RNA m6A modification affects tumorigenesis and development of hematological malignancies / 中华检验医学杂志

N6-Methyladenosine (m6A) is one of the most prevalent RNA modifications in mammals. The m6A modification is catalyzed by m6A writers or erasers and involved in various RNA metabolic processes with the recognition by m6A readers. Recently, emerging studies have shown m6A modification is pivotal in fundamental bioprocesses including cell homeostasis and oxidative stress, programmed cell death, cell metabolism, and immune regulation, and accounts for tumoral occurrence and development. To date, abnormal m6A levels and dysregulated related enzymes participate in tumorigenesis and chemoresistance among acute leukemias, chronic myeloid leukemia, multiple myeloma, lymphomas, thus influencing patient prognosis. The mechanisms of m6A modification are sophisticated and varied in different types of malignancies or subtypes. Screening appropriate patients to apply m6A-targeted inhibitors is instructive to the precise treatment of hematological malignancies.

Mechanism of platelet aggregation induced by Streptococcus suis serotype 2 muramidase-released pro-tein (MRP) / 中华微生物学和免疫学杂志

Objective To study the mechanism of platelet aggregation induced by Streptococcus suis serotype 2 muramidase-released protein (MRP) and to provide scientific proof and theoretical basis for clinical treatment of patients with Streptococcus suis infection. Methods Nickel column affinity chromatogra-phy was used to purify recombinant proteins of MRP-N and MRP-C. Platelet aggregometer, thromboelastog-raphy (TEG) and scanning electron microscope were used to observe the platelet aggregation induced by MRP. Results Streptococcus suis 2 wild type strain,but not the mutant strain ΔMRP,could induce platelet aggregation. It was MRP-N but not MRP-C that induced platelet aggregation. GPRP,an inhibitor of β2inte-grin receptor,could significantly inhibit the platelet aggregation induced by MRP. Conclusion Streptococ-cus suis 2 MRP induces platelet aggregation through β2integrin receptor pathway.

Pathogenicity of ABC transporter SSU05 0946 of Streptococcus suis serotype 2 / 中华微生物学和免疫学杂志

Objective To construct a mutant strain of Streptococcus suis type 2 05ZYH33 express-ing ABC transporter SSU05 0946 and to study the pathogenicity of ABC transporter SSU05 0946 for better understanding the immune evasion strategies by Streptococcus suis. Methods Genome of the Streptococcus suis type 2 05ZYH33 strain was extracted and used as a template to amplify SSU05 0946 upstream and down-stream homeodomains. Chloramphenicol-resistance gene was amplified by using pSET1 plasmid as the tem-plate. These three amplified fragments were fused and integrated with the thermo-sensitive plasmid pSET4s by using overlap extension PCR. Homologous recombination method was used to construct the mutant strain 05ZYH33Δ0946. Differences between the mutant and wild type strains were evaluated through bacterial ad-hesion assay,whole blood killing assay and challenge test in mice and piglets. Results The mutant strain 05ZYH33Δ0946 was successfully constructed. Results of bacterial adhesion assay demonstrated that SSU05 0946 was not involved in the adherence of Streptococcus suis to human epithelial cells. SSU05 0946 was an ovel anti-phagocytic factor and virulence factor of Streptococcus suis. Conclusion Streptococcus suis type 2 ABC transporter SSU05 0946 is a newly discovered virulence factor of Streptococcus suis, playing an impor-tant role in the evasion of host innate immunity by Streptococcus suis.

Method comparison and accuracy of 15 commercial serum total protein assays / 中华检验医学杂志

Objective To evaluate the difference of Doumas′method and 15 commercial serum total protein ( TP ) methods based on EP9-A3.Methods Serum panels were quantified for TP with Doumas′method and measured in parallel with 15 commercial methods.The linear regression analyses were performed, followed by calculating relative deviation and 95%CI between commercial method and Doumas′method at three different medical decision levels (45 g/L, 60 g/L, 80 g/L).We also calculated relative deviation, 95% limit of agreement ( LoA ) and 95% CI based on classical and improved Bland-Altman method at three different medical decision levels.If both the relative deviation and 95%CI were within 5%, we conside red the commercial serum total protein method was comparable to Doumas′method.Results (1) All assays presented high correlation ( r>0.975, P<0.001) with the Doumas′method.All assays showed that the relative deviations and 95%CIs were within the biological total error goal (5%) at medical decision levels based on regression analysis.(2) Based on classical and improved Bland-Altman method, fourteen of 15 commercial methods showed that the relative deviations and 95%CIs were within +/-5%. Conclusions All commercial assays are comparable to Doumas′method at medical deviation levels.There is no difference between regression analysis and Bland-Altman method for comparison study.

