The expression of paired immunoglobin-like receptors A (PIR-A) and B (PIR-B) and their relationship with tolerogenic dendritic cells (T-DC) in mice were investigated. The mouse DCs line, DC2.4 cells were cultured with...The expression of paired immunoglobin-like receptors A (PIR-A) and B (PIR-B) and their relationship with tolerogenic dendritic cells (T-DC) in mice were investigated. The mouse DCs line, DC2.4 cells were cultured with the recombinant murine interleukin-10 (IL-10) and recombinant human transforming growth factor β1 (TGF-β1) respectively to develop the T-DC and stimulated with lipopolysaccharide (LPS) for 48 h to induce the mature dendritic cells (LPS-DC). Special small interfering RNAs (siRNA) molecule for PIR-B was chemically synthesized and transfected into DC2.4 cells (Si-DC) by lip2000. The expression of PIRs on DC2.4 cells were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), flow cytometry (FCM) and Western blot. Realtime reverse transcriptase-polymerase chain reaction (Realtime-PCR) was ap- plied for measurement of PIR-A and CD80, CD86, MHC-Ⅱ mRNA expression. The allogeneic stimulating capacity of DCs was measured by mixed lymphocyte reaction (MLR) using 3H-thymidine incorporation test. The concentration of IFN-γ in supernatants of MLR from distinct groups was ana- lyzed by ELISA. The results showed that PIR positive rate was (28.65±8.12)% examined by FCM on DC2.4 cells. PIR positive rate was increased dramatically to (54.21±6.34)%, (58.78±4.70)%, (48.24±6.75)% respectively for IL-10, TGF-β1 and LPS induction (P<0.01), but there was no sig- nificantly different among the three groups (P>0.01). The semi-quantitative RT-PCR and Western blot revealed that IL-10 and TGF-β1 induced the higher PIR-B level and lower PIR-A level. On the contrary, the LPS down-regulated the PIR-B expression and up-regulated the PIR-A expression. Realtime PCR examination demonstrated that PIR-A and co-stimulating molecules such as CD80, CD86 and MHC-Ⅱ were increased significantly after stimulation with LPS. Compared with the DC2.4 cells and the LPS-DC, the T-DCs inhibited alloactived T cell proliferation and down-regulated the IFN-γ secretion in MLR supernatant. Si-DC promoted the T cell proliferation (P<0.01) and en- hanced the IFN-γ secretion (P<0.01). It was concluded that up-regulating the PIR-B and down-regulating the PIR-A expression were the general feature of phenotype and constructed the new targets for dendritic cells to acquire immune tolerance in mice. Overexpression of PIR-B can inhibit the up-regulation of the PIR-A, CD80, CD86 and MHC-Ⅱ expression, which might be the molecular mechanism for the T-DC.展开更多
目的探讨高氧及配对免疫球蛋白样受体B(Pir B)对大鼠体外少突前体细胞(OPCs)的影响,以及17β雌二醇(E2)的保护作用。方法用逆转录定量酶联反应(RT-q PCR)检测OPCs的Pir B m RNA和蛋白表达,用si RNA沉默OPCs的Pir B,高氧刺激后再用E2处理...目的探讨高氧及配对免疫球蛋白样受体B(Pir B)对大鼠体外少突前体细胞(OPCs)的影响,以及17β雌二醇(E2)的保护作用。方法用逆转录定量酶联反应(RT-q PCR)检测OPCs的Pir B m RNA和蛋白表达,用si RNA沉默OPCs的Pir B,高氧刺激后再用E2处理,通过3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)比色法和流式细胞术测定OPCs细胞存活率和凋亡率。结果高氧导致OPCs凋亡增加和活性降低,E2能够明显下调Pir B的表达,E2处理或沉默Pir B能够明显减少高氧所致的OPCs凋亡、增加细胞存活率。结论高氧导致OPCs的凋亡,而E2能够保护高氧所致的OPCs凋亡。展开更多
基金a grant from National Natu-ral Sciences Foundation of China (No. 30571755)
文摘The expression of paired immunoglobin-like receptors A (PIR-A) and B (PIR-B) and their relationship with tolerogenic dendritic cells (T-DC) in mice were investigated. The mouse DCs line, DC2.4 cells were cultured with the recombinant murine interleukin-10 (IL-10) and recombinant human transforming growth factor β1 (TGF-β1) respectively to develop the T-DC and stimulated with lipopolysaccharide (LPS) for 48 h to induce the mature dendritic cells (LPS-DC). Special small interfering RNAs (siRNA) molecule for PIR-B was chemically synthesized and transfected into DC2.4 cells (Si-DC) by lip2000. The expression of PIRs on DC2.4 cells were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), flow cytometry (FCM) and Western blot. Realtime reverse transcriptase-polymerase chain reaction (Realtime-PCR) was ap- plied for measurement of PIR-A and CD80, CD86, MHC-Ⅱ mRNA expression. The allogeneic stimulating capacity of DCs was measured by mixed lymphocyte reaction (MLR) using 3H-thymidine incorporation test. The concentration of IFN-γ in supernatants of MLR from distinct groups was ana- lyzed by ELISA. The results showed that PIR positive rate was (28.65±8.12)% examined by FCM on DC2.4 cells. PIR positive rate was increased dramatically to (54.21±6.34)%, (58.78±4.70)%, (48.24±6.75)% respectively for IL-10, TGF-β1 and LPS induction (P<0.01), but there was no sig- nificantly different among the three groups (P>0.01). The semi-quantitative RT-PCR and Western blot revealed that IL-10 and TGF-β1 induced the higher PIR-B level and lower PIR-A level. On the contrary, the LPS down-regulated the PIR-B expression and up-regulated the PIR-A expression. Realtime PCR examination demonstrated that PIR-A and co-stimulating molecules such as CD80, CD86 and MHC-Ⅱ were increased significantly after stimulation with LPS. Compared with the DC2.4 cells and the LPS-DC, the T-DCs inhibited alloactived T cell proliferation and down-regulated the IFN-γ secretion in MLR supernatant. Si-DC promoted the T cell proliferation (P<0.01) and en- hanced the IFN-γ secretion (P<0.01). It was concluded that up-regulating the PIR-B and down-regulating the PIR-A expression were the general feature of phenotype and constructed the new targets for dendritic cells to acquire immune tolerance in mice. Overexpression of PIR-B can inhibit the up-regulation of the PIR-A, CD80, CD86 and MHC-Ⅱ expression, which might be the molecular mechanism for the T-DC.
文摘目的探讨高氧及配对免疫球蛋白样受体B(Pir B)对大鼠体外少突前体细胞(OPCs)的影响,以及17β雌二醇(E2)的保护作用。方法用逆转录定量酶联反应(RT-q PCR)检测OPCs的Pir B m RNA和蛋白表达,用si RNA沉默OPCs的Pir B,高氧刺激后再用E2处理,通过3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)比色法和流式细胞术测定OPCs细胞存活率和凋亡率。结果高氧导致OPCs凋亡增加和活性降低,E2能够明显下调Pir B的表达,E2处理或沉默Pir B能够明显减少高氧所致的OPCs凋亡、增加细胞存活率。结论高氧导致OPCs的凋亡,而E2能够保护高氧所致的OPCs凋亡。
