FcαR内吞及脱落机制研究
发布时间：2018-07-17 06:48
【摘要】： 本论文分为两部分,第一部分研究了FcaR (CD89)的内吞机制,第二部分研究了FcaR的脱落机制。另外,2006年3月至2007年2月期间,本人参与了本课题组尹娜博士的研究课题,这部分研究内容和结果未纳入本论文,具体内容详见尹娜博士的博士论文以及发表文章。 第一部分 FcaR是IgA的Fc受体,在IgA介导的免疫反应中发挥着重要作用。研究发现,IgA以及IgA免疫复合物结合FcaR后,会被FcaR迅速内吞入细胞内。但是,FcaR介导IgA和IgA免疫复合物内吞的机制还不清楚。在本研究中,我们使用天然表达FcaR的U937细胞以及转染的CHO、COS-7和Hela细胞,系统性地研究了FcaR的内吞机制。通过使用能特异性抑制不同内吞途径的化学抑制剂、过表达Eps15负显性突变体以及运用RNA干扰技术抑制内源性笼型蛋白重链(CHC)的表达水平,我们证明了FcαR是通过笼型蛋白途径内吞的。内吞后FcaR进入Rab5以及Rab11阳性的内体中。但是,过表达Rab5的负显性突变体并不能抑制FcaR的内吞。同时,通过过表达Dynamin的负显性突变体,我们发现FcaR内吞依赖于Dynamin o我们还研究了FcαR的内吞基序,但意外地发现FcaR的内吞序列并不存在于FcαR的胞内区。综上所述,我们证明了FcaR内吞依赖于笼型蛋白和Dynamin,但不受Rab5调控,而且FcaR的内吞基序不在膜内区。 第二部分 FcaR在IgA介导的免疫反应中发挥着重要作用。研究发现,人血清和表达FcaR细胞的培养液上清中存在着可溶性的FcaR (sFcaR)。进一步研究证实,sFcaR是FcaR的膜外区被酶切后脱落产生的,即所谓的受体脱落。但是,FcaR脱落的具体机制目前还不清楚。因此,我们研究了FcaR的脱落机制并找到了酶切FcaR膜外区产生sFcaR的蛋白酶。在使用化学抑制剂进行的实验中,EDTA、EGTA和广谱金属蛋白酶抑制GM6001能显著抑制FcaR脱落,表明FcaR是被某一种或几种金属蛋白酶酶切的。在293T细胞中过表达ADAM (a disintegrin andmetalloproteinase)10和ADAM 17的负显性突变体能明显抑制FcaR脱落,表明FcaR脱落与这两种金属蛋白酶有关。最后,通过RNA干扰技术在天然表达FcaR的U937细胞中抑制内源性ADAM10和ADAM 17的表达水平,我们证明了ADAM 10和ADAM17都参与了FcaR脱落。该研究鉴定出了导致FcaR脱落的蛋白酶,阐明了sFcaR产生的分子机制,这有助于我们进一步研究sFcaR的生理和病理作用。
[Abstract]:This thesis is divided into two parts: the first part studies the endocytosis mechanism of FCAR (CD89) and the second part studies the mechanism of FCAR exfoliation. In addition, from March 2006 to February 2007, I participated in the research project of Dr. Yin Na, my research group. This part of the research content and results were not included in this thesis. The first part, FCAR, is the FC receptor of IgA, which plays an important role in the immune response mediated by IgA. It has been found that IgA and IgA immune complexes bind to FCAR and are rapidly ingested into cells by FCAR. However, the mechanism of FCAR mediated endocytosis of IgA and IgA immune complex remains unclear. In this study, we systematically studied the endocytosis mechanism of FcaR by using U937 cells expressing FCAR and transfected CHOCOS-7 and Hela cells. Overexpression of EPS15 negative dominant mutant and inhibition of the expression of endogenous caged protein heavy chain (CHC) by the use of chemical inhibitors that specifically inhibit different endocytosis pathways, and RNA interference were used to inhibit the expression of EPS15 negative dominant mutants. We have proved that FC 伪 R is endocytosis through cage protein pathway. After endocytosis, FCAR entered Rab5 and Rab11 positive innervation. However, negative dominant mutants expressing Rab5 did not inhibit the endocytosis of FCAR. At the same time, by over-expressing the negative dominant mutant of dynamic, we found that the endocytosis of FCAR is dependent on dynamics o. We also studied the endocytosis motif of FC 伪 R, but accidentally found that the endocytosis sequence of FCAR does not exist in the intracellular region of FC 伪 R. In conclusion, we prove that the endocytosis of FCAR is dependent on cage protein and dynamic, but not regulated by Rab5, and the endocytosis motif of FCAR is not in the intramembrane region. In the second part, FCAR plays an important role in IgA mediated immune response. It was found that soluble FCAR (sFcaR) existed in human serum and the supernatant of FCAR cells. Further studies confirmed that the extracellular region of FCAR was exfoliated by enzyme, the so-called receptor exfoliation. However, the specific mechanism of FCAR abscission is unclear. Therefore, we studied the exfoliation mechanism of FCAR and found the protease of sFcaR produced by enzyme digestion of the extracellular region of FCAR. In the experiments using chemical inhibitors, EDTAEGTA and broad-spectrum metalloproteinases inhibited FCAR shedding significantly, indicating that FCAR was cut by one or more kinds of metalloproteinases. The negative dominant mutations overexpressing Adam (a disintegrin andmetalloproteinase) 10 and ADAM17 in 293T cells could significantly inhibit FCA R exfoliation, suggesting that FCAR exfoliation was related to these two metalloproteinases. Finally, the expression levels of endogenous ADAM10 and Adam17 were inhibited by RNA interference in U937 cells expressing FCAR. We proved that both ADAM10 and Adam17 were involved in FcaR exfoliation. This study identified the protease that caused the exfoliation of FcaR and elucidated the molecular mechanism of sFcaR, which is helpful to further study the physiological and pathological role of sFcaR.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392.1
