AIM: To investigate anti-hypersensitive effects of α2δ-1 ligands in non-inflammatory and inflammationassociated colonic hypersensitivity(CHS) mouse models.METHODS: To induce an inflammation-associated CHS, 1% dextra...AIM: To investigate anti-hypersensitive effects of α2δ-1 ligands in non-inflammatory and inflammationassociated colonic hypersensitivity(CHS) mouse models.METHODS: To induce an inflammation-associated CHS, 1% dextran sulfate sodium(DSS) was administered to C57Bl/6J male mice, in drinking water, for 14 d. Regarding the non-inflammatory neonatal maternal separation(NMS)-induced CHS model, wild-type C57BI/6J pups were isolated from their mother from day 2 to day 14(P2 to P14), three hours per day(from 9:00 a.m. to 12:00 p.m.). Colorectal distension was performed by inflating distension probe from 20 μL to 100 μL by 20 μL increment step every 10 s. After a first colorectal distension(CRD), drugs were administered subcutaneously, in a cumulative manner,(Gabapentin at 30 mg/kg and 100 mg/kg; Pregabalin at 10 mg/kg and 30 mg/kg; Carbamazepine at 10 mg/kg and 30 mg/kg) and a second CRD was performed one hour after each injection.RESULTS: The visceromotor response(VMR) to CRD was increased by our NMS paradigm protocol in comparison to non-handled(NH) mice, considering the highest distension volumes(80 μL: 0.783 ± 0.056 mV /s vs 0.531 ± 0.034 m V/s, P < 0.05 and 100 μL: 1.087 ± 0.056 m V/s vs 0.634 ± 0.038 m V/s, P < 0.05 for NMS and NH mice, respectively). In the inflammationassociated CHS, DSS-treated mice showed a dramatic and significant increase in VMR at 60 and 80 μL distension volumes when compared to control mice(60 μL: 0.920 ± 0.079 m V/s vs 0.426 ± 0.100 m V/s P < 0.05 and 80 μL: 1.193 ± 0.097 mV /s vs 0.681 ± 0.094 mV /s P < 0.05 for DSS- and Water-treated mice, respectively). Carbamazepine failed to significantly reduce CHS in both models. Gabapentin significantly reduced CHS in the DSS-induced model for both subcutaneous injections at 30 or 100 mg/kg. Pregabalin s i g n i f i c a n t l y r e d u c e d V M R t o C R D i n t h e n o n-inflammatory NMS-induced CHS model for the acute subcutaneous administration of the highest cumulative dose(30 mg/kg) and significantly reduced CHS in lowdose DSS-treated mice in a dose-dependent manner. Finally, the percent decrease of AUC induced by acute GBP or Pregabalin treatment were higher in the inflammatory DSS-induced CHS model in comparison to the non-inflammatory NMS-induced CHS model.CONCLUSION: This preclinical study demonstrates α2δ-1 ligands efficacy on inflammation-associated CHS, highlighting their potential clinical interest in patients with chronic abdominal pain and moderate intestinal inflammation.展开更多
The new ligand bis-（1,4-sodium thiolactate） butane （L）-O2CCH3S-（CH2）4SCHCH3CO2- has been prepared from the reaction of disodium salt of thiolactic acid and 1,4-dichlorobutane, while the disodium thiolactate was ...The new ligand bis-（1,4-sodium thiolactate） butane （L）-O2CCH3S-（CH2）4SCHCH3CO2- has been prepared from the reaction of disodium salt of thiolactic acid and 1,4-dichlorobutane, while the disodium thiolactate was prepared instanteously through the reaction of thiolactic acid with NaOH. Mono and dinuclear complexes were obtained by direct reaction of the above ligands with H[AuCI4] in 1 ： 1, 2：1, 1：2, 2：2 and 3：1 ligands to metal molar ratio. The prepared complexes were characterized by elemental analysis, spectral studies FTIR （Fourier transform Infrared） and UV-Vis, magnetic measurement, conductivity measurement and IHNMR for the ligand （L） and some of the complexes. The conductance data indicate that the complexes of the formulas Na[Au（SCH3CHCOO）2], Na[Au（SCH3CHCOO）（OOCCHCH3SH）2] and [Au（L）]Cl are 1：1 electrolyte. Electronic spectra and magnetic moment values indicate the presence of square planner geometry around Au（III） ions.展开更多
Objective: To obtain recombinant human SDF-1β expressed in E. colt and purify SDF-lfi with bio-logical activity from the bacterium. Methods: A thioredoxin-SDF-1β fusion protein (26×103) composed of230 amino aci...Objective: To obtain recombinant human SDF-1β expressed in E. colt and purify SDF-lfi with bio-logical activity from the bacterium. Methods: A thioredoxin-SDF-1β fusion protein (26×103) composed of230 amino acid residues was expressed in E. coli AD494 (DE3)pLysS under the induction of IPTG whenpET32a( + )-SDF-1β was used as an expression vector. Purified SDF-lfi was produced through following pro-cedures: Bacteria lysis, metal-chelated affinity chromatography (MAC), enterokinase digestion to separateSDF-lfi from fusion protein, cation exchange chromatography (CEC) and reverse-phase high performance liq-uid chromatography (RP-HPLC). Western blot with anti-SDF-1β monoclonal antibody (mAb), N-terminalamino acid sequencing, ligand-binding assay and cytosensor/microphysiometry were used to investigate thebiochemical characters and biological activities of the purified SDF-1β. Results: From 10% to 15% of totalbacterium protein was expressed as fusion protein. Approximately 400 fig purified SDF-1β (7. 