Class Ⅱ Transactivator(CIITA)作为重要的MHCⅡ类分子反式激活因子在抗原递呈细胞的成熟和功能实现过程中发挥关键作用,为建立一种快速检测猪源CIITA基因的方法,本研究根据GenBank中猪源CIITA基因序列设计引物,从猪肺泡巨噬细胞的总c...Class Ⅱ Transactivator(CIITA)作为重要的MHCⅡ类分子反式激活因子在抗原递呈细胞的成熟和功能实现过程中发挥关键作用,为建立一种快速检测猪源CIITA基因的方法,本研究根据GenBank中猪源CIITA基因序列设计引物,从猪肺泡巨噬细胞的总cDNA中扩增CIITA基因,并克隆到pCold-TF载体中获得重组质粒pCold-TF-CIITA。利用重组质粒pCold-TF-CIITA作为标准品建立SYBR Green Ⅰ荧光定量PCR检测方法。试验结果显示:该方法在1.68×10^(1)~1.68×10^(7) copies/μL范围内呈现出良好的线性关系,检测灵敏度可达16.8 copies/μL,组内和组间变异系数均小于1%。利用该方法对不同细胞中的CIITA基因进行检测,结果显示该方法仅能够检测到猪源细胞中的CIITA基因。本试验结果表明,利用pCold-TF-CIITA作为标准品建立的CIITA基因SYBR Green Ⅰ荧光定量PCR检测方法具有良好的特异性、敏感性和重复性,可用于CIITA基因的定量检测。展开更多
Increased accumulation and/or impaired utilization of fatty acid in extra-adipose tissues are implicated in the pathogenesis of insulin resistance and type 2 diabetes. Pyruvate dehydrogenase kinase 4 （Pdk4） is a key...Increased accumulation and/or impaired utilization of fatty acid in extra-adipose tissues are implicated in the pathogenesis of insulin resistance and type 2 diabetes. Pyruvate dehydrogenase kinase 4 （Pdk4） is a key enzyme involved in fatty oxidation and energy expenditure, and its expression can be repressed by pro-inflammatory stimuli. Previously, we have shown that class II transactivator （CIITA） mediates the adverse effect of interferon gamma （IFN-7） in skeletal muscle cells by cooperating with hypermethylated in cancer 1 （HIC1） to repress silent informa- tion regulator 1 （SIRT1） transcription. Building upon this finding, we report here that CIITA interacted with HIC1 via the GTP-binding domain （GBD） while HIC1 interacted with CIITA via the BTB/POZ domain. The GBD domain was required for CIITA to repress SIRT1 transcription probably acting as a bridge for CIITA to bind to HIC1 and consequently to bind to the SIRT1 promoter. IFN-7 stimulation, CIITA over-expression, or HIC1 over- expression repressed Pdk4 promoter activity while silencing either CIITA or HIC1 normalized Pdk4 expression in the presence of IFN-7. An increase in SIRT1 expression or activity partially rescued Pdk4 expression in the pre- sence of CIITA, but SIRT1 inhibition abrogated Pdk4 normalization even in the absence of CIITA. Taken together, our data have identified a HIC1-CIITA-SIRT1 axis that regulates Pdk4 transcription in response to IFN-7 stimula- tion.展开更多
目的:构建携带小鼠MHCII类分子反式激活因子(MHC class II molecule transactivator,CIITA)突变体基因的腺病毒,并观察腺病毒介导该基因的表达情况以及表达产物的体外功能。方法:采用常规分子生物学方法从IFN-γ诱导后的BALB/c小鼠腹腔...目的:构建携带小鼠MHCII类分子反式激活因子(MHC class II molecule transactivator,CIITA)突变体基因的腺病毒,并观察腺病毒介导该基因的表达情况以及表达产物的体外功能。方法:采用常规分子生物学方法从IFN-γ诱导后的BALB/c小鼠腹腔巨噬细胞中获得IV型CIITA cDNA;利用重叠延伸PCR法构建CIITA突变体基因,并克隆入表达载体pIRES;采用pAdEasy-1系统获得具有感染能力的、携带CIITA突变体基因的缺陷型重组腺病毒(Ad-CIITAm)和空载对照病毒(Ad-GFP),并经大量扩增、纯化及滴度测定;将Ad-CIITAm和Ad-GFP分别感染HeLa细胞和Raji细胞,流式细胞术观察对诱导型和组成型HLA-DR分子表达的影响。结果:成功克隆了小鼠CIITA突变体基因,并构建了携带小鼠CIITA突变体基因的重组腺病毒Ad-CIITAm;经流式细胞术证实感染Ad-CIITAm的Hela和Raji细胞较感染Ad-GFP的细胞,其表面HLA-DR分子的表达均受到明显的抑制。结论:本实验证实了重组腺病毒介导表达的小鼠CIITA突变体在体外能够有效地抑制MHCII类分子的表达。展开更多
To study the effect of the major histocompatibility complex class Ⅱ (MHC Ⅱ ) transactivator (CIITA) antisense RNA on the expression of the human leukemia (HLA) class Ⅱ molecules, 5’ end cDNA sequence of CIITA gene...To study the effect of the major histocompatibility complex class Ⅱ (MHC Ⅱ ) transactivator (CIITA) antisense RNA on the expression of the human leukemia (HLA) class Ⅱ molecules, 5’ end cDNA sequence of CIITA gene was cloned, and antisense RNA expression vector pcDNA-Ⅱ was constructed. HeLa cells transfected with pcDNA-Ⅱ and pcDNA3 were induced by IFN-γ for 3 d. The expression of HLA class Ⅱ molecules on HeLa/pcDNA-Ⅱ cells was significantly decreased, while it has no effect on the expression of HLA class Ⅰ molecules. This result suggests that the CIITA antisense RNA can inhibit the expression of HLA class Ⅱ molecules in HeLa cells. It also implies a promising approach to generate immune tolerance in qraft transplantation.展开更多