本文编号：2129471
[Abstract]:This thesis is divided into two parts: the first part studies the endocytosis mechanism of FCAR (CD89) and the second part studies the mechanism of FCAR exfoliation. In addition, from March 2006 to February 2007, I participated in the research project of Dr. Yin Na, my research group. This part of the research content and results were not included in this thesis. The first part, FCAR, is the FC receptor of IgA, which plays an important role in the immune response mediated by IgA. It has been found that IgA and IgA immune complexes bind to FCAR and are rapidly ingested into cells by FCAR. However, the mechanism of FCAR mediated endocytosis of IgA and IgA immune complex remains unclear. In this study, we systematically studied the endocytosis mechanism of FcaR by using U937 cells expressing FCAR and transfected CHOCOS-7 and Hela cells. Overexpression of EPS15 negative dominant mutant and inhibition of the expression of endogenous caged protein heavy chain (CHC) by the use of chemical inhibitors that specifically inhibit different endocytosis pathways, and RNA interference were used to inhibit the expression of EPS15 negative dominant mutants. We have proved that FC 伪 R is endocytosis through cage protein pathway. After endocytosis, FCAR entered Rab5 and Rab11 positive innervation. However, negative dominant mutants expressing Rab5 did not inhibit the endocytosis of FCAR. At the same time, by over-expressing the negative dominant mutant of dynamic, we found that the endocytosis of FCAR is dependent on dynamics o. We also studied the endocytosis motif of FC 伪 R, but accidentally found that the endocytosis sequence of FCAR does not exist in the intracellular region of FC 伪 R. In conclusion, we prove that the endocytosis of FCAR is dependent on cage protein and dynamic, but not regulated by Rab5, and the endocytosis motif of FCAR is not in the intramembrane region. In the second part, FCAR plays an important role in IgA mediated immune response. It was found that soluble FCAR (sFcaR) existed in human serum and the supernatant of FCAR cells. Further studies confirmed that the extracellular region of FCAR was exfoliated by enzyme, the so-called receptor exfoliation. However, the specific mechanism of FCAR abscission is unclear. Therefore, we studied the exfoliation mechanism of FCAR and found the protease of sFcaR produced by enzyme digestion of the extracellular region of FCAR. In the experiments using chemical inhibitors, EDTAEGTA and broad-spectrum metalloproteinases inhibited FCAR shedding significantly, indicating that FCAR was cut by one or more kinds of metalloproteinases. The negative dominant mutations overexpressing Adam (a disintegrin andmetalloproteinase) 10 and ADAM17 in 293T cells could significantly inhibit FCA R exfoliation, suggesting that FCAR exfoliation was related to these two metalloproteinases. Finally, the expression levels of endogenous ADAM10 and Adam17 were inhibited by RNA interference in U937 cells expressing FCAR. We proved that both ADAM10 and Adam17 were involved in FcaR exfoliation. This study identified the protease that caused the exfoliation of FcaR and elucidated the molecular mechanism of sFcaR, which is helpful to further study the physiological and pathological role of sFcaR.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392.1
【共引文献】
相关期刊论文 前1条
1 任继辉,石敏鹿,张伟;人IgA Fc受体(CD89)的功能部位[J];中国医学科学院学报;2000年06期
相关博士学位论文 前4条
1 薛婧;FcαR的糖基化及其异形体的功能效应[D];北京协和医学院;2011年
2 张圆;小鼠免疫抑制性受体LAIR-1分子的分布及功能的研究[D];第四军医大学;2006年
3 徐刚;人IgA Fc受体FcαRI（CD89）的结构和功能分析[D];中国协和医科大学;2004年
4 尹娜;FcαR在中性粒细胞的亚细胞定位和FcαR剪接体表达分析&依赖于FcαR的双特异性分子在自身免疫疾病治疗中的初步探索[D];中国协和医科大学;2007年
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