本文選題：肝星狀細(xì)胞　切入點(diǎn)：酸敏感離子通道　出處：《安徽醫(yī)科大學(xué)》2014年碩士論文

【摘要】：酸敏感離子通道(ASICs)是一類配體門控性離子通道，能被胞外H+激活，開放的通道對(duì)Na+、Ca2+具有通透性。研究表明，ASICs參與一系列以組織酸化為特征的疾病，如炎癥、腦缺血、腫瘤等。前期實(shí)驗(yàn)結(jié)果研究表明在大鼠肝纖維化的肝星狀細(xì)胞上存在ASIC1a的高表達(dá)，并參與肝星狀細(xì)胞的活化過程，但具體機(jī)制尚不清楚。為此，本研究選用大鼠肝星狀細(xì)胞為研究對(duì)象，采用胞外血小板衍生因子(Platelet derived growth factor, PDGF)刺激肝星狀細(xì)胞活化體外模型，探討ASIC1a在PDGF誘導(dǎo)的肝星狀細(xì)胞活化中發(fā)揮的作用及其可能的分子機(jī)制。目的：本研究旨在觀察ASIC1a對(duì)血小板衍生生長(zhǎng)因子(Platelet derived growth factor,PDGF)誘導(dǎo)肝星狀細(xì)胞活化的影響，并探討其作用的分子機(jī)制。 內(nèi)容： 1.觀察PDGF刺激HSCs過程中，ASIC1a和CaMKII的表達(dá)變化； 2.利用ASIC1a siRNA建立體外ASIC1a的表達(dá)沉默模型并驗(yàn)證其沉默效率； 3.觀察沉默或阻滯ASIC1a后，肝星狀細(xì)胞活化指標(biāo)的改變； 4.探討ASIC1a在PDGF誘導(dǎo)肝星狀細(xì)胞活化過程中作用的分子機(jī)制； 方法： 1. PDGF(0、5、10、20ng/mL)，(0、12、24、48h)作用于大鼠肝星狀細(xì)胞后，通過Western-blot法檢測(cè)ASIC1a和CaMKII的表達(dá)。 2.采用特異性ASIC1a siRNA建立體外ASIC1a的表達(dá)沉默，采用熒光倒置顯微鏡、RT-PCR、實(shí)時(shí)熒光定量PCR(q-RT-PCR)及Western-Blot法檢測(cè)特異性siRNA對(duì)ASIC1a的沉默效率及其對(duì)ASIC1a mRNA和蛋白的抑制作用； 3.選擇HSC-T6細(xì)胞株為實(shí)驗(yàn)對(duì)象，設(shè)正常組、PDGF模型組(10ng/mL)、電壓門控性Ca2+通道阻滯劑組(5M nimodipine,3M ω-conotoxin MVIIC)、PcTx1組（100ng/mL）、特異性ASIC1a SiRNA沉默組、ASIC1a SiRNA沉默的陰性對(duì)照組，激光共聚焦技術(shù)檢測(cè)胞內(nèi)[Ca2+]i水平；MTT和Transwell法檢測(cè)細(xì)胞增殖和趨化能力；實(shí)時(shí)熒光定量PCR及Western-Blot法檢測(cè)肝星狀細(xì)胞活化相關(guān)基因-SMA,TGF-, Col-1, MMP-13, TIMP-1的含量，比較其差異性。 4.將PDGF作用于肝星狀細(xì)胞24h后，采用Western-blot法檢測(cè)各組細(xì)胞p38、ERK1/2、JNK的磷酸化水平。 結(jié)果： 1. PDGF誘導(dǎo)肝星狀活化過程中，ASIC1a和CaMKII蛋白表達(dá)呈時(shí)間和劑量依賴性，且均在24h,10ng/mL時(shí)二者表達(dá)量達(dá)到最大水平； 2.化學(xué)合成的特異性ASIC1a siRNA可成功轉(zhuǎn)染入大鼠肝星狀細(xì)胞中，且明顯下調(diào)ASIC1a mRNA和蛋白表達(dá)量； 3.基因水平干預(yù)ASIC1a表達(dá)或阻斷ASIC1a可導(dǎo)致肝星狀細(xì)胞胞內(nèi)Ca2+濃度降低，，CaMKII的表達(dá)降低，且可抑制PDGF誘導(dǎo)的大鼠肝星狀細(xì)胞活化，具體表現(xiàn)為：細(xì)胞增殖和趨化能力降低，炎癥因子的表達(dá)量下降，膠原沉積減少，基質(zhì)金屬蛋白酶及其抑制劑的比值升高； 4.沉默或阻斷ASIC1a可抑制PDGF誘導(dǎo)的ERK1/2及JNK磷酸化水平的升高。 結(jié)論： 1. ASIC1a和CaMKII參與PDGF誘導(dǎo)肝星狀細(xì)胞活化過程； 2. ASIC1a siRNA轉(zhuǎn)染可成功構(gòu)建大鼠肝星狀細(xì)胞ASIC1a表達(dá)沉默模型； 3.沉默或抑制ASIC1a抑制PDGF誘導(dǎo)Ca2+超載，延緩肝星狀細(xì)胞的活化； 4. ASIC1a通過調(diào)節(jié)ERK1/2及JNK信號(hào)通路發(fā)揮抑制PDGF誘導(dǎo)的大鼠肝星狀細(xì)胞活化的作用；
[Abstract]:Objective : To investigate the effect of ASIC1a on platelet derived growth factor ( PDGF ) in hepatic stellate cells and to investigate the molecular mechanism of ASIC1a on platelet derived growth factor ( PDGF ) induced hepatic stellate cell activation .

