本文選題：肝星狀細(xì)胞 + 自噬�。� 參考：《河北醫(yī)科大學(xué)》2014年碩士論文

【摘要】：肝纖維化(hepatic fibrosis, HF)是嗜肝病毒、酒精、藥物和毒物等致病因素引起各種慢性肝病進(jìn)而逐漸發(fā)展為肝硬化的必經(jīng)病理過程，是機(jī)體對各種持續(xù)的急慢性肝損傷產(chǎn)生的一種損傷修復(fù)反應(yīng)。肝星狀細(xì)胞(hepaticstellate cells, HSCs)在肝纖維化的形成過程中發(fā)揮著關(guān)鍵作用。HSCs在正常情況時處于靜止?fàn)顟B(tài)具有大脂滴結(jié)構(gòu)，合成少量膠原，活化后的HSCs轉(zhuǎn)化為肌成纖維樣細(xì)胞，大脂滴結(jié)構(gòu)消失同時自身增殖和膠原合成增加，這個活化過程與自噬的發(fā)生有關(guān)。自噬(autophagy)是細(xì)胞將自身損傷或過剩的結(jié)構(gòu)通過溶酶體機(jī)制而被分解使之可再利用的過程。自噬的發(fā)生包括粗面內(nèi)質(zhì)網(wǎng)的無核糖體附著區(qū)、高爾基體等來源的雙層磷脂結(jié)構(gòu)的膜包裹部分胞質(zhì)、細(xì)胞內(nèi)需降解的細(xì)胞器和衰老或錯誤折疊的蛋白質(zhì)等成分形成自噬體(autophagosome)，再通過與溶酶體融合利用其內(nèi)的水解酶降解內(nèi)容物，這個融合后的狀態(tài)叫做自噬溶酶體(autolysosome)。在組織細(xì)胞受到各種理化因素?fù)p傷時，自噬性溶酶體大量增加，使細(xì)胞能夠適應(yīng)環(huán)境變化同時修復(fù)損傷。 C-Jun氨基末端激酶(c-Jun N-terminal kinase, JNK)是絲裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)家族中的一員。JNK的激活通過三級激酶模式，包括MAP kinase kinase kinases (MAP3Ks orMKKKs)，MAP kinase kinases (MAP2Ks or MKKs)和MAP kinases (JNKs)。JNK可以被許多刺激激活，包括細(xì)胞因子、活性氧(ROS)、病原體、毒素、藥物、內(nèi)質(zhì)網(wǎng)應(yīng)激、游離脂肪酸和代謝環(huán)境改變。血小板衍生生長因子(platelet-derived growth factor, PDGF)是HSCs最強(qiáng)有力的促有絲分裂因子。肝纖維化時PDGF及其受體表達(dá)量均有增加，PDGF可以與其受體結(jié)合后激活受體，活化的PDGF受體與許多能夠激活RaS的信號分子有很強(qiáng)的親和力，通過間接激活RaS從而可以進(jìn)一步激活下游的Raf，進(jìn)而激活包括MAPKs在內(nèi)的多條信號通路。目前已知的JNK下游蛋白至少有40種。其中，c-Jun（一種轉(zhuǎn)錄調(diào)節(jié)因子，屬亮氨酸拉鏈家族成員）是特征性的下游蛋白，可以與JunB、JunD或者Fos結(jié)合參與轉(zhuǎn)錄因子AP-1的形成，實現(xiàn)包括細(xì)胞代謝、增殖、分化等在內(nèi)的多種生物學(xué)效應(yīng)。 迄今，關(guān)于JNK與HSCs的自噬之間的關(guān)系的研究甚少，對于JNK的干預(yù)能否成為抗纖維化治療的一個靶點？為此，我們運用JNK抑制劑SP600125特異性阻斷JNK活性，同時在PDGF誘導(dǎo)下，對JNK在HSCs自噬中的作用做了初步研究。 目的：通過應(yīng)用JNK抑制劑SP600125探討JNK在PDGF誘導(dǎo)肝星狀細(xì)胞自噬及活化中的作用。 方法：應(yīng)用體外HSCs培養(yǎng)技術(shù)，利用PDGF-BB刺激人肝星狀細(xì)胞系HSC-LX2后，以一定濃度的SP600125阻斷JNK通路。分組如下①對照組；②P DGF組；③S P600125組；④SP600125+PDGF組。采用免疫細(xì)胞化學(xué)方法檢測HSCs中α-SMA的表達(dá)，Westem bloting方法檢測JNK、p-JNK、c-Jun、p-c-Jun、α-SMA、LC3BⅡ（檢測自噬發(fā)生的標(biāo)志性蛋白）、LC3BⅠ、Agt5和Beclin1（自噬起始階段的兩個關(guān)鍵蛋白）蛋白表達(dá)，，RT-PCR法檢測Agt5和Beclin1的基因水平的表達(dá)；采用吖啶橙染色熒光顯微鏡觀察HSCs自噬情況。 結(jié)果： 1SP600125對PDGF誘導(dǎo)的HSCs的JNK和c-Jun磷酸化具有抑制作用。應(yīng)用Western blot技術(shù)檢測各組JNK和p-JNK、c-Jun和p-c-Jun蛋白的表達(dá)情況。統(tǒng)計p-JNK與JNK、p-c-Jun與c-Jun蛋白灰度的比值，PDGF組較對照組JNK、c-Jun磷酸化水平明顯升高(0.77±0.05vs0.34±0.02,0.92±0.52vs0.51±0.05)(p0.01)，SP600125+PDGF組和SP600125組分別較PDGF組和對照組JNK (0.39±0.04vs0.77±0.05,0.24±0.02vs0.34±0.02)、c-Jun (0.21±0.04vs0.92±0.52,0.14±0.01vs0.51±0.05)磷酸化水平顯著降低(p0.05)。 2SP600125對PDGF誘導(dǎo)的HSCs的自噬具有抑制作用。