Human bone marrow derived mesenchymal stem cells regulate Th17 cells of peripheral blood in vitro / 白血病·淋巴瘤

Objective To investigate the in vitro effects of human bone marrow derived mesenchymal stem cells (hBMSC) on Th17 cells of the human peripheral blood.Methods The density gradient centrifugation combined with lymphocyte separation medium was used to isolate hBMSC,which were then cultured.Cytokine IL-17 in the peripheral blood from a healthy person was measured by enzyme-linked immunosorbent assay (ELISA).Proportion of Th17 cells was evaluated by flow cytometry.Results The expression level of IL-17 in spent culture supernatant of the healthy person PBMC and AML hBMSC was (292.32±37.25) pg/ml,and was significantly higher than that of the healthy person PBMC and healthy hBMSC [(169.64±17.47) pg/ml,P ＜ 0.01].There was no significant difference between the expression level of IL-17 in spent culture supernatant of the healthy person PBMC and ALL hBMSC [(159.89±23.71) pg/ml] and the level of the healthy person PBMC and hBMSC.The percentages of Th17 cells of co-culture systems from hBMSC,ALL-hBMSC,and AML-hBMSC and PBMC were (10.13±2.19) ％,(13.77±4.04) ％ and (21.53±5.05) ％,respectively,with the result between AML patients and healthy people being statistically different (P ＜ 0.01).ALL patients and healthy people showed no difference (P ＞ 0.05).Conclusion AML-hBMSC promotes the CD~ T cells to generate Th17 cells,which suggests that the MSC from AML marrow may play a role in the regulation of immune suppression.

Splicing factor proline/glutamine-rich contributes to the sensitivity of acute myeloid leukemia HL-60 cells to doxorubicin / 白血病·淋巴瘤

Objective To investigate the anti-leukemia adriamycin (ADR) effect of membrane protein Splicing factor proline/glutamine-rich (SFPQ).Methods HL-60 and its adriamycin-resistant cells HL-60/ADR were cultured in vitro.Expression of SFPQ on HL-60 and HL-60/ADR cell membranes were examined by immunofluorescence.McAb 5D12 was used to block membrane SFPQ protein activity.ADR susceptibility and cell proliferation were analyzed by MTT assay.IC50 values of ADR in HL-60 and HL-60/ADR cell lines and up-regulations in cell proliferation induced by 5D12 were calculated.Intracellular accumulation of rhodamine in HL-60 and HL-60/ADR cells were measured using fluorescence-activated cell sorting.Results Expression of SFPQ on cell membrane was higher in HL-60 cells compared to the HL-60/ADR cell line.After membraneblocking with 5D12,ADR sensitivity was decreased in vitro compared with the untreated cells [the 48 h IC50 value,HL-60 cell line (0.19±0.03) μg/ml vs (0.95±0.13) μg/ml,HL-60/ADR cell line (14.41±2.42) μg/ml vs (21.33±4.26) μg/ml].Blocking of membrane SFPQ by 5D12 did not affect the intracellular accumulation of rhodamine in these cells,however,5D12 induced HL-60 and HL-60/ADR cell proliferation,following 1,3,5,8 and 12 μg/ml 5D12 treatment for 96 h were (9.12±2.02) ％,(16.63±0.92) ％,(19.04±0.25) ％,(24.17±0.53) ％,(34.04±3.20) ％ (HL-60),and (7.40±2.23) ％,(8.72±2.38) ％,(10.47±3.78) ％,(11.57±1.49) ％,(13.97±0.91) ％ (HL-60/ADR),respectively.Conclusion Nuclear protein-SFPQ contributes to HL-60′ ssensitivity to adriamycin by increasing its surface expression and promoting cell proliferation,but the protein has no significant effect on the intracellular accumulation of the drug.

The new method for transfection of macromolecules into cells using protein transductions domain / 白血病·淋巴瘤

Objective To construct a fusion protein to transfect some cell lines that were difficult to be transfected such as neoplastic cells, nerve cells, stem cells. Methods PCR was performed to amplified protein transductions domain(PTD),G4S and streptavidin (Strep).Enzymatic digestion and ligation were used to construct pAYZ-PTD-Strep plasmid. Fusion protein was induced to express by AP5 medium and was isolated by E-tag affinity chromatography. Fusion protein was identified by Western blot. eGFP was trasfected into U937 cells by pAYZ-PTD-Strep. FACS was performed to detect transfection percentage. Results Fusion protein PTD-G4S-Strep was expressed as soluble protein.The concentration of fusion protein was about 0.7 mg/L,and purity was over 90 ％. The protein could carry plasmid into a suspended cell line, U937 cells. The transfection-efficiency of protein was higher than monometer PTD.Conclusion The protein PAYZ-PTD-Strep could carry macromolecules into blood tumor cells,and its biological activity may be expected to develop into a highly efficient and reliable transfection method.