8×103) con-sisting of 71 amino acid residues were produced from 1 L of fermented bacteria. Western blot showed that an-ti-SDF-1β mAb bound with the purified SDF-1β specifically. N-terminal amino acid sequencing indicates thatN-terminus of purified SDF-1β possessed as the same amino acid sequence as nature one. Purified SDF-1β notonly had the binding activity with CXCR4 expressing cells [Kd= (12. 20±2. 99) mnol/L], but also activatedCXCR4 expressing cell signaling specifically in a dose-dependence manner. Conclusion: The purified recombi-nant human SDF-1β produced with this method possesses biochemical characters and biological activities assame as those nature human SDF-1β.展开更多
Previous animal studies have shown that stromal cell-derived factor-1 （SDF-1）/CXC chemokine receptor-4 （CXCR4） signaling pathway plays an important role in the targeted migration of bone marrow-derived mesenchymal...Previous animal studies have shown that stromal cell-derived factor-1 （SDF-1）/CXC chemokine receptor-4 （CXCR4） signaling pathway plays an important role in the targeted migration of bone marrow-derived mesenchymal stem cells （BMSCs） to the injured area. In the present study, we aimed to investigate the potential role of chemotactic SDF-1/CXCR4 signaling pathway in the homing of transplanted BMSCs to the injured cochlea after noise-induced hearing loss （NIHL） in a rat model. White noise exposure （110 dB） paradigm was used for hearing loss induction in male rats for 6 hours in 5 days. Distortion-product otoacoustic emission （DPOAE） responses were recorded before the experiment and post noise exposure.Hoechst 33342-labeled BMSCs and CXCR4 antagonist （AMD3100）-treated BMSCs were injected into the rat cochlea through the round window. SDF-1 protein expression in the cochlear tissue was assayed using western blot assay. The number of labeled BMSCs reaching the endolymph was determined after 24 hours.SDF-1 was significantly increased in the cochlear tissue of rats in the noise exposure group than in the control group. The number of Hoechst 33342-labeled BMSCs reaching the endolymph of the cochlea was significantly smaller in the AMD3100-treated BMSCs group than in the normal BMSCs group. Our present findings suggest that the SDF-1/CXCR4 signaling pathway has a critical role in BMSCs migration to the injured cochlea in a rat model of noise-induced hearing loss.展开更多
Objective:This paper is to explore a method of transferring human SDF-1 and its mutant SDF-154 intrakine gene into COS-7 cells for determining their expression and subcelluar localization of the fusion protein.This co...Objective:This paper is to explore a method of transferring human SDF-1 and its mutant SDF-154 intrakine gene into COS-7 cells for determining their expression and subcelluar localization of the fusion protein.This could offer feasibility for inhibiting the metastasis of malignant tumors by phonotypic knockout for blocking functional expression of receptor on the cell-surface.Methods:Amplify the target gene with PCR from the constructed plasimid SDF-WT-Gly×4-DecPET-30a(+) with a C-terminal retention signal fragment KDEL. After the pcDNA3.1 SDF-1KDEL,pcDNA3.1 SDF-154KDEL,pEGFPSDF-1KDEL and pEGFPSDF-154KDEL eukaryotic expression vectors were constructed and the DNA sequence was accurate,they were transferred into COS-7 cells with liposome . The exogenous expressions were observed, fusion protein SDF-1His and SDF-154His were confirmed by Western blot,and the SDF-1EGFP and SDF-154EGFP were determined by Laser Scanning Confocal Microscopy. Results:Four expression vectors were constructed successfully, the fusion protein SDF-1 KDELHis and SDF-1 54 KDELHis expressed in COS-7 cells.Subcelluar localization analysis showed that SDF-1KDELEGFP and SDF-154KDELEGFP were located mainly in endoplasmic reticulum.Conclusion:Four expression vectors pcDNA3.1 SDF-1KDEL, pcDNA3.1SDF-154KDEL, pEGFPSDF-1KDEL and pEGFPSDF-154KDEL were constructed successfully, which could express in eukaryotic cell and locate mainly in the endoplasmic reticulum .展开更多