應(yīng)用吖啶橙染色方法結(jié)果顯示，PDGF組較對照組紅色熒光面積明顯升高(130.0±24.1vs54.6±8.3)(p0.05)，SP600125+PDGF組較PDGF組紅色熒光的面積顯著降低(50.2±6.6vs130.0±24.1)(p0.05)；應(yīng)用Western blot技術(shù)檢測各組LC3BⅡ、LC3BⅠ、Beclin-1和Atg-5蛋白的表達(dá)情況，統(tǒng)計LC3BⅡ與LC3BⅠ的比值和Beclin-1和Atg-5蛋白表達(dá)量。PDGF組較對照組LC3BⅡ與LC3BⅠ的比值、Beclin-1和Atg-5蛋白的表達(dá)均明顯升高(1.29±0.21vs0.66±0.05,746.10±46.33vs310.20±31.80,1778.00±130.00vs562.90±60.90)(p0.05)。SP600125+PDGF組和SP600125組分別較PDGF組和對照組LC3BⅡ與LC3BⅠ的比值(0.35±0.03vs1.29±0.21,0.33±0.05vs0.66±0.05)、Beclin-1蛋白的表達(dá)(197.50±26.99vs746.10±46.33,133.90±17.96vs310.20±31.80)均顯著降低(p0.05)。SP600125+PDGF組較PDGF組Atg-5蛋白的表達(dá)顯著降低(714.20±47.75vs1778.00±130.00)(p0.01)，而SP600125組較對照組Atg-5蛋白的表達(dá)量無顯著性差異(p0.05)；應(yīng)用Real-time PCR方法檢測Beclin-1mRNA和Atg-5mRNA的表達(dá)，其結(jié)果與Western blot結(jié)構(gòu)一致。 3SP600125能夠抑制HSCs α-SMA的表達(dá)。應(yīng)用免疫細(xì)胞化學(xué)和Western blot檢測α-SMA蛋白的表達(dá)情況。PDGF組較對照組α-SMA蛋白的表達(dá)水平明顯升高(1566.00±116.50vs757.70±72.87)(p0.01)，SP600125+PDGF組較PDGF組α-SMA蛋白的表達(dá)顯著降低(582.10±60.48vs1566.00±116.50)(p0.01)。 結(jié)論：特異性地阻斷JNK通路能夠抑制PDGF誘導(dǎo)的肝星狀細(xì)胞的自噬，抑制肝星狀細(xì)胞活化。
[Abstract]:Hepatic fibrosis (HF) is an essential pathological process that causes various chronic liver diseases, such as hepatoviruses, alcohol, drugs and toxicants, and then gradually develops into liver cirrhosis. It is a kind of injury repair response to various persistent acute and chronic liver damage. The hepatic stellate cells (hepaticstellate cells, HSCs) are in the liver fiber. In the process of formation,.HSCs plays a key role in the normal condition with a large lipid droplet structure, a small amount of collagen is synthesized, and the activated HSCs is converted into myofibroblast like cells. The large fat droplet structure disappears and increases in self proliferation and collagen synthesis. This activation process is related to the occurrence of autophagy. Autophagy (autophagy) is thin. The process of decomposing the structure of its own damage or excess through the lysosome mechanism. The occurrence of autophagy includes the unribosomal attachment area of the rough endoplasmic reticulum, the membrane of the bilayer phospholipid structure, such as the Golgi body, the part of the cytoplasm, the cellular organelles that degrade in the cells, and the aging or erroneous folding proteins. The formation of autophagosome (autophagosome) is formed by fusion of lysosomes with lysosomes to degrade content. The fusion state is called autophagic lysosome (autolysosome). When tissue cells are damaged by various physical and chemical factors, autophagic lysosomes are greatly increased to enable cells to adapt to environmental changes and repair damage.
