A human B cell line （3D5） that responds specifically to B cell growth factor （BCGF） hasbeen developed by a sequence of Staphylococcus aureus Cowen I activation,EB virus im-mortalization,and cloning.Proliferative r...A human B cell line （3D5） that responds specifically to B cell growth factor （BCGF） hasbeen developed by a sequence of Staphylococcus aureus Cowen I activation,EB virus im-mortalization,and cloning.Proliferative response to PHA-stimulated T cell supernatant（PHA-T-Sup） and nonresponsiveness to rIL-2 stimulation were factors used to screen positivecells.Phenotype analysis with a flow cytometer indicated that:1) 3D5 is a B cell line:100% of the cells were positive for B1 marker and 59% were positive for sIg,while T3and Mo 1 were negative:2) 3D5 is an activated B cell line:both Tac and 4F2 markersof activated （but not of resting） B cells were 100% positive:3) 3D5 expresses high molecularweight BCGF （HMW-BCGF） receptor-associated epitope BA5.3D5 cells proliferated inresponse to cpBCGF stimulation in a dose-dependent manner.HMW-BCGF also induced3D5 cells to proliferate.Interestingly.no proliferation could be detected in the presenceof rIL-2,rIL-4,or rIFN-r.The data show that 3D5 cells are specifically BCGF-responsiveB cells.Using 3D5 cells as target,BCGF activity was detected in crude BCGF preparationsedimented by 85% （NH<sub>4</sub>）<sub>2</sub>SO<sub>4</sub> and chromatographed in a DEAE-Sephadex A-25 column fromPHA-T-Sup.T24 cell supernatant with B cell differentiation factor （BCDF） activity couldnot induce 3D5 cells to differentiate into immunoglobulin-secreting cells.展开更多
AIM:To study the effects of hypoxia-inducible factor1α(HIF-1α) silencing on the proliferation of hypoxic CBRH-7919 rat hepatoma cells.METHODS:The CBRH-7919 rat hepatoma cell line was used in this study and the hypox...AIM:To study the effects of hypoxia-inducible factor1α(HIF-1α) silencing on the proliferation of hypoxic CBRH-7919 rat hepatoma cells.METHODS:The CBRH-7919 rat hepatoma cell line was used in this study and the hypoxic model was constructed using CoCl2.The HIF-1α-specific RNAi sequences were designed according to the gene coding sequence of rat HIF-1α obtained from GeneBank.The secondary structure of the HIF-1α gene sequence was analyzed using RNA draw software.The small interfering RNA(siRNA) transfection mixture was produced by mixing the siRNA and Lipofectamine2000TM,and transfected into the hypoxic hepatoma cells.Real time reverse transcription-polymerase chain reaction(RTPCR) and Western blotting assay were used to detect the expression levels of mRNA and protein.HIF-1α and vascular endothelial growth factor(VEGF) mRNA was determined using real time RT-PCR;the protein expression levels of AKT,p-AKT,p21 and cyclinD1 were determined using Western blotting.The proliferation of hepatoma cells was observed using the methyl thiazolyl tetrazolium(MTT) assay and the bromodeoxyuridine(BrdU) incorporation cell proliferation assay.RESULTS:Under induced hypoxia,the viability of the hepatoma cells reached a minimum at 800 μmol/L CoCl2;the viability of the cells was relatively high at CoCl2 concentrations between 100 μmol/L and 200 μmol/L.Under hypoxia,the mRNA and protein expression levels of HIF-1α and VEGF were significantly higher than that of hepatoma cells that were cultured in normaxia.HIF-1α-specific RNAi sequences