The title complex [Cd（IPA）L（H2O）]n（1,H2IPA = isophthalic acid,L = 1,4-bis（pyra-zole-1-ylmethyl）benzene） has been hydrothermally synthesized,and characterized by elemental analyses,IR spectroscopy,TGA and X-ray...The title complex [Cd（IPA）L（H2O）]n（1,H2IPA = isophthalic acid,L = 1,4-bis（pyra-zole-1-ylmethyl）benzene） has been hydrothermally synthesized,and characterized by elemental analyses,IR spectroscopy,TGA and X-ray single-crystal diffraction.It crystallizes in the triclinic system,space group P1 with a = 9.3060（6）,b = 10.2374（7）,c = 11.9706（8） ？,α = 73.804（6）,β = 77.883（5）,γ = 85.942（5）°,V = 1070.7（1） ？3,Z = 2,C22H20CdN4O5,Mr = 532.82,Dc = 1.653 g/cm3,μ = 1.062 mm-1,λ（MoKα） = 0.71073 ？,F（000） = 536,R = 0.0392 and wR = 0.0640 for 3751 observed reflections with I 2σ（I）.Crystal structure analysis showed that complex 1 has a 1D double chain structure,which is assembled together through strong O-H...O hydrogen bonding interactions between coordinated water molecules and carboxylate groups of isophthalate to form a 2D supramolecular network.In addition,the solid-state fluorescent spectrum of 1 exhibits strong emission at 364 nm.展开更多
HIV-1 infection requires the expression of CD4＋ molecules in colligation with C-C chemokine receptor type 5 （CCR5） and C-X-C chemokine receptor type 4 （CXCR4） as the major coreceptors. The role of SNP in 3＇ untr...HIV-1 infection requires the expression of CD4＋ molecules in colligation with C-C chemokine receptor type 5 （CCR5） and C-X-C chemokine receptor type 4 （CXCR4） as the major coreceptors. The role of SNP in 3＇ untranslated region ofSDF-1 （SDF1-3 ＇A） and low copy number （CN） of the CCL3L1 gene is reported to confer increased resistance to HIV-1 infection. The aim of the present study was to analyze the combinatorial effect of both the variations in protection towards HIV-1 infection in Indian population. The combinatorial effect of genetic variation in terms of SNP in SDF-1 gene and CCL3L1 CN was investigated in 105 healthy individuals and 78 HIV-I patients. Genotyping of SDF-1 was performed by RFLP-PCR and CCL3L1 by real-time PCR using TaqMan chemistry. The genotype frequency distribution of SDF-1 was found to be （SDF-1/SDF-I： 65.4%, SDF-1/SDF1-3＇A： 29.5% and SDFI-3＇A/SDF1-3＇A- 5.1%） in HIV patients as compared to （SDF-1/SDF-I： 64.8%, SDF-1/SDF1-3＇A： 30.5% and SDF1-3 ＇A/SDF1-3 ＇A： 4.7%） in healthy individuals, whereas a range of 1 to 6 copies per diploid genome was observed for CCL3L1 gene.展开更多
Objective ： To investigate the mechanism of phenotypic knockout of CXCR4 on T-cell leukemia cell line Molt-4 via SDF-1α/54/KDEL intrakine technology, which the mutant SDF-1α/54, human stromal cell-derived Faceor-1 ...Objective ： To investigate the mechanism of phenotypic knockout of CXCR4 on T-cell leukemia cell line Molt-4 via SDF-1α/54/KDEL intrakine technology, which the mutant SDF-1α/54, human stromal cell-derived Faceor-1 （SDF-1α） was deleted its C- terminal α-helix and attached with a endoplasimc reticulum retention signal 4-peptide- KDEL encoding gene, so that retain the newly synthesized receptor CXCR4 within the Molt-4 cells endoplasmic reticulum. Methods： The recombinant vector pEGFP-C3/SDF- 1α/54/KDEL were transfected into Cos-7 cells by liposome, SDF-1α/54/KDEL fusion protein was confirmed with western blot. The recombinant plasmids were transfected transiently into Molt-4 by electroporation. Results：Western blot confirmed SDF-1α/54/KDEL expression in Cos-7. A dramatic downregulation of CXCR4 expression on Molt-4 was demonstrated by flow cytometric （FCM） analysis. Conelusion：SDF-1α/54/KDEL and SDF- 1αKDEL have no significant deviation for phenotypic knockout of CXCR4. These suggest that the phenotypic knockout effects of SDF-1α/54 against CXCR4 are not influenced by deleting of SDF-1α helix in the C-terminal.展开更多
Cell therapy has shown beneficial effects on ventricular function and tissue regeneration in patients with acute and chronic myocardial infarction, although with diverse grades of variability in the results, possibly ...Cell therapy has shown beneficial effects on ventricular function and tissue regeneration in patients with acute and chronic myocardial infarction, although with diverse grades of variability in the results, possibly by proportion, subtype and cell cycle status. Objective: Identify and phenotypically characterize, via CXCR4 and SDF-1 expression, the bone marrow cell subpopulations that are mobilized into the bloodstream in patients with Acute Myocardial Infarction (AMI) and Acute Ischemia (AI) such as acute angina and Chronic Ischemia (CI) such as chronic stable angina, and also determine the cell cycle status of these cells. Method: Patients with AMI and AI were recruited in the ICCU, and patients with CI in the departments of cardiology and cardiovascular surgery. The quantification of