FcαR內(nèi)吞及脫落機(jī)制研究
發(fā)布時(shí)間：2018-07-17 06:48
【摘要】： 本論文分為兩部分,第一部分研究了FcaR (CD89)的內(nèi)吞機(jī)制,第二部分研究了FcaR的脫落機(jī)制。另外,2006年3月至2007年2月期間,本人參與了本課題組尹娜博士的研究課題,這部分研究內(nèi)容和結(jié)果未納入本論文,具體內(nèi)容詳見尹娜博士的博士論文以及發(fā)表文章。 第一部分 FcaR是IgA的Fc受體,在IgA介導(dǎo)的免疫反應(yīng)中發(fā)揮著重要作用。研究發(fā)現(xiàn),IgA以及IgA免疫復(fù)合物結(jié)合FcaR后,會被FcaR迅速內(nèi)吞入細(xì)胞內(nèi)。但是,FcaR介導(dǎo)IgA和IgA免疫復(fù)合物內(nèi)吞的機(jī)制還不清楚。在本研究中,我們使用天然表達(dá)FcaR的U937細(xì)胞以及轉(zhuǎn)染的CHO、COS-7和Hela細(xì)胞,系統(tǒng)性地研究了FcaR的內(nèi)吞機(jī)制。通過使用能特異性抑制不同內(nèi)吞途徑的化學(xué)抑制劑、過表達(dá)Eps15負(fù)顯性突變體以及運(yùn)用RNA干擾技術(shù)抑制內(nèi)源性籠型蛋白重鏈(CHC)的表達(dá)水平,我們證明了FcαR是通過籠型蛋白途徑內(nèi)吞的。內(nèi)吞后FcaR進(jìn)入Rab5以及Rab11陽性的內(nèi)體中。但是,過表達(dá)Rab5的負(fù)顯性突變體并不能抑制FcaR的內(nèi)吞。同時(shí),通過過表達(dá)Dynamin的負(fù)顯性突變體,我們發(fā)現(xiàn)FcaR內(nèi)吞依賴于Dynamin o我們還研究了FcαR的內(nèi)吞基序,但意外地發(fā)現(xiàn)FcaR的內(nèi)吞序列并不存在于FcαR的胞內(nèi)區(qū)。綜上所述,我們證明了FcaR內(nèi)吞依賴于籠型蛋白和Dynamin,但不受Rab5調(diào)控,而且FcaR的內(nèi)吞基序不在膜內(nèi)區(qū)。 第二部分 FcaR在IgA介導(dǎo)的免疫反應(yīng)中發(fā)揮著重要作用。研究發(fā)現(xiàn),人血清和表達(dá)FcaR細(xì)胞的培養(yǎng)液上清中存在著可溶性的FcaR (sFcaR)。進(jìn)一步研究證實(shí),sFcaR是FcaR的膜外區(qū)被酶切后脫落產(chǎn)生的,即所謂的受體脫落。但是,FcaR脫落的具體機(jī)制目前還不清楚。因此,我們研究了FcaR的脫落機(jī)制并找到了酶切FcaR膜外區(qū)產(chǎn)生sFcaR的蛋白酶。在使用化學(xué)抑制劑進(jìn)行的實(shí)驗(yàn)中,EDTA、EGTA和廣譜金屬蛋白酶抑制GM6001能顯著抑制FcaR脫落,表明FcaR是被某一種或幾種金屬蛋白酶酶切的。在293T細(xì)胞中過表達(dá)ADAM (a disintegrin andmetalloproteinase)10和ADAM 17的負(fù)顯性突變體能明顯抑制FcaR脫落,表明FcaR脫落與這兩種金屬蛋白酶有關(guān)。最后,通過RNA干擾技術(shù)在天然表達(dá)FcaR的U937細(xì)胞中抑制內(nèi)源性ADAM10和ADAM 17的表達(dá)水平,我們證明了ADAM 10和ADAM17都參與了FcaR脫落。該研究鑒定出了導(dǎo)致FcaR脫落的蛋白酶,闡明了sFcaR產(chǎn)生的分子機(jī)制,這有助于我們進(jìn)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 偽 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 偽 R, but accidentally found that the endocytosis sequence of FCAR does not exist in the intracellular region of FC 偽 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.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R392.1
本文編號：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 偽 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 偽 R, but accidentally found that the endocytosis sequence of FCAR does not exist in the intracellular region of FC 偽 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.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R392.1
【共引文獻(xiàn)】
相關(guān)期刊論文 前1條
1 任繼輝,石敏鹿,張偉;人IgA Fc受體(CD89)的功能部位[J];中國醫(yī)學(xué)科學(xué)院學(xué)報(bào);2000年06期
相關(guān)博士學(xué)位論文 前4條
1 薛婧;FcαR的糖基化及其異形體的功能效應(yīng)[D];北京協(xié)和醫(yī)學(xué)院;2011年
2 張圓;小鼠免疫抑制性受體LAIR-1分子的分布及功能的研究[D];第四軍醫(yī)大學(xué);2006年
3 徐剛;人IgA Fc受體FcαRI（CD89）的結(jié)構(gòu)和功能分析[D];中國協(xié)和醫(yī)科大學(xué);2004年
4 尹娜;FcαR在中性粒細(xì)胞的亞細(xì)胞定位和FcαR剪接體表達(dá)分析&依賴于FcαR的雙特異性分子在自身免疫疾病治療中的初步探索[D];中國協(xié)和醫(yī)科大學(xué);2007年
，本文編號：2129471
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