were successfully transfected into hepatoma cells.The transfection of specific siRNAs significantly inhibited the mRNA and protein expression levels of HIF-1α and VEGF,along with the protein expression levels of p-AKT and cyclinD1;the protein expression of p21 was significantly increased,and there was no significant difference in the expression of AKT.The MTT assay showed that the amount of hepatoma cells in S phase in the siRNA transfection group was obviously smaller than that in the control group;in the siRNA transfection group,the amount of hepatoma cells in G1 phase was more than that in the control group.The BrdU incorporation assay showed that the number of BrdU positive hepatoma cells in the siRNA transfection group was less than that in the control group.The data of the MTT assay and BrdU incorporation assay suggested that HIF-1α silencing using siRNAs significantly inhibited the proliferation of hepatoma cells.CONCLUSION:Hypoxia increases the expression of HIF-1α,and HIF-1α silencing significantly inhibits the proliferation of hypoxic CBRH-7919 rat hepatoma cells.展开更多
目的:观察Graves病(GD)小鼠模型脾脏调节性B细胞(regulatory B cells,Bregs)及相关细胞因子表达量的变化,探讨Bregs在GD发生、发展中的免疫调节作用。方法:将6周龄BALB/c雌性小鼠随机分为对照组(肌注空载腺病毒)、GD组(肌注TSHR-A亚单位...目的:观察Graves病(GD)小鼠模型脾脏调节性B细胞(regulatory B cells,Bregs)及相关细胞因子表达量的变化,探讨Bregs在GD发生、发展中的免疫调节作用。方法:将6周龄BALB/c雌性小鼠随机分为对照组(肌注空载腺病毒)、GD组(肌注TSHR-A亚单位-腺病毒建立GD小鼠模型)。每3周免疫1次,第3次免疫3周后处死。留取小鼠血清,化学发光法检测FT4水平,细胞培养生物学方法检测TSAb活性。取甲状腺,制备石蜡切片行HE染色。取脾脏,部分制成单个核细胞悬液,行三标记免疫荧光染色、流式细胞分析CD1dhiCD5+CD19+Bregs比例;部分脾脏提取总RNA,实时定量RT-PCR检测Bregs相关细胞因子白细胞介素10(IL-10)mRNA和转化生长因子-β(TGF-β)mRNA的表达水平。结果:发生GD的小鼠甲状腺滤泡细胞增生肥大,且血清FT4及TSAb水平升高。小鼠血清TSAb与FT4水平呈显著正相关(r=0.90,P<0.01);TSHR-A亚单位-腺病毒免疫组发生GD的小鼠脾脏CD19+B细胞较对照组明显升高(P<0.05),B细胞中CD1dhiCD5+CD19+Bregs所占比例和脾组织IL-10 mRNA、TGF-βmRNA表达量较对照组明显降低(P<0.05);且血清TSAb活性与CD1dhiCD5+CD19+Bregs占脾脏B细胞比例、脾组织IL-10 mRNA、TGF-βmRNA表达量均呈显著负相关(P<0.05)。结论:TSHR-A亚单位-腺病毒免疫建立的GD模型中Bregs表达存在缺陷,提示Bregs表达可能在GD的发生及发展中发挥免疫调节作用,且GD疾病严重程度可能与Bregs缺乏程度有关。展开更多
文摘A human B cell line （3D5） that responds specifically to B cell growth factor （BCGF） hasbeen developed by a sequence of Staphylococcus aureus Cowen I activation,EB virus im-mortalization,and cloning.Proliferative response to PHA-stimulated T cell supernatant（PHA-T-Sup） and nonresponsiveness to rIL-2 stimulation were factors used to screen positivecells.Phenotype analysis with a flow cytometer indicated that:1) 3D5 is a B cell line:100% of the cells were positive for B1 marker and 59% were positive for sIg,while T3and Mo 1 were negative:2) 3D5 is an activated B cell line:both Tac and 4F2 markersof activated （but not of resting） B cells were 100% positive:3) 3D5 expresses high molecularweight BCGF （HMW-BCGF） receptor-associated epitope BA5.3D5 cells proliferated inresponse to cpBCGF stimulation in a dose-dependent manner.HMW-BCGF also induced3D5 cells to proliferate.Interestingly.no proliferation could be detected in the presenceof rIL-2,rIL-4,or rIFN-r.The data show that 3D5 cells are specifically BCGF-responsiveB cells.Using 3D5 cells as target,BCGF activity was detected in crude BCGF preparationsedimented by 85% （NH<sub>4</sub>）<sub>2</sub>SO<sub>4</sub> and chromatographed in a DEAE-Sephadex A-25 column fromPHA-T-Sup.T24 cell supernatant with B cell differentiation factor （BCDF） activity couldnot induce 3D5 cells to differentiate into immunoglobulin-secreting cells.