MHC class II expression is controlled mainly at transcriptional level by class II transactivator(CIITA),which is a non-DNA binding coactivator and serves as a master control factor for MHC class II genes expression.He...MHC class II expression is controlled mainly at transcriptional level by class II transactivator(CIITA),which is a non-DNA binding coactivator and serves as a master control factor for MHC class II genes expression.Here,we describe the function of a novel splice-isoform of CIITA,DC-expressed caspase inhibitory isoform of CIITA(or DC-CASPIC),and we show that the expression of DCCASPIC in DC is upregulated upon lipopolysaccharides(LPS)induction.DC-CASPIC localizes to mitochondria,and protein-protein interaction study demonstrates that DC-CASPIC interacts with caspases and inhibits its activity in DC.Consistently,DC-CASPIC suppresses caspases-induced degradation of nitric oxide synthase-2(NOS2)and subsequently promotes the synthesis of nitric oxide(NO).NO is an essential regulatory molecule that modulates the capability of DC in stimulating T cell proliferation/activation in vitro;hence,overexpression of DC-CASPIC in DC enhances this stimulation.Collectively,our findings reveal that DC-CASPIC is a key molecule that regulates caspases activity and NO synthesis in DC.展开更多
In a recent paper in Science,Bruchez et al.1 provide new insights how host cells can fight virus infection.They show that an MHC class II transactivator(CIITA)—which normally operates as part of the interferon(IFN)-s...In a recent paper in Science,Bruchez et al.1 provide new insights how host cells can fight virus infection.They show that an MHC class II transactivator(CIITA)—which normally operates as part of the interferon(IFN)-stimulated immune response—is also involved in an antiviral response,beyond its well-known function of antigen presentation.展开更多
文摘Class Ⅱ Transactivator(CIITA)作为重要的MHCⅡ类分子反式激活因子在抗原递呈细胞的成熟和功能实现过程中发挥关键作用,为建立一种快速检测猪源CIITA基因的方法,本研究根据GenBank中猪源CIITA基因序列设计引物,从猪肺泡巨噬细胞的总cDNA中扩增CIITA基因,并克隆到pCold-TF载体中获得重组质粒pCold-TF-CIITA。利用重组质粒pCold-TF-CIITA作为标准品建立SYBR Green Ⅰ荧光定量PCR检测方法。试验结果显示:该方法在1.68×10^(1)~1.68×10^(7) copies/μL范围内呈现出良好的线性关系,检测灵敏度可达16.8 copies/μL,组内和组间变异系数均小于1%。利用该方法对不同细胞中的CIITA基因进行检测,结果显示该方法仅能够检测到猪源细胞中的CIITA基因。本试验结果表明,利用pCold-TF-CIITA作为标准品建立的CIITA基因SYBR Green Ⅰ荧光定量PCR检测方法具有良好的特异性、敏感性和重复性,可用于CIITA基因的定量检测。
基金supported,in part,by the National Natural Science Foundation of China(31200645)the Natural Science Foundation of Jiangsu Province(BK20141498)a grant from Jiangsu Jiankang Vocational University(JK201405)
文摘Increased accumulation and/or impaired utilization of fatty acid in extra-adipose tissues are implicated in the pathogenesis of insulin resistance and type 2 diabetes. Pyruvate dehydrogenase kinase 4 （Pdk4） is a key enzyme involved in fatty oxidation and energy expenditure, and its expression can be repressed by pro-inflammatory stimuli. Previously, we have shown that class II transactivator （CIITA） mediates the adverse effect of interferon gamma （IFN-7） in skeletal muscle cells by cooperating with hypermethylated in cancer 1 （HIC1） to repress silent informa- tion regulator 1 （SIRT1） transcription. Building upon this finding, we report here that CIITA interacted with HIC1 via the GTP-binding domain （GBD） while HIC1 interacted with CIITA via the BTB/POZ domain. The GBD domain was required for CIITA to repress SIRT1 transcription probably acting as a bridge for CIITA to bind to HIC1 and consequently to bind to the SIRT1 promoter. IFN-7 stimulation, CIITA over-expression, or HIC1 over- expression repressed Pdk4 promoter activity while silencing either CIITA or HIC1 normalized Pdk4 expression in the presence of IFN-7. An increase in SIRT1 expression or activity partially rescued Pdk4 expression in the pre- sence of CIITA, but SIRT1 inhibition abrogated Pdk4 normalization even in the absence of CIITA. Taken together, our data have identified a HIC1-CIITA-SIRT1 axis that regulates Pdk4 transcription in response to IFN-7 stimula- tion.
