發(fā)布時間：2018-04-05 14:09

  本文選題：肝星狀細胞　切入點：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。前期實驗結(jié)果研究表明在大鼠肝纖維化的肝星狀細胞上存在ASIC1a的高表達，并參與肝星狀細胞的活化過程，但具體機制尚不清楚。為此，本研究選用大鼠肝星狀細胞為研究對象，采用胞外血小板衍生因子(Platelet derived growth factor, PDGF)刺激肝星狀細胞活化體外模型，探討ASIC1a在PDGF誘導(dǎo)的肝星狀細胞活化中發(fā)揮的作用及其可能的分子機制。目的：本研究旨在觀察ASIC1a對血小板衍生生長因子(Platelet derived growth factor,PDGF)誘導(dǎo)肝星狀細胞活化的影響，并探討其作用的分子機制。 內(nèi)容： 1.觀察PDGF刺激HSCs過程中，ASIC1a和CaMKII的表達變化； 2.利用ASIC1a siRNA建立體外ASIC1a的表達沉默模型并驗證其沉默效率； 3.觀察沉默或阻滯ASIC1a后，肝星狀細胞活化指標的改變； 4.探討ASIC1a在PDGF誘導(dǎo)肝星狀細胞活化過程中作用的分子機制； 方法： 1. PDGF(0、5、10、20ng/mL)，(0、12、24、48h)作用于大鼠肝星狀細胞后，通過Western-blot法檢測ASIC1a和CaMKII的表達。 2.采用特異性ASIC1a siRNA建立體外ASIC1a的表達沉默，采用熒光倒置顯微鏡、RT-PCR、實時熒光定量PCR(q-RT-PCR)及Western-Blot法檢測特異性siRNA對ASIC1a的沉默效率及其對ASIC1a mRNA和蛋白的抑制作用； 3.選擇HSC-T6細胞株為實驗對象，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沉默的陰性對照組，激光共聚焦技術(shù)檢測胞內(nèi)[Ca2+]i水平；MTT和Transwell法檢測細胞增殖和趨化能力；實時熒光定