本文選題：BECN1 + DEFA1　； 參考：《延邊大學(xué)》2012年碩士論文

【摘要】：目的：為了觀察大自噬和抗菌肽在細(xì)胞內(nèi)的相互作用關(guān)系,兩者都在生物細(xì)胞的抗菌作用中起著關(guān)鍵性的作用。本實(shí)驗(yàn)選擇了抗菌肽的一個(gè)代表DEFAl,通過(guò)觀察它和大自噬的相關(guān)蛋白BECNl之間的相互作用說(shuō)明DEFA1是如何參與和調(diào)節(jié)大白噬的過(guò)程的。 方法：應(yīng)用免疫共沉淀、蛋白免疫印跡和共聚焦顯微鏡等方法,同時(shí)采用可以調(diào)節(jié)大自噬過(guò)程的處理方法并在不同的時(shí)間窗來(lái)觀察BECN1和DEFA1蛋白以及大自噬的變化。將BECN1-myc和DEFA1-flag質(zhì)粒分別以及合并轉(zhuǎn)染給人胚腎293細(xì)胞進(jìn)行免疫共沉淀實(shí)驗(yàn),同時(shí)將其轉(zhuǎn)染給人宮頸癌細(xì)胞進(jìn)行熒光染色以及將帶有不同熒光質(zhì)粒的BECN1和DEFA1質(zhì)粒轉(zhuǎn)染給人宮頸癌細(xì)胞制成樣本,采用共聚焦顯微鏡觀察其結(jié)果以印證免疫共沉淀結(jié)果；另外,將轉(zhuǎn)染了BECN1和DEFA1質(zhì)粒的細(xì)胞進(jìn)行誘導(dǎo)和抑制大自噬的處理在不同時(shí)間點(diǎn)觀察兩個(gè)目的蛋白以及大自噬過(guò)程的變化。同時(shí)為了檢測(cè)兩個(gè)蛋白在其他蛋白降解途徑中的變化,用蛋白酶抑制劑處理兩種蛋白轉(zhuǎn)染后的細(xì)胞并檢測(cè)其變化。其中蛋白免疫印跡實(shí)驗(yàn)的結(jié)果量化分析后加以統(tǒng)計(jì)學(xué)分析各個(gè)量之間的統(tǒng)計(jì)學(xué)差異,P0.05表示差異具有顯著性。 結(jié)果： 1. DEFA1和BECN1在細(xì)胞內(nèi)可互相結(jié)合。 2. BECNl和DEFA1都可與溶酶體結(jié)合,并使溶酶體向細(xì)胞核聚攏；細(xì)胞共轉(zhuǎn)染BECN1和DEFA1后溶酶體和DEFA1的結(jié)合率明顯增高。 3.在饑餓引發(fā)的大自噬過(guò)程中,BECNl隨著饑餓時(shí)間的延長(zhǎng)沒(méi)有顯著的形態(tài)學(xué)變化；DEFA1在饑餓2小時(shí)時(shí)即圍成環(huán)狀位于近核區(qū)并維持這個(gè)形態(tài)持續(xù)至饑餓12小時(shí)；LC3在未受饑餓處理時(shí)是散在狀和點(diǎn)狀混合存在的,而在饑餓2小時(shí)時(shí)散在狀和點(diǎn)狀形態(tài)分開(kāi),饑餓4到12小時(shí)只剩點(diǎn)狀形態(tài)的LC3。在BECN1和DEFA1共轉(zhuǎn)染的細(xì)胞中,BECN1在2小時(shí)時(shí)出現(xiàn)略微的網(wǎng)狀結(jié)構(gòu)；BECN1和LC3共轉(zhuǎn)染的細(xì)胞中LC3受BECN1的保護(hù)在饑餓4個(gè)小時(shí)的情況下也沒(méi)出現(xiàn)顯著的形態(tài)變化；DEFA1和LC3共轉(zhuǎn)染細(xì)胞中LC3在饑餓4小時(shí)時(shí)受到DEFA1的影響其形態(tài)與DEFA1相似。而在BECN1,DEFA1和LC3共轉(zhuǎn)染的細(xì)胞中LC3受BECN1和DEFA1影響信號(hào)顯示非常弱,在饑餓2小時(shí)時(shí)各個(gè)蛋白均呈現(xiàn)散狀和點(diǎn)狀混合存在,而在饑餓4個(gè)小時(shí)時(shí)形態(tài)與LC3和DEFA1共轉(zhuǎn)染4個(gè)小時(shí)的形態(tài)幾乎相同,BECN1卻沒(méi)有顯著的形態(tài)變化。 4.通過(guò)免疫印跡實(shí)驗(yàn)驗(yàn)證BECN1, DEFA1和LC3(內(nèi)源)在饑餓2小時(shí)和4小時(shí)條件下的互相影響,在此過(guò)程中DEFA1增強(qiáng)了BECN1的降解,而B(niǎo)ECN1卻保護(hù)了DEFA1不被降解。轉(zhuǎn)染了BECN1, DEFA1和BECN1DEFA1質(zhì)粒的細(xì)胞中LC3-Ⅱ/LC3-Ⅰ比值有所增加。在饑餓誘導(dǎo)大自噬后,除了轉(zhuǎn)染BECN1的細(xì)胞其余所有的LC3-Ⅱ/LC3-Ⅰ比值都增加了。 5.巴弗洛霉素保護(hù)DEFA1不被降解的作用比對(duì)BECN1的強(qiáng)。DEFA1能增強(qiáng)巴弗洛霉素對(duì)BECN1降解的保護(hù)作用,而B(niǎo)ECN1能增強(qiáng)巴弗洛霉素對(duì)DEFA1降解的保護(hù)作用僅僅是在巴弗洛霉素的濃度為200μM時(shí),在100μM的巴弗洛霉素中DEFA1降解受保護(hù)的作用則不被BECN1影響。