本文選題：Akt + 細(xì)胞自噬　； 參考：《安徽大學(xué)》2014年碩士論文

【摘要】：Akt是一種絲氨酸/蘇氨酸激酶,又稱蛋白激酶B (Protein kinase B, PKB),它位于mTOR的上游,在自噬調(diào)控中通常認(rèn)為起負(fù)調(diào)控作用。而細(xì)胞自噬(Autophagy)是真核生物用于清除胞內(nèi)聚集物、損傷細(xì)胞器而維持其穩(wěn)態(tài)平衡的一種溶酶體降解途徑。細(xì)胞自噬不僅在細(xì)胞生長發(fā)育、成熟分化等過程中起重要作用,且與多種疾病發(fā)生、病毒感染與免疫等密切相關(guān)。我們用電鏡、激光共聚焦顯微鏡、免疫印跡檢測發(fā)現(xiàn)穩(wěn)定表達(dá)HBV基因組的HepG2.215細(xì)胞自噬較HepG2細(xì)胞顯著增強(qiáng),說明HBV感染能夠增強(qiáng)細(xì)胞基礎(chǔ)自噬,同時(shí)發(fā)現(xiàn)在HepG2.215細(xì)胞中Akt磷酸化水平異常高表達(dá),這暗示著Akt在細(xì)胞自噬和HBV復(fù)制中起著重要作用,我們用API-2抑制Akt磷酸化后發(fā)現(xiàn)HepG2.215細(xì)胞自噬體減少,LC3脂酰化降低。通過進(jìn)一步的研究發(fā)現(xiàn),API-2引起的自噬降低是通過減少自噬基因Beclinl的表達(dá),降低正調(diào)控自噬基因Erk和GSK的磷酸化實(shí)現(xiàn)的。這一結(jié)果表明Akt正調(diào)控HepG2.215細(xì)胞自噬。同時(shí)進(jìn)一步豐富了Akt信號通路在細(xì)胞自噬中的理論研究。 乙肝病毒(Hepatitis B viru, HBV)感染是嚴(yán)重危害我國人口健康的常見傳染病之一。統(tǒng)計(jì)顯示,目前我國HBV感染者達(dá)1.2億。由于長期的病毒感染,病情反復(fù)發(fā)作,使得肝功能受損,有很多病人最終會發(fā)展成肝細(xì)胞癌。目前,無論對乙肝病毒感染者還是肝癌病人均需要更有效的治療手段,因此對其病因?qū)W以及新的治療手段的研究顯得尤為重要�；衔颎liocladicillin C (C77)是分離的Gliocladium sp的發(fā)酵產(chǎn)物中獲得的Epipolythiodioxopiperazines (ETPs)類分子。該類化合物能抑制腫瘤細(xì)胞增殖,對細(xì)菌、病毒等病原體有殺傷作用。我們發(fā)現(xiàn)該類化合物早期能引起細(xì)胞自噬,而自噬又是HBV復(fù)制所必須的。那C77是否通過自噬對HBV復(fù)制產(chǎn)生一定影響呢?通過定量PCR檢測表明C77是通過降低自噬基因的表達(dá)來抑制HBV-X基因的轉(zhuǎn)錄量,從而影響HBV復(fù)制。 Akt促進(jìn)細(xì)胞自噬而自噬又與HBV復(fù)制關(guān)系密切,那Akt是否與HBV復(fù)制有關(guān)系呢?我們用Akt抑制劑API-2發(fā)現(xiàn)既可以抑制HBV的復(fù)制,也可以降低細(xì)胞自噬。激光共聚焦顯微鏡顯示API-2可以減少HepG2.215自噬體的數(shù)量。敲降自噬相關(guān)基因LC3、p62和Beclinl抑制HBV的復(fù)制,而敲降A(chǔ)kt減少了LC3-Ⅱ水平,抑制了病毒的復(fù)制。所以我們的實(shí)驗(yàn)結(jié)論：C77是通過降低自噬相關(guān)基因表達(dá)來抑制病毒復(fù)制的。Akt信號是維持HepG2.215細(xì)胞高水平基礎(chǔ)自噬自噬所必須的,Akt促進(jìn)HBV的復(fù)制是通過調(diào)節(jié)細(xì)胞自噬來實(shí)現(xiàn)的。
[Abstract]:Akt is a serine / threonine kinase, also known as protein kinase B (PKB). It is located upstream of mTOR and plays a negative role in autophagy regulation. Autophagy (Autophagy) is a lysosomal degradation pathway which is used by eukaryotes to clear intracellular aggregates and damage organelles to maintain its homeostasis. Autophagy not only plays an important role in cell development, maturation and differentiation, but also is closely related to many diseases, virus infection and immunity. Electron microscopy, laser confocal microscopy and Western blot analysis showed that the autophagy of HepG2.215 cells expressing HBV genome was significantly higher than that of HepG2 cells, indicating that HBV infection could enhance the basic autophagy of HepG2.215 cells. It was also found that the Akt phosphorylation level in HepG2.215 cells was abnormal, which suggested that Akt plays an important role in autophagy and HBV replication. We found that API-2 inhibited Akt phosphorylation in HepG2.215 cells and decreased lipoacylation of LC3 in HepG2.215 cells. It was found that the decrease of autophagy induced by API-2 was achieved by reducing the expression of the autophagy gene Beclinl and decreasing the phosphorylation of the positive regulated autophagy genes Erk and GSK. These results suggest that Akt is regulating autophagy of HepG 2.215 cells. At the same time, it further enriches the theoretical study of Akt signaling pathway in autophagy. Hepatitis B virus (HBV) infection is one of the most common infectious diseases in China. According to statistics, the number of HBV infected people in China is 0. 120 million. Because of long-term viral infection, repeated attacks, damage to liver function, many patients will eventually develop hepatocellular carcinoma. At present, both hepatitis B virus infection and liver cancer patients need more effective treatment, so it is particularly important to study its etiology and new treatment methods. The compound Gliocladicillin C (C77) is an Epipolythiodioxopiperazines (ETPs) molecule obtained from the fermentation products of isolated Gliocladium sp. These compounds can inhibit the proliferation of tumor cells and kill pathogens such as bacteria and viruses. We found that these compounds can induce autophagy at an early stage, and autophagy is necessary for HBV replication. Does C77 have an effect on HBV replication through autophagy? The results of quantitative PCR showed that C77 inhibited the transcription of HBV-X gene by reducing the expression of autophagy gene, thus affecting HBV replication. Akt promoted autophagy and autophagy was closely related to HBV replication. Is Akt related to HBV replication? We found that Akt inhibitor API-2 can inhibit HBV replication as well as reduce autophagy. Laser confocal microscopy showed that API-2 could reduce the number of HepG 2. 215 autophagy. Knock down autophagy associated genes LC3p62 and Beclinl inhibited HBV replication, while knock down Akt decreased LC3- 鈪，

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