PSF/SFPQ relocated on cell membrane in hematologic neoplasia, a potential MDR target of hematologic tumors / 白血病·淋巴瘤

Objective To identify multidrug resistance (MDR) associated cell surface antigen in hematologic neoplasia and to investigate the universality of membrane-relocated expression of this antigen in hematologic neoplasia.Methods The membrane antigen was isolated and precipitated by SDS-PAGE and co-immunoprecipitation (co-IP),then was identified by mass spectrum (MS).Specific siRNA was used to interfere with gene expression,laser confocal microsopy was used to validate the results involved in antigen information.FACS was performed to analyse relocated expression of the antigen in hematologic neoplasia.Results Co-IP and MS show that a nuclear factor PSF was the antigen of 5D12,a leukemia-MDR associated McAb,and this antigen could relocate on HL-60 cell membrane.A series of experiences further confirmed that PSF overexpressed on HL-60 cell membrane compared with HL-60/ADR.The binding percentages of 5D12 to many hematologic tumor cells were observed,HL-60 (78.56±0.76) ％,K562 (26.54±4.42) ％,Nomalwa (38.10±5.11) ％,U937 (64.03±7.96) ％,Jurkat (29.12±5.58) ％,Raji (74.92±3.41) ％,CEM (12.18±3.21) ％.Conclusion Nuclear protein,PSF relocalizes on cell surfaces in hematologic tumor cells and contributes to cell sensitivity.PSF is a potential target of MDR prediction in hematologic neoplasia.

Preparation of a toxicity fusion protein targeting to CD123 on leukemia stem cell / 白血病·淋巴瘤

Objective To construct a fusion protein that used for treatment of resistance and palindromia in leukemia and studied its biological activity. Methods IL-3 and LP gene fragments were amplified by PCR. After enzymatic digestion and T4 ligation, the fusion gene was cloned into expression vector pAYZ. The product was purified by exchange chromatography and anti-Etag affinity chromatography. IL3-G4SLP fusion protein was analyzed by SDS-PAGE and Western blot. Protein biological activity was detected by FACS. Results The fusion protein was expressed as soluble protein by E.Coli 16C9. The protein expression level was about 1 mg/L, its purity was over 95 %, and the expression level was about 1 mg/L. The fusion protein can combined specificely with CD123 on leukemia stem cells. Conclusion Fusion protein IL-3-G4S-LP can target on leukemia stem cells and maybe as a potential drug used for treatment of resistance and palindromia in leukemia.

Mechanism of synergistic antitumor effects of EBB and doxorubicin on multidrug resistant MCF-7/ADR / 中国药理学通报

Aim To study the mechanism of synergistic antitumor of EBB and doxorubicin in doxorubicin-resistant MCF-7/ADR breast carcinoma cells.Methods The antitumor activity of doxorubiein alone and its combination with EBB were measured by MTT assay in MCF-7/ADR and MCF-7cells. The rate of doxorubicin-induced apoptosis and the protein and mRNA levels of P-glycoprotein(P-gp) were determined in MCF-7/ADR treated with EBB by flow cytometry (FACS), respectively.Laser scanning confocal microscopy was used to detect the intracellular accumulation of drug in EBB-treated MCF-7 and MCF-7/ADR cells.Results EBB had antitumor effects for MCF-7 and MCF-7/ADR.It could potentiate the antitumor effect of dororubicin with CDI of 0.73 and 0.49 for MCF-7 and MCF-7/ADR,respectively.EBB and doxorubicin acted synergistically in elevating apoptosis of MCF-7/ADR and downregulating the expression of P-gp in a dose-dependent manner in MCF-7/ADR.EBB restored the intracellular accumulation of doxorubicin in MCF-7/ADR cells in a dose-dependent manner.After pretreatment with EBB for 24 h and 48 h,the intracellular accumulation of doxorubicin and Rh123 was obviousely restored in MCF-7/ADR cells compared with control in a time-dependent manner.Conclusion EBB is a potential agent which has strong inhibitory effect on both multidrug resistant cells and their parental cells.EBB can significantly potentiate the antitumor effects of dororubicin in MCF-7/ADR cells by blocking the function of P-glycoprotein and inhibiting the expression of P-glycoprotein.

