Akt磷酸化对细胞自噬的调节及真菌次级代谢产物C77影响细胞自噬和HBV复制

发布时间：2018-06-22 09:52

  本文选题：Akt + 细胞自噬　； 参考：《安徽大学》2014年硕士论文

【摘要】：Akt是一种丝氨酸/苏氨酸激酶,又称蛋白激酶B (Protein kinase B, PKB),它位于mTOR的上游,在自噬调控中通常认为起负调控作用。而细胞自噬(Autophagy)是真核生物用于清除胞内聚集物、损伤细胞器而维持其稳态平衡的一种溶酶体降解途径。细胞自噬不仅在细胞生长发育、成熟分化等过程中起重要作用,且与多种疾病发生、病毒感染与免疫等密切相关。我们用电镜、激光共聚焦显微镜、免疫印迹检测发现稳定表达HBV基因组的HepG2.215细胞自噬较HepG2细胞显著增强,说明HBV感染能够增强细胞基础自噬,同时发现在HepG2.215细胞中Akt磷酸化水平异常高表达,这暗示着Akt在细胞自噬和HBV复制中起着重要作用,我们用API-2抑制Akt磷酸化后发现HepG2.215细胞自噬体减少,LC3脂酰化降低。通过进一步的研究发现,API-2引起的自噬降低是通过减少自噬基因Beclinl的表达,降低正调控自噬基因Erk和GSK的磷酸化实现的。这一结果表明Akt正调控HepG2.215细胞自噬。同时进一步丰富了Akt信号通路在细胞自噬中的理论研究。 乙肝病毒(Hepatitis B viru, HBV)感染是严重危害我国人口健康的常见传染病之一。统计显示,目前我国HBV感染者达1.2亿。由于长期的病毒感染,病情反复发作,使得肝功能受损,有很多病人最终会发展成肝细胞癌。目前,无论对乙肝病毒感染者还是肝癌病人均需要更有效的治疗手段,因此对其病因学以及新的治疗手段的研究显得尤为重要。化合物Gliocladicillin C (C77)是分离的Gliocladium sp的发酵产物中获得的Epipolythiodioxopiperazines (ETPs)类分子。该类化合物能抑制肿瘤细胞增殖,对细菌、病毒等病原体有杀伤作用。我们发现该类化合物早期能引起细胞自噬,而自噬又是HBV复制所必须的。那C77是否通过自噬对HBV复制产生一定影响呢?通过定量PCR检测表明C77是通过降低自噬基因的表达来抑制HBV-X基因的转录量,从而影响HBV复制。 Akt促进细胞自噬而自噬又与HBV复制关系密切,那Akt是否与HBV复制有关系呢?我们用Akt抑制剂API-2发现既可以抑制HBV的复制,也可以降低细胞自噬。激光共聚焦显微镜显示API-2可以减少HepG2.215自噬体的数量。敲降自噬相关基因LC3、p62和Beclinl抑制HBV的复制,而敲降Akt减少了LC3-Ⅱ水平,抑制了病毒的复制。所以我们的实验结论：C77是通过降低自噬相关基因表达来抑制病毒复制的。Akt信号是维持HepG2.215细胞高水平基础自噬自噬所必须的,Akt促进HBV的复制是通过调节细胞自噬来实现的。
[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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