在巴弗洛霉素和饑餓共同處理的情況下,100μM巴弗洛霉素對(duì)BECN1降解的保護(hù)作用比對(duì)DEFA1的強(qiáng),當(dāng)巴弗洛霉素的濃度為200μM時(shí)間為6小時(shí)時(shí)和100μM時(shí)相同,而12小時(shí)時(shí)卻相反。 6.溶酶體抑制劑(E64d/Pepstatin A)和饑餓條件共同作用于BECN1和DEFA1時(shí),溶酶體抑制劑在12小時(shí)時(shí)對(duì)BECN1降解的保護(hù)作用比對(duì)DEFA1的強(qiáng)。DEFA1使溶酶體抑制劑對(duì)BECN1降解的保護(hù)作用減弱,而B(niǎo)ECN1卻增強(qiáng)了溶酶體抑制劑對(duì)DEFA1的保護(hù)作用。 7. BECN1和DEFA1在細(xì)胞內(nèi)的降解都受到蛋白酶抑制劑MG132的保護(hù)。 結(jié)論：DEFA1引發(fā)大自噬的發(fā)生,并且可以通過(guò)結(jié)合自噬的相關(guān)蛋白BECN1和LC3參與和調(diào)節(jié)大自噬的生理過(guò)程。除了大自噬的降解途徑,BECN1和DEFA1還通過(guò)蛋白酶降解途徑降解。
[Abstract]:Objective: To observe the interaction between the large autophagy and the antibacterial peptide in the cell, both of them play a key role in the antibacterial action of the biological cells. A representative DEFAl of the antimicrobial peptide was selected in this experiment, and the interaction between it and the related protein BECNl of the large autophagy showed how the DEFA1 was involved and regulated. The process of white ophagy.
Methods: the methods of immunoprecipitation, Western blot and confocal microscopy were used. At the same time, the process of autophagy could be regulated and the changes of BECN1 and DEFA1 protein and autophagy were observed at different time windows. The BECN1-myc and DEFA1-flag plasmids were immunized and transfected to 293 cells of human embryonic kidney, respectively. The co precipitation experiment was used to transfect it to human cervical cancer cells by fluorescence staining and transfection of BECN1 and DEFA1 plasmids with different fluorescent plasmids to human cervical cancer cells. The results were observed by confocal microscopy, and the cells transfected with BECN1 and DEFA1 plasmids were induced to be induced. The treatment of large autophagy was observed at different time points and the changes in the process of autophagy were observed at different time points. In order to detect the changes of the two proteins in other protein degradation pathways, the changes of the transfected cells after the treatment of the two proteins were detected by protease inhibitors. The results of the immunoblot test of the two proteins were quantified. After statistical analysis of the statistical difference between the various quantities, P0.05 indicated that the difference was significant.
Result錛，

本文編號(hào)：1955879