Production of discrepant monoclonal antibody against HL60 and HL60/ADR by SI technique / 中国免疫学杂志

Objective:To prepare and characterize specific and discrepant mouse hybridoma antibodies on membrane of HL60 and HL60/ADR cell lines.Methods:BALB/c mice were immunized by subtractive immunization induced Cp(Cyclophosphamide).McAbs were prepared by hybridoma technique,screened and detected by FACS and LSCM.Results:51 candidates and discrepant antibodies were found,and one of them (5F6) was purified and identified.Conclusion:Combination of SI with discrepant screening method should facilitate the preparing and identifying discrepant McAbs for identifying antibodies that can distinguish the differences in proteins expressed in HL60 and HL60/ADR,which is a significative and potential method in the research and target therapy associated drug-resistance.

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.

In vitro cytotoxity and reversal effects of PHⅡ-7 in human multidrug-resistant breast cancer MCF-7/ ADR cells / 中国癌症杂志

Background and purpose:Multidrug resistance(MDR)is one of the major causes of progressive breast cancer chemotherapy failure.One of the major mechanisms of MDR is the overexpression of P-glycoprotein (P-gp).Therefore,the identification of novel agents which can inhibit the drug transporter function of P-gp or its expression is of utmost interest in cancer research.The aim of this study was to explore the antitumor and reversal effect of PHⅡ-7,natural products from traditional Chinese medicine(TCM).Methods:The cytotoxicity of PHⅡ7 alone and combined application of PHⅡ-7 and adriamycin(ADR)on breast cancer cells were determined using MTT assay.Annexin V–FITC/PI apoptosis detection kit was used to observe the apoptosis-inducing effect of PHⅡ-7 in MCF-7 and MCF-7/ADR cells.The effect of PHⅡ-7 on mdr1 mRNA was determined by reverse transcription PCR and real time PCR,flow cytometer was used to measure the intracellular ADR accumulation.Results:PHⅡ-7 alone inhibited cell growth of MCF-7 and MCF-7/ADR cells with the IC 50 (6.07?0.85),(5.51?1.22)?mol/L,respectively when combined with ADR,PHⅡ-7 enhanced the cytotoxicity of ADR toward MCF-7/ADR cells.In addition,PHⅡ-7 induced apoptosis both on MCF-7 and MCF-7/ADR cells;PHⅡ-7 reversed the drug resistance to ADR in MCF-7/ADR cells by inhibiting mdr1 mRNA transcription and increasing the intracellular ADR accumulation.Conclusion:PHⅡ-7 displayed significant anti-proliferative and apoptosis-inducing effect on sensitive and multidrug resistant breast cells in vitro.PHⅡ-7 reversed effectively MDR by blocking the drugs to be pumped out by inhibiting P-gp expression and function pathway.

Construction and expression of anti-CD3?anti-CD19 bispecific diabody and analyzing its specific binding activity / 中国癌症杂志

Background and purpose:Bispecific antibody is a special pattern of genetic engineering antibody.It possesses 2 antigen binding sites, one directed at the effecter cells and the other at the target cells which are always the tumor cells.This enables the effecter cells to recruit to the targets efficiently and exert cytotoxic effect.The experiment was aimed to construct the expression vector of anti-CD3?anti-CD19 bispecific antibody, purification and analyzing the activity of the diabody.Methods:Applyed PCR and overlap PCR to construct the expression vector of anti-CD3?anti-CD19 bispecific antibodies, transformed it into E.coli 16C9 for expression.Purified the diabody by His-tag affinity chromatography and confirmed by 12% SDS-PAGE and Western blot.Antigen binding activity of the diabody was analyzed by FACS.Results:The DNA sequence of the expression vector containing anti-CD3?anti-CD19 fragment was comfirmed.The yield of purified diabody was up to 5 mg/L.The molecular weight of diabody was correctly indicated by SDS-PAGE and Western blot.The diabody could specifically bind to CD19+ Raji cells and CD3+ Jurkat cells while competitively inhibit binding of HIT3a and HIT19a to the cells.Conclusion:Successfully constructed the expression vector of anti-CD3?anti-CD19 bispecific antibodies, the diabody acquired by the method could be produced with high level expression and also had high specific affinity with CD19+ Raji cells and CD3+ Jurkat cells.