自身免疫调节因子对RAW264.7细胞中CCL22表达影响的研究
摘要
自身免疫调节因子(autoimmune regulator, Aire)是一种转录调节因子,其基因的突变可以导致自身免疫性多腺体综合征,因此,被认为是维持自身免疫耐受的重要分子。Aire在多种淋巴器官中都有表达,尤其在胸腺中表达较高。胸腺中,Aire主要表达在胸腺髓质上皮细胞(mTEC)上,其功能是调节mTEC外周组织抗原的表达、清除自身反应性T细胞,在胸腺的阴性选择、维持中枢自身免疫耐受过程中发挥重要作用。并且,Aire还可通过促进mTEC表达趋化因子CCL22趋化胸腺细胞在胸腺中迁移参与阴性选择。
然而,Aire在外周免疫器官中表达的生物学意义还不清楚。我们推测,Aire在外周免疫器官也可能通过调节趋化因子的表达,进而趋化特定的免疫耐受细胞群,在维持外周免疫耐受中发挥作用。
为此,本研究采用Aire转染RAW264.7细胞的方法,从以下两个方面探讨了Aire对RAW264.7细胞中CCL22表达的影响:
1.首先研究瞬时转染Aire的RAW264.7细胞中CCL22表达变化,研究结果表明转染Aire后CCL22表达水平上调。
2.通过建立稳定表达GFP及GFP-Aire融合蛋白的RAW264.7细胞系,研究稳定表达Aire的RAW264.7中CCL22表达变化,结果显示该细胞系中CCL22表达增加。
本研究通过探讨Aire对趋化因子CCL22的影响,为进一步研究Aire能否在外周免疫器官通过调节趋化因子的表达,进而趋化特定的免疫耐受细胞群,如调节性T细胞(regulatory T cell, Treg),奠定了基础。为揭示Aire在外周免疫耐受中的作用及其基因突变导致相关自身免疫病的发病机理,开辟了新的思路。
Autoimmune regulator (Aire) is a transcriptional regulator, which is considered as an important factor in maintenance of self-tolerance because its mutations could cause autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Studies have revealed that Aire is primarily expressed in lymphoid organs, especially in thymus. Within the thymus, it is expressed predominantly in medullary thymic epithelia (mTEC). Aire expressed in mTEC could play the function in central tolerance by regulating the expression of peripheral tissue-specific antigens (PTAs) and deleting the self-reactive T cells. But, the function of above perhaps is not the only role of Aire in maintenance of central tolerance. Other studies have demonstrated that mTEC can express many chemokines regulated by Aire, including CCL22, and these chemokines can attract thymocytes towards mTEC to finish negative selection.
Studies have revealed that Aire is not only expressed in thymus, but also in peripheral lymphoid organs, such as lymph nodes and spleen. Recent data have also shown that Aire is expressed in appendix and embryo in low level. However, the function of Aire expressed in peripheral lymphoid organs is not clear so far. The facts that Aire in thymus can regulate the expression of CCL22 to induce central tolerance and some APCs in peripheral can secrete CCL22 to attract CD4+CD25+Treg make the possibility that in peripheral the expression of CCL22 can be regulated by Aire to maintain immune tolerance. To test this hypothesis, we investigated whether Aire could regulate the expression of CCL22 in APCs.
In this study, RAW264.7 cells which belong to macrophage cell line were used to establish two kinds of cell models: transiently and stable transfected cells with Aire.
1 Expression changes of CCL22 in RAW264.7 cells transfected with Aire transiently
In this research, RAW264.7 cell models transiently transfected with pEGFP-C1 or pEGFP-C1-Aire were established to study whether CCL22 could be regulated by Aire. Firstly, to identify whether the plasmids were the right ones, the plasmids were digested by enzymes: pEGFP-C1 was digested by one enzyme and pEGFP-C1-Aire was digested by one and two kinds of enzymes respectively. Results showed that the products of digestion were the same size as expected, indicting the plasmids were the ones needed. Secondly, RAW264.7 cells were transfected with pEGFP-C1 or pEGFP-C1-Aire transiently respectively, and then transfection effects were identified by fluorescence microscope and analyzed expression level of Aire mRNA. The results revealed that the transfection was successful. Thirdly, mRNA expression level of CCL22 (48h after transfection) was analyzed by RT-PCR. The results showed that compared with vector group, CCL22 of Aire group was up-regulated and statistical significance (P<0.05), making the possibility that expression level of CCL22 in RAW264.7 cells could be up-regulated by Aire.
2 Expression changes of CCL22 in RAW264.7 cells transfected with Aire stably
In order to better understand the role that Aire plays on the expression level of CCL22 and avoid stochastic factors, stable transfected cell lines expressing GFP and GFP-Aire were established. The stable transfected cell lines were generated using selection marker G418.The positive cell clones were identified by RT-PCR, fluorescence microscope and immunohistochemistry. The results showed that the stable transfected cell lines were achieved successfully. After that, mRNA expression level of CCL22 was analyzed by RT-PCR. The results revealed that CCL22 expression level in Aire group was significantly up-regulated and statistically significant (P<0.05), showing that the expression level of CCL22 could be up-regulated in RAW264.7 stable transfected cell lines by Aire.