基金Supported by Natural Science Foundation of Guangdong Province People’s Republic of China,No. 10151008901000182
文摘AIM:To study the effects of hypoxia-inducible factor1α(HIF-1α) silencing on the proliferation of hypoxic CBRH-7919 rat hepatoma cells.METHODS:The CBRH-7919 rat hepatoma cell line was used in this study and the hypoxic model was constructed using CoCl2.The HIF-1α-specific RNAi sequences were designed according to the gene coding sequence of rat HIF-1α obtained from GeneBank.The secondary structure of the HIF-1α gene sequence was analyzed using RNA draw software.The small interfering RNA(siRNA) transfection mixture was produced by mixing the siRNA and Lipofectamine2000TM,and transfected into the hypoxic hepatoma cells.Real time reverse transcription-polymerase chain reaction(RTPCR) and Western blotting assay were used to detect the expression levels of mRNA and protein.HIF-1α and vascular endothelial growth factor(VEGF) mRNA was determined using real time RT-PCR;the protein expression levels of AKT,p-AKT,p21 and cyclinD1 were determined using Western blotting.The proliferation of hepatoma cells was observed using the methyl thiazolyl tetrazolium(MTT) assay and the bromodeoxyuridine(BrdU) incorporation cell proliferation assay.RESULTS:Under induced hypoxia,the viability of the hepatoma cells reached a minimum at 800 μmol/L CoCl2;the viability of the cells was relatively high at CoCl2 concentrations between 100 μmol/L and 200 μmol/L.Under hypoxia,the mRNA and protein expression levels of HIF-1α and VEGF were significantly higher than that of hepatoma cells that were cultured in normaxia.HIF-1α-specific RNAi sequences were successfully transfected into hepatoma cells.The transfection of specific siRNAs significantly inhibited the mRNA and protein expression levels of HIF-1α and VEGF,along with the protein expression levels of p-AKT and cyclinD1;the protein expression of p21 was significantly increased,and there was no significant difference in the expression of AKT.The MTT assay showed that the amount of hepatoma cells in S phase in the siRNA transfection group was obviously smaller than that in the control group;in the siRNA transfection group,the amount of hepatoma cells in G1 phase was more than that in the control group.The BrdU incorporation assay showed that the number of BrdU positive hepatoma cells in the siRNA transfection group was less than that in the control group.The data of the MTT assay and BrdU incorporation assay suggested that HIF-1α silencing using siRNAs significantly inhibited the proliferation of hepatoma cells.CONCLUSION:Hypoxia increases the expression of HIF-1α,and HIF-1α silencing significantly inhibits the proliferation of hypoxic CBRH-7919 rat hepatoma cells.
文摘目的:观察Graves病(GD)小鼠模型脾脏调节性B细胞(regulatory B cells,Bregs)及相关细胞因子表达量的变化,探讨Bregs在GD发生、发展中的免疫调节作用。方法:将6周龄BALB/c雌性小鼠随机分为对照组(肌注空载腺病毒)、GD组(肌注TSHR-A亚单位-腺病毒建立GD小鼠模型)。每3周免疫1次,第3次免疫3周后处死。留取小鼠血清,化学发光法检测FT4水平,细胞培养生物学方法检测TSAb活性。取甲状腺,制备石蜡切片行HE染色。取脾脏,部分制成单个核细胞悬液,行三标记免疫荧光染色、流式细胞分析CD1dhiCD5+CD19+Bregs比例;部分脾脏提取总RNA,实时定量RT-PCR检测Bregs相关细胞因子白细胞介素10(IL-10)mRNA和转化生长因子-β(TGF-β)mRNA的表达水平。结果:发生GD的小鼠甲状腺滤泡细胞增生肥大,且血清FT4及TSAb水平升高。小鼠血清TSAb与FT4水平呈显著正相关(r=0.90,P<0.01);TSHR-A亚单位-腺病毒免疫组发生GD的小鼠脾脏CD19+B细胞较对照组明显升高(P<0.05),B细胞中CD1dhiCD5+CD19+Bregs所占比例和脾组织IL-10 mRNA、TGF-βmRNA表达量较对照组明显降低(P<0.05);且血清TSAb活性与CD1dhiCD5+CD19+Bregs占脾脏B细胞比例、脾组织IL-10 mRNA、TGF-βmRNA表达量均呈显著负相关(P<0.05)。结论:TSHR-A亚单位-腺病毒免疫建立的GD模型中Bregs表达存在缺陷,提示Bregs表达可能在GD的发生及发展中发挥免疫调节作用,且GD疾病严重程度可能与Bregs缺乏程度有关。