cellular subpopulations was made by cytofluorometry with a FACS caliburcyto fluorometry (Becton Dickinson) with specific FITC-labeled anti human monoclonal antibodies against CD34, CD133, CD117, CD48, CXCR4, SDF-1 and Ki67 (Becton Dickinson). Serum concentration of IL-6 and IL-8 were determined by a sequential solid phase chemiluminescent assay performed in a SIEMENS IMMULITE 1000 Analyzer. Statistical analysis was made with the SPSS version 20.0 for Windows. A p value 3/ml) than that in AI (9.2 ± 1.3 × 103/ml) and CI (6.6 ± 1.1 × 103/ml) patients (p p = 0.22 to 0.39), but interestingly in AMI and AI patients, cells were CXCR4+ in almost half of these mobilized cells, although the proportion was significantly higher in AMI patients (46.8% ± 7.1% to 55.7% ± 6.3% vs 23% ± 1.6% to 28.4% ± 2.1%, p = 0.03 to 0.05). A similar behavior was observed with the Ki67 antibody (29.9% ± 2.1% to 36.1% ± 6.3% vs 10% ± 1.2% to 24% ± 1.1%, p = 0.001 to 0.05). Bivariate analysis of the results showed a significant correlation of the cell proportion in AMI but not in AI and CI patients (p = 0.001 to 0.05;0.12 to 0.87 and 0.17 to 0.92 respectively). The amount of myocardial tissue infarcted did not show any correlation with the amount of cellular subpopulations mobilized to peripheral blood (r = 0.10 to 0.20;p = 0.21 to 0.64) from the bone marrow. Conclusion: The proportion of cellular subpopulations with regenerative potential mobilized to circulation during an event of Acute Myocardial Infarction is significantly higher than during an event of acute angina and chronic stable angina, with a significant proportion of mobilized cells that expressed CXCR4, most of which were already in some of the cell cycle phases.展开更多
Background Autoimmune hepatitis （AIH） is a chronic inflammatory liver disease with unknown etiology. Programmed death 1 （PD-1） and its ligands （PD-L1 and PD-L2）, BT-H1/PD-L1 and B7-DC/PD-L2, are new CD28-B7 fami...Background Autoimmune hepatitis （AIH） is a chronic inflammatory liver disease with unknown etiology. Programmed death 1 （PD-1） and its ligands （PD-L1 and PD-L2）, BT-H1/PD-L1 and B7-DC/PD-L2, are new CD28-B7 family members that are involved in the regulation of immune responses. Previous observation suggests that PD-1 system plays an inhibitory role in regulating peripheral blood T cells, B cells and myeloid cells, thus their abnormality may be related to autoimmune diseases. This study aimed to explore the role of PD-1/PD-L1, L2 system in the pathogenesis of AIH. Methods The mice model of experimental autoimmune hepatitis （EAH） was established in C57BL/6 mice and the expression levels of PD-1 and PD-L1, L2 in the murine liver and the cytokines, including interferon （IFN）-γ, tumor necrosis factor （TNF）-α and interleukin （IL）-4 in the spleen were detected using reverse transcription-polymerase chain reaction （RT-PCR）, and the results were compared with those of normal controls. Results The expression levels of PD-1, PD-L1, PD-L2 mRNA were higher in EAH compared with normal controls （P 〈0.05）, the PD-L2/PD-1 ratio was relatively lower in EAH （EAH -0.08±0.35, normal controls 0.52±0.07, P=0.009）. In the EAH, the expression of the three cytokines were all upregulated compared with normal controls. PD-L1 had a positive correlation with the expression of IFN-γ （r =0.289, P 〈0.05）, while PD-L2 showed a positive correlation with both expressions of IL-4 （r=0.378, P 〈0.01） and IFN-γ （r =0.261, P 〈0.05）. While TNF-α showed no correlation with PD-L1 （r=0.044, P=0.736） or PD-L2 （r=0.127, P=0.335）. Conclusions The expression of PD-1/PD-L1, L2 is upregulated in EAH and regulated by IFN-γ and IL-4. PD-1 system may play an important role in the pathogenesis of AIH.展开更多
BACKGROUND Bevacizumab,an anti-vascular endothelial growth factor(VEGF)monoclonal antibody,inhibits angiogenesis and reduces tumor growth.Serum VEGF-C,lactate dehydrogenase,and inflammatory markers have been reported ...BACKGROUND Bevacizumab,an anti-vascular endothelial growth factor(VEGF)monoclonal antibody,inhibits angiogenesis and reduces tumor growth.Serum VEGF-C,lactate dehydrogenase,and inflammatory markers have been reported as predictive markers related to bevacizumab treatment.Programmed cell death ligand 1(PD-L1)could act upon VEGF receptor 2 to induce cancer cell angiogenesis and metastasis.AIM To investigate the efficacy of bevacizumab-containing chemotherapy in patients with metastatic colorectal cancer(CRC)according to the expression of PD-L1.METHODS This analysis included CRC patients who received bevacizumab plus FOLFOX or FOLFIRI as first-line therapy between June 24,2014 and February 28,2022,at Samsung Medical