This study demonstrated that CCL22 expression level can be up-regulated by Aire in RAW264.7 cells and brought possibility that CCL22 could be up-regulated by Aire in peripheral APCs. This study provided experimental basis for studying relationship between Aire and CD4+CD25+Treg, and so offered a new point of view to understand function and significance of Aire in peripheral immune tolerance.
然而,Aire在外周免疫器官中表达的生物学意义还不清楚。我们推测,Aire在外周免疫器官也可能通过调节趋化因子的表达,进而趋化特定的免疫耐受细胞群,在维持外周免疫耐受中发挥作用。
为此,本研究采用Aire转染RAW264.7细胞的方法,从以下两个方面探讨了Aire对RAW264.7细胞中CCL22表达的影响:
1.首先研究瞬时转染Aire的RAW264.7细胞中CCL22表达变化,研究结果表明转染Aire后CCL22表达水平上调。
2.通过建立稳定表达GFP及GFP-Aire融合蛋白的RAW264.7细胞系,研究稳定表达Aire的RAW264.7中CCL22表达变化,结果显示该细胞系中CCL22表达增加。
本研究通过探讨Aire对趋化因子CCL22的影响,为进一步研究Aire能否在外周免疫器官通过调节趋化因子的表达,进而趋化特定的免疫耐受细胞群,如调节性T细胞(regulatory T cell, Treg),奠定了基础。为揭示Aire在外周免疫耐受中的作用及其基因突变导致相关自身免疫病的发病机理,开辟了新的思路。
Autoimmune regulator (Aire) is a transcriptional regulator, which is considered as an important factor in maintenance of self-tolerance because its mutations could cause autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Studies have revealed that Aire is primarily expressed in lymphoid organs, especially in thymus. Within the thymus, it is expressed predominantly in medullary thymic epithelia (mTEC). Aire expressed in mTEC could play the function in central tolerance by regulating the expression of peripheral tissue-specific antigens (PTAs) and deleting the self-reactive T cells. But, the function of above perhaps is not the only role of Aire in maintenance of central tolerance. Other studies have demonstrated that mTEC can express many chemokines regulated by Aire, including CCL22, and these chemokines can attract thymocytes towards mTEC to finish negative selection.
Studies have revealed that Aire is not only expressed in thymus, but also in peripheral lymphoid organs, such as lymph nodes and spleen. Recent data have also shown that Aire is expressed in appendix and embryo in low level. However, the function of Aire expressed in peripheral lymphoid organs is not clear so far. The facts that Aire in thymus can regulate the expression of CCL22 to induce central tolerance and some APCs in peripheral can secrete CCL22 to attract CD4+CD25+Treg make the possibility that in peripheral the expression of CCL22 can be regulated by Aire to maintain immune tolerance. To test this hypothesis, we investigated whether Aire could regulate the expression of CCL22 in APCs.
In this study, RAW264.7 cells which belong to macrophage cell line were used to establish two kinds of cell models: transiently and stable transfected cells with Aire.
1 Expression changes of CCL22 in RAW264.7 cells transfected with Aire transiently
In this research, RAW264.7 cell models transiently transfected with pEGFP-C1 or pEGFP-C1-Aire were established to study whether CCL22 could be regulated by Aire. Firstly, to identify whether the plasmids were the right ones, the plasmids were digested by enzymes: pEGFP-C1 was digested by one enzyme and pEGFP-C1-Aire was digested by one and two kinds of enzymes respectively. Results showed that the products of digestion were the same size as expected, indicting the plasmids were the ones needed. Secondly, RAW264.7 cells were transfected with pEGFP-C1 or pEGFP-C1-Aire transiently respectively, and then transfection effects were identified by fluorescence microscope and analyzed expression level of Aire mRNA. The results revealed that the transfection was successful. Thirdly, mRNA expression level of CCL22 (48h after transfection) was analyzed by RT-PCR. The results showed that compared with vector group, CCL22 of Aire group was up-regulated and statistical significance (P<0.05), making the possibility that expression level of CCL22 in RAW264.7 cells could be up-regulated by Aire.
2 Expression changes of CCL22 in RAW264.7 cells transfected with Aire stably
In order to better understand the role that Aire plays on the expression level of CCL22 and avoid stochastic factors, stable transfected cell lines expressing GFP and GFP-Aire were established. The stable transfected cell lines were generated using selection marker G418.The positive cell clones were identified by RT-PCR, fluorescence microscope and immunohistochemistry. The results showed that the stable transfected cell lines were achieved successfully. After that, mRNA expression level of CCL22 was analyzed by RT-PCR. The results revealed that CCL22 expression level in Aire group was significantly up-regulated and statistically significant (P<0.05), showing that the expression level of CCL22 could be up-regulated in RAW264.7 stable transfected cell lines by Aire.
This study demonstrated that CCL22 expression level can be up-regulated by Aire in RAW264.7 cells and brought possibility that CCL22 could be up-regulated by Aire in peripheral APCs. This study provided experimental basis for studying relationship between Aire and CD4+CD25+Treg, and so offered a new point of view to understand function and significance of Aire in peripheral immune tolerance.
引文
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