Content :

1 . Observe the change of expression of ASIC1a and CaMKII during PDGF stimulated HSCs ;


2 . Using ASIC1a siRNA to establish an in vitro ASIC1a expression silencing model and verify its silencing efficiency ;


3 . Observe the change of hepatic stellate cell activation index after observing the silence or blocking ASIC1a ;


4 . To investigate the molecular mechanism of ASIC1a in the activation of hepatic stellate cells induced by PDGF ;


Method :

1 . PDGF ( 0 , 5 , 10 , 20 ng / mL ) , ( 0 , 12 , 24 , 48 h ) was used to detect the expression of ASIC1a and CaMKII by Western - blot .

2 . The expression silencing of ASIC1a in vitro was established by using specific ASIC1a siRNA . RT - PCR , real - time fluorescence quantitative PCR ( q - RT - PCR ) and Western - Blot were used to detect the silencing efficiency of ASIC1a and its inhibitory effect on ASIC1a mRNA and protein .


3 . The HSC - T6 cell line was selected as the experimental subject , the normal group , the PDGF model group ( 10ng / mL ) , the voltage - gated Ca2 + channel blocker group ( 5M ) , 3M 蠅 - lymphotoxin MVIIC ) , the Tx1 group ( 100ng / mL ) , the specific ASIC1a SiRNA silencing group , ASIC1a SiRNA silencing negative control group , the laser confocal technique were used to detect the intracellular Ca2 + level ;
MTT and Transwell assay were used to detect cell proliferation and chemotropism ;
The content of SMA , TGF - , Col - 1 , MMP - 13 and TIMP - 1 in hepatic stellate cells were detected by real - time fluorescence quantitative PCR and Western - Blot .

4 . After 24 hours of PDGF acting on hepatic stellate cells , the phosphorylation level of p38 , ERK 1 / 2 and P 1 in each group was detected by Western - blot .

Results :

1 . During the activation of hepatic stellate cells , the expression of ASIC1a and CaMKII protein was time - dependent and dose - dependent , and the expression level of ASIC1a and CaMKII protein reached the maximum level at 24h and 10ng / mL .


2 . The specific ASIC1a siRNA was successfully transfected into hepatic stellate cells of rat , and the expression of ASIC1a mRNA and protein was significantly downregulated .


3 . The expression of Ca 2 + in hepatic stellate cells decreased and the expression of CaMKII decreased , and the expression level of inflammatory factor decreased , collagen deposition decreased , matrix metalloproteinases and its inhibitors increased .


4 . Silence or blockade of ASIC1a inhibited PDGF - induced increase in the level of 1 / 2 and phosphorylation .

Conclusion :

1 . ASIC1a and CaMKII participate in PDGF - induced hepatic stellate cell activation ;


2 . The expression silencing model of ASIC1a in rat liver stellate cells was successfully constructed by ASIC1a siRNA transfection .


3 . silencing or inhibiting ASIC1a inhibited PDGF - induced Ca2 + overload and delayed activation of hepatic stellate cells ;


4 . ASIC1a plays a role in inhibiting the activation of PDGF - induced rat hepatic stellate cells by regulating the ERK pathway .

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R575.2

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