Center(Seoul,South Korea).Analysis of patient data included evaluation of PD-L1 expression by the combined positive score(CPS).We analyzed the efficacy of bevacizumab according to PD-L1 expression status in patients with CRC.RESULTS A total of 124 patients was included in this analysis.Almost all patients were treated with bevacizumab plus FOLFIRI or FOLFOX as the first-line chemotherapy.While 77%of patients received FOLFOX,23%received FOLFIRI as backbone first-line chemotherapy.The numbers of patients with a PD-L1 CPS of 1 or more,5 or more,or 10 or more were 105(85%),64(52%),and 32(26%),respectively.The results showed no significant difference in progression-free survival(PFS)and overall survival(OS)with bevacizumab treatment between patients with PDL1 CPS less than 1 and those with PD-L1 CPS of 1 or more(PD-L1<1%vs PD-L1≥1%;PFS:P=0.93,OS:P=0.33),between patients with PD-L1 CPS less than 5 and of 5 or more(PD-L1<5%vs PD-L1≥5%;PFS:P=0.409,OS:P=0.746),and between patients with PD-L1 CPS less than 10 and of 10 or more(PD-L1<10%vs PD-L1≥10%;PFS:P=0.529,OS:P=0.568).CONCLUSION Chemotherapy containing bevacizumab can be considered as first-line therapy in metastatic CRC irrespective of PD-L1 expression.展开更多
文摘AIM: To investigate anti-hypersensitive effects of α2δ-1 ligands in non-inflammatory and inflammationassociated colonic hypersensitivity(CHS) mouse models.METHODS: To induce an inflammation-associated CHS, 1% dextran sulfate sodium(DSS) was administered to C57Bl/6J male mice, in drinking water, for 14 d. Regarding the non-inflammatory neonatal maternal separation(NMS)-induced CHS model, wild-type C57BI/6J pups were isolated from their mother from day 2 to day 14(P2 to P14), three hours per day(from 9:00 a.m. to 12:00 p.m.). Colorectal distension was performed by inflating distension probe from 20 μL to 100 μL by 20 μL increment step every 10 s. After a first colorectal distension(CRD), drugs were administered subcutaneously, in a cumulative manner,(Gabapentin at 30 mg/kg and 100 mg/kg; Pregabalin at 10 mg/kg and 30 mg/kg; Carbamazepine at 10 mg/kg and 30 mg/kg) and a second CRD was performed one hour after each injection.RESULTS: The visceromotor response(VMR) to CRD was increased by our NMS paradigm protocol in comparison to non-handled(NH) mice, considering the highest distension volumes(80 μL: 0.783 ± 0.056 mV /s vs 0.531 ± 0.034 m V/s, P < 0.05 and 100 μL: 1.087 ± 0.056 m V/s vs 0.634 ± 0.038 m V/s, P < 0.05 for NMS and NH mice, respectively). In the inflammationassociated CHS, DSS-treated mice showed a dramatic and significant increase in VMR at 60 and 80 μL distension volumes when compared to control mice(60 μL: 0.920 ± 0.079 m V/s vs 0.426 ± 0.100 m V/s P < 0.05 and 80 μL: 1.193 ± 0.097 mV /s vs 0.681 ± 0.094 mV /s P < 0.05 for DSS- and Water-treated mice, respectively). Carbamazepine failed to significantly reduce CHS in both models. Gabapentin significantly reduced CHS in the DSS-induced model for both subcutaneous injections at 30 or 100 mg/kg. Pregabalin s i g n i f i c a n t l y r e d u c e d V M R t o C R D i n t h e n o n-inflammatory NMS-induced CHS model for the acute subcutaneous administration of the highest cumulative dose(30 mg/kg) and significantly reduced CHS in lowdose DSS-treated mice in a dose-dependent manner. Finally, the percent decrease of AUC induced by acute GBP or Pregabalin treatment were higher in the inflammatory DSS-induced CHS model in comparison to the non-inflammatory NMS-induced CHS model.CONCLUSION: This preclinical study demonstrates α2δ-1 ligands efficacy on inflammation-associated CHS, highlighting their potential clinical interest in patients with chronic abdominal pain and moderate intestinal inflammation.
文摘The new ligand bis-（1,4-sodium thiolactate） butane （L）-O2CCH3S-（CH2）4SCHCH3CO2- has been prepared from the reaction of disodium salt of thiolactic acid and 1,4-dichlorobutane, while the disodium thiolactate was prepared instanteously through the reaction of thiolactic acid with NaOH. Mono and dinuclear complexes were obtained by direct reaction of the above ligands with H[AuCI4] in 1 ： 1, 2：1, 1：2, 2：2 and 3：1 ligands to metal molar ratio. The prepared complexes were characterized by elemental analysis, spectral studies FTIR （Fourier transform Infrared） and UV-Vis, magnetic measurement, conductivity measurement and IHNMR for the ligand （L） and some of the complexes. The conductance data indicate that the complexes of the formulas Na[Au（SCH3CHCOO）2], Na[Au（SCH3CHCOO）（OOCCHCH3SH）2] and [Au（L）]Cl are 1：1 electrolyte. Electronic spectra and magnetic moment values indicate the presence of square planner geometry around Au（III） ions.