The C-Jun amino terminal kinase (c-Jun N-terminal kinase, JNK) is a member of the mitogen activated protein kinase (mitogen-activated protein kinase, MAPK) family, which is activated by the three stage kinase mode, including MAP kinase. Many stimuli are activated, including cytokines, reactive oxygen species (ROS), pathogens, toxins, drugs, endoplasmic reticulum stress, free fatty acids and metabolic environment changes. Platelet-derived growth factor (PDGF) is the most powerful fibroblast stimulating factor of HSCs. The expression of PDGF and its receptor in liver fibrosis is increased, PDGF can be increased. Activating the receptor after binding to its receptor, the activated PDGF receptor has a strong affinity with many signal molecules that can activate RaS. By activating the RaS indirectly, the downstream Raf can be activated further, and then multiple signal pathways, including MAPKs, are activated. At least 40 known downstream proteins of the JNK are known, of which, c-Jun (a transcription) Regulatory factor, a member of the leucine zipper family) is a characteristic downstream protein that can be combined with JunB, JunD or Fos to participate in the formation of transcription factor AP-1, and to achieve a variety of biological effects, including cell metabolism, proliferation, and differentiation.
So far, there is little research on the relationship between autophagy between JNK and HSCs. Can the intervention of JNK be a target for anti fibrosis therapy? To this end, we use JNK inhibitor SP600125 to specifically block the activity of JNK. At the same time, we have made a preliminary study on the use of JNK in HSCs autophagy under the induction of PDGF.
Objective: To explore the role of JNK in PDGF induced autophagy and activation of hepatic stellate cells by using JNK inhibitor SP600125.
Methods: using the HSCs culture technique in vitro and using PDGF-BB to stimulate the human hepatic stellate cell line HSC-LX2, the JNK pathway was blocked with a certain concentration of SP600125. The groups were grouped as follows: the control group, (2) P DGF group; (3) S P600125 group; (4) SP600125+PDGF group. The expression of alpha -SMA in HSCs was detected by immunocytochemical method. P-JNK, c-Jun, p-c-Jun, alpha -SMA, LC3B II (detection of autophagy), LC3B I, Agt5 and Beclin1 (two key proteins at the beginning of autophagy) protein expression, RT-PCR method for detecting the expression of Agt5 and Beclin1 gene levels, and using acridine orange staining fluorescence microscopy to observe the status of HSCs autophagy.
Result錛

本文編號：1896522