文摘目的:构建携带小鼠MHCII类分子反式激活因子(MHC class II molecule transactivator,CIITA)突变体基因的腺病毒,并观察腺病毒介导该基因的表达情况以及表达产物的体外功能。方法:采用常规分子生物学方法从IFN-γ诱导后的BALB/c小鼠腹腔巨噬细胞中获得IV型CIITA cDNA;利用重叠延伸PCR法构建CIITA突变体基因,并克隆入表达载体pIRES;采用pAdEasy-1系统获得具有感染能力的、携带CIITA突变体基因的缺陷型重组腺病毒(Ad-CIITAm)和空载对照病毒(Ad-GFP),并经大量扩增、纯化及滴度测定;将Ad-CIITAm和Ad-GFP分别感染HeLa细胞和Raji细胞,流式细胞术观察对诱导型和组成型HLA-DR分子表达的影响。结果:成功克隆了小鼠CIITA突变体基因,并构建了携带小鼠CIITA突变体基因的重组腺病毒Ad-CIITAm;经流式细胞术证实感染Ad-CIITAm的Hela和Raji细胞较感染Ad-GFP的细胞,其表面HLA-DR分子的表达均受到明显的抑制。结论:本实验证实了重组腺病毒介导表达的小鼠CIITA突变体在体外能够有效地抑制MHCII类分子的表达。
文摘To study the effect of the major histocompatibility complex class Ⅱ (MHC Ⅱ ) transactivator (CIITA) antisense RNA on the expression of the human leukemia (HLA) class Ⅱ molecules, 5’ end cDNA sequence of CIITA gene was cloned, and antisense RNA expression vector pcDNA-Ⅱ was constructed. HeLa cells transfected with pcDNA-Ⅱ and pcDNA3 were induced by IFN-γ for 3 d. The expression of HLA class Ⅱ molecules on HeLa/pcDNA-Ⅱ cells was significantly decreased, while it has no effect on the expression of HLA class Ⅰ molecules. This result suggests that the CIITA antisense RNA can inhibit the expression of HLA class Ⅱ molecules in HeLa cells. It also implies a promising approach to generate immune tolerance in qraft transplantation.
基金the Academic Research Council-Singapore Ministry of Education(R182-000-099-112 to S.H.W)NMRC(R182-000-137-213 to S.H.W)grants.
文摘MHC class II expression is controlled mainly at transcriptional level by class II transactivator(CIITA),which is a non-DNA binding coactivator and serves as a master control factor for MHC class II genes expression.Here,we describe the function of a novel splice-isoform of CIITA,DC-expressed caspase inhibitory isoform of CIITA(or DC-CASPIC),and we show that the expression of DCCASPIC in DC is upregulated upon lipopolysaccharides(LPS)induction.DC-CASPIC localizes to mitochondria,and protein-protein interaction study demonstrates that DC-CASPIC interacts with caspases and inhibits its activity in DC.Consistently,DC-CASPIC suppresses caspases-induced degradation of nitric oxide synthase-2(NOS2)and subsequently promotes the synthesis of nitric oxide(NO).NO is an essential regulatory molecule that modulates the capability of DC in stimulating T cell proliferation/activation in vitro;hence,overexpression of DC-CASPIC in DC enhances this stimulation.Collectively,our findings reveal that DC-CASPIC is a key molecule that regulates caspases activity and NO synthesis in DC.
基金supported by the“Excellence Initiative—Research University”program at the Nicolaus Copernicus UniversityK.P.is supported by the subsidy from the Polish Ministry of Science and Higher Education for the research on the SARS-CoV-2 and a grant from the National Science Center UMO-2017/27/B/NZ6/02488C.S.v.B.is supported by grant GM103554 from the National Institutes of Health.
文摘In a recent paper in Science,Bruchez et al.1 provide new insights how host cells can fight virus infection.They show that an MHC class II transactivator(CIITA)—which normally operates as part of the interferon(IFN)-stimulated immune response—is also involved in an antiviral response,beyond its well-known function of antigen presentation.