文摘Objective: To obtain recombinant human SDF-1β expressed in E. colt and purify SDF-lfi with bio-logical activity from the bacterium. Methods: A thioredoxin-SDF-1β fusion protein (26×103) composed of230 amino acid residues was expressed in E. coli AD494 (DE3)pLysS under the induction of IPTG whenpET32a( + )-SDF-1β was used as an expression vector. Purified SDF-lfi was produced through following pro-cedures: Bacteria lysis, metal-chelated affinity chromatography (MAC), enterokinase digestion to separateSDF-lfi from fusion protein, cation exchange chromatography (CEC) and reverse-phase high performance liq-uid chromatography (RP-HPLC). Western blot with anti-SDF-1β monoclonal antibody (mAb), N-terminalamino acid sequencing, ligand-binding assay and cytosensor/microphysiometry were used to investigate thebiochemical characters and biological activities of the purified SDF-1β. Results: From 10% to 15% of totalbacterium protein was expressed as fusion protein. Approximately 400 fig purified SDF-1β (7. 8×103) con-sisting of 71 amino acid residues were produced from 1 L of fermented bacteria. Western blot showed that an-ti-SDF-1β mAb bound with the purified SDF-1β specifically. N-terminal amino acid sequencing indicates thatN-terminus of purified SDF-1β possessed as the same amino acid sequence as nature one. Purified SDF-1β notonly had the binding activity with CXCR4 expressing cells [Kd= (12. 20±2. 99) mnol/L], but also activatedCXCR4 expressing cell signaling specifically in a dose-dependence manner. Conclusion: The purified recombi-nant human SDF-1β produced with this method possesses biochemical characters and biological activities assame as those nature human SDF-1β.
基金financially supported by the Hearing Disorders Research Center of Shahid Beheshti University of Medical Sciences
文摘Previous animal studies have shown that stromal cell-derived factor-1 （SDF-1）/CXC chemokine receptor-4 （CXCR4） signaling pathway plays an important role in the targeted migration of bone marrow-derived mesenchymal stem cells （BMSCs） to the injured area. In the present study, we aimed to investigate the potential role of chemotactic SDF-1/CXCR4 signaling pathway in the homing of transplanted BMSCs to the injured cochlea after noise-induced hearing loss （NIHL） in a rat model. White noise exposure （110 dB） paradigm was used for hearing loss induction in male rats for 6 hours in 5 days. Distortion-product otoacoustic emission （DPOAE） responses were recorded before the experiment and post noise exposure.Hoechst 33342-labeled BMSCs and CXCR4 antagonist （AMD3100）-treated BMSCs were injected into the rat cochlea through the round window. SDF-1 protein expression in the cochlear tissue was assayed using western blot assay. The number of labeled BMSCs reaching the endolymph was determined after 24 hours.SDF-1 was significantly increased in the cochlear tissue of rats in the noise exposure group than in the control group. The number of Hoechst 33342-labeled BMSCs reaching the endolymph of the cochlea was significantly smaller in the AMD3100-treated BMSCs group than in the normal BMSCs group. Our present findings suggest that the SDF-1/CXCR4 signaling pathway has a critical role in BMSCs migration to the injured cochlea in a rat model of noise-induced hearing loss.
基金This work was supported by grants from the National Natural Science Foundation of China(30170270)
文摘Objective:This paper is to explore a method of transferring human SDF-1 and its mutant SDF-154 intrakine gene into COS-7 cells for determining their expression and subcelluar localization of the fusion protein.This could offer feasibility for inhibiting the metastasis of malignant tumors by phonotypic knockout for blocking functional expression of receptor on the cell-surface.Methods:Amplify the target gene with PCR from the constructed plasimid SDF-WT-Gly×4-DecPET-30a(+) with a C-terminal retention signal fragment KDEL. After the pcDNA3.1 SDF-1KDEL,pcDNA3.1 SDF-154KDEL,pEGFPSDF-1KDEL and pEGFPSDF-154KDEL eukaryotic expression vectors were constructed and the DNA sequence was accurate,they were transferred into COS-7 cells with liposome . The exogenous expressions were observed, fusion protein SDF-1His and SDF-154His were confirmed by Western blot,and the SDF-1EGFP and SDF-154EGFP were determined by Laser Scanning Confocal Microscopy. Results:Four expression vectors were constructed successfully, the fusion protein SDF-1 KDELHis and SDF-1 54 KDELHis expressed in COS-7 cells.Subcelluar localization analysis showed that SDF-1KDELEGFP and SDF-154KDELEGFP were located mainly in endoplasmic reticulum.Conclusion:Four expression vectors pcDNA3.1 SDF-1KDEL, pcDNA3.1SDF-154KDEL, pEGFPSDF-1KDEL and pEGFPSDF-154KDEL were constructed successfully, which could express in eukaryotic cell and locate mainly in the endoplasmic reticulum .
