxicity to Raji cells with IC_(50) values of 0.9×10~(-10) mol·L~(-1).Conclusions Anti-CD20Fab-LDP with high level expression was successfully obtained and could bind to Raji cells cells.Anti-CD20Fab-LDM showed specific killing activity to Raji cells in vitro.

Reversal multi-drug resistance by reducing the expression of CK8 and BCRP / 中国药理学通报

Aim To determine whether membrane cytokeratin 8(CK8 )and BCRP expression cooperatively contributed to multidrug resistance(MDR)in MCF-7/MX cells.Methods MCF-7/MX cells were transfected with specific anti CK8-siRNAs and anti BCRP-siRNAs via LipofectAMINE2000.The expression of CK8 and BCRP was determined using Western blot,and membrane staining was observed by laser confocal microscopy.Sensitivity to chemical drugs was examined by Sulforhodamine B method.Results The expression levels of cell surface CK8 and BCRP were obviously reduced by siRNAs,and inhibition of CK8 and BCRP expression could effectively restore the sensitivity to drugs and reverse MDR phenotype of MCF-7/MX cells.Conclusions CK8 together with BCRP may play significant roles in conferring the multifactorial MDR phenotype of MCF-7/MX cells,but may act independently via potentially different mechanisms.Combinational approaches that target multiple drug-resistance-related molecules/pathways in cancer cells may represent more efficacious strategies to overcome MDR.

Study on the construction and expression of anti-CD3/anti-CD20 Diabody / 中国免疫学杂志

Objective:To construct and express anti CD3/anti CD20 Diabody and identify its biological activity.Methods:PCR and overlap PCR were used to construct anti CD3/anti CD20 Diabody.DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide.The product was purified by affinity chromatography and analyzed by both the detection of Western blot and size exclusion chromatography;its antigen binding activity was examined by FACS and rosetting assay.Results:The data of DNA sequence showed that the anti CD3/anti CD20 Diabody was corrected.The Diabody was recovered in high yield(up to 1 mg/ml) after E taq purification and predominantly(90%) as a dimer.The Diabody binded Jurkat cells(CD3 +) and Daudi cells (CD20 +),respectively.Furthermore,the Diabody was capable of simultaneous binding to Jurkat cells and Daudi cells as shown by cellular rosetting.Conclusion:The anti CD3/anti CD20 BsF(ab') 2 was first recast into the Diabody format and succeeded to obtain high level expression.The results of some biological activity experiments indicated that the Diabody could bind to Jurkat cells and Daudi cells.

The metastasis suppressing ability and its mechanism of calmodulin antagonist EBB in human metastatic ovarian clear cell line ES-2 / 中国药理学通报

Aim To investigate the potential suppressing effect of calmodulin antagonist EBB on metastasis-associated properties of human metastatic ovarian clear cell adenocarcinoma cell ES-2.Methods MTT assay was used to assess the growth inhibition of EBB on ES-2 cells.The invasive capacity and motility potential were determined by Transwell chamber assay and Wound assay.[Ca2+]i was observed under the laser scanning confocal microscopy.Results EBB inhibited the proliferation of ES-2 cells in vitro.The IC50 on ES-2 cells was(13.67?1.56)?mol?L-1.The invasive ability and motility potential of ES-2 cells were decreased after exposure to 3,7 and 14 ?mol?L-1 EBB respectively(P