文摘The title complex [Cd（IPA）L（H2O）]n（1,H2IPA = isophthalic acid,L = 1,4-bis（pyra-zole-1-ylmethyl）benzene） has been hydrothermally synthesized,and characterized by elemental analyses,IR spectroscopy,TGA and X-ray single-crystal diffraction.It crystallizes in the triclinic system,space group P1 with a = 9.3060（6）,b = 10.2374（7）,c = 11.9706（8） ？,α = 73.804（6）,β = 77.883（5）,γ = 85.942（5）°,V = 1070.7（1） ？3,Z = 2,C22H20CdN4O5,Mr = 532.82,Dc = 1.653 g/cm3,μ = 1.062 mm-1,λ（MoKα） = 0.71073 ？,F（000） = 536,R = 0.0392 and wR = 0.0640 for 3751 observed reflections with I 2σ（I）.Crystal structure analysis showed that complex 1 has a 1D double chain structure,which is assembled together through strong O-H...O hydrogen bonding interactions between coordinated water molecules and carboxylate groups of isophthalate to form a 2D supramolecular network.In addition,the solid-state fluorescent spectrum of 1 exhibits strong emission at 364 nm.
文摘HIV-1 infection requires the expression of CD4＋ molecules in colligation with C-C chemokine receptor type 5 （CCR5） and C-X-C chemokine receptor type 4 （CXCR4） as the major coreceptors. The role of SNP in 3＇ untranslated region ofSDF-1 （SDF1-3 ＇A） and low copy number （CN） of the CCL3L1 gene is reported to confer increased resistance to HIV-1 infection. The aim of the present study was to analyze the combinatorial effect of both the variations in protection towards HIV-1 infection in Indian population. The combinatorial effect of genetic variation in terms of SNP in SDF-1 gene and CCL3L1 CN was investigated in 105 healthy individuals and 78 HIV-I patients. Genotyping of SDF-1 was performed by RFLP-PCR and CCL3L1 by real-time PCR using TaqMan chemistry. The genotype frequency distribution of SDF-1 was found to be （SDF-1/SDF-I： 65.4%, SDF-1/SDF1-3＇A： 29.5% and SDFI-3＇A/SDF1-3＇A- 5.1%） in HIV patients as compared to （SDF-1/SDF-I： 64.8%, SDF-1/SDF1-3＇A： 30.5% and SDF1-3 ＇A/SDF1-3 ＇A： 4.7%） in healthy individuals, whereas a range of 1 to 6 copies per diploid genome was observed for CCL3L1 gene.
基金Grant sponsor:National Natural Science Foundation of ChinaGrant number:30572209
文摘Objective ： To investigate the mechanism of phenotypic knockout of CXCR4 on T-cell leukemia cell line Molt-4 via SDF-1α/54/KDEL intrakine technology, which the mutant SDF-1α/54, human stromal cell-derived Faceor-1 （SDF-1α） was deleted its C- terminal α-helix and attached with a endoplasimc reticulum retention signal 4-peptide- KDEL encoding gene, so that retain the newly synthesized receptor CXCR4 within the Molt-4 cells endoplasmic reticulum. Methods： The recombinant vector pEGFP-C3/SDF- 1α/54/KDEL were transfected into Cos-7 cells by liposome, SDF-1α/54/KDEL fusion protein was confirmed with western blot. The recombinant plasmids were transfected transiently into Molt-4 by electroporation. Results：Western blot confirmed SDF-1α/54/KDEL expression in Cos-7. A dramatic downregulation of CXCR4 expression on Molt-4 was demonstrated by flow cytometric （FCM） analysis. Conelusion：SDF-1α/54/KDEL and SDF- 1αKDEL have no significant deviation for phenotypic knockout of CXCR4. These suggest that the phenotypic knockout effects of SDF-1α/54 against CXCR4 are not influenced by deleting of SDF-1α helix in the C-terminal.
文摘Cell therapy has shown beneficial effects on ventricular function and tissue regeneration in patients with acute and chronic myocardial infarction, although with diverse grades of variability in the results, possibly by proportion, subtype and cell cycle status. Objective: Identify and phenotypically characterize, via CXCR4 and SDF-1 expression, the bone marrow cell subpopulations that are mobilized into the bloodstream in patients with Acute Myocardial Infarction (AMI) and Acute Ischemia (AI) such as acute angina and Chronic Ischemia (CI) such as chronic stable angina, and also determine the cell cycle status of these cells. Method: Patients with AMI and AI were recruited in the ICCU, and patients with CI in the departments of cardiology and cardiovascular surgery. The quantification of cellular subpopulations was made by cytofluorometry with a FACS caliburcyto fluorometry (Becton Dickinson) with specific FITC-labeled anti human monoclonal antibodies against CD34, CD133, CD117, CD48, CXCR4, SDF-1 and Ki67 (Becton Dickinson). Serum concentration of IL-6 and IL-8 were determined by a sequential solid phase chemiluminescent assay performed in a SIEMENS IMMULITE 1000 Analyzer. Statistical analysis was made with the SPSS version 20.0 for Windows. A p value 3/ml) than that in AI (9.2 ± 1.3 × 103/ml) and CI (6.6 ± 1.1 × 103/ml) patients (p p = 0.22 to 0.39), but interestingly in AMI and AI patients, cells were CXCR4+ in almost half of these mobilized cells, although the proportion was significantly higher in AMI patients (46.8% ± 7.1% to 55.7% ± 6.3% vs 23% ± 1.6% to 28.4% ± 2.1%, p = 0.03 to 0.05). A similar behavior was observed with the Ki67 antibody (29.9% ± 2.1% to 36.1% ± 6.3% vs 10% ± 1.2% to 24% ± 1.1%, p = 0.001 to 0.05). Bivariate analysis of the results showed a significant correlation of the cell proportion in AMI but not in AI and CI patients (p = 0.001 to 0.05;0.12 to 0.87 and 0.17 to 0.92 respectively). The amount of myocardial tissue infarcted did not show any correlation with the amount of cellular subpopulations mobilized to peripheral blood (r = 0.10 to 0.20;p = 0.21 to 0.64) from the bone marrow. Conclusion: The proportion of cellular subpopulations with regenerative potential mobilized to circulation during an event of Acute Myocardial Infarction is significantly higher than during an event of acute angina and chronic stable angina, with a significant proportion of mobilized cells that expressed CXCR4, most of which were already in some of the cell cycle phases.
文摘Background Autoimmune hepatitis （AIH） is a chronic inflammatory liver disease with unknown etiology. Programmed death 1 （PD-1） and its ligands （PD-L1 and PD-L2）, BT-H1/PD-L1 and B7-DC/PD-L2, are new CD28-B7 family members that are involved in the regulation of immune responses. Previous observation suggests that PD-1 system plays an inhibitory role in regulating peripheral blood T cells, B cells and myeloid cells, thus their abnormality may be related to autoimmune diseases. This study aimed to explore the role of PD-1/PD-L1, L2 system in the pathogenesis of AIH. Methods The mice model of experimental autoimmune hepatitis （EAH） was established in C57BL/6 mice and the expression levels of PD-1 and PD-L1, L2 in the murine liver and the cytokines, including interferon （IFN）-γ, tumor necrosis factor （TNF）-α and interleukin （IL）-4 in the spleen were detected using reverse transcription-polymerase chain reaction （RT-PCR）, and the results were compared with those of normal controls. Results The expression levels of PD-1, PD-L1, PD-L2 mRNA were higher in EAH compared with normal controls （P 〈0.05）, the PD-L2/PD-1 ratio was relatively lower in EAH （EAH -0.08±0.35, normal controls 0.52±0.07, P=0.009）. In the EAH, the expression of the three cytokines were all upregulated compared with normal controls. PD-L1 had a positive correlation with the expression of IFN-γ （r =0.289, P 〈0.05）, while PD-L2 showed a positive correlation with both expressions of IL-4 （r=0.378, P 〈0.01） and IFN-γ （r =0.261, P 〈0.05）. While TNF-α showed no correlation with PD-L1 （r=0.044, P=0.736） or PD-L2 （r=0.127, P=0.335）. Conclusions The expression of PD-1/PD-L1, L2 is upregulated in EAH and regulated by IFN-γ and IL-4. PD-1 system may play an important role in the pathogenesis of AIH.
文摘BACKGROUND Bevacizumab,an anti-vascular endothelial growth factor(VEGF)monoclonal antibody,inhibits angiogenesis and reduces tumor growth.Serum VEGF-C,lactate dehydrogenase,and inflammatory markers have been reported as predictive markers related to bevacizumab treatment.Programmed cell death ligand 1(PD-L1)could act upon VEGF receptor 2 to induce cancer cell angiogenesis and metastasis.AIM To investigate the efficacy of bevacizumab-containing chemotherapy in patients with metastatic colorectal cancer(CRC)according to the expression of PD-L1.METHODS This analysis included CRC patients who received bevacizumab plus FOLFOX or FOLFIRI as first-line therapy between June 24,2014 and February 28,2022,at Samsung Medical Center(Seoul,South Korea).Analysis of patient data included evaluation of PD-L1 expression by the combined positive score(CPS).We analyzed the efficacy of bevacizumab according to PD-L1 expression status in patients with CRC.RESULTS A total of 124 patients was included in this analysis.Almost all patients were treated with bevacizumab plus FOLFIRI or FOLFOX as the first-line chemotherapy.While 77%of patients received FOLFOX,23%received FOLFIRI as backbone first-line chemotherapy.The numbers of patients with a PD-L1 CPS of 1 or more,5 or more,or 10 or more were 105(85%),64(52%),and 32(26%),respectively.The results showed no significant difference in progression-free survival(PFS)and overall survival(OS)with bevacizumab treatment between patients with PDL1 CPS less than 1 and those with PD-L1 CPS of 1 or more(PD-L1<1%vs PD-L1≥1%;PFS:P=0.93,OS:P=0.33),between patients with PD-L1 CPS less than 5 and of 5 or more(PD-L1<5%vs PD-L1≥5%;PFS:P=0.409,OS:P=0.746),and between patients with PD-L1 CPS less than 10 and of 10 or more(PD-L1<10%vs PD-L1≥10%;PFS:P=0.529,OS:P=0.568).CONCLUSION Chemotherapy containing bevacizumab can be considered as first-line therapy in metastatic CRC irrespective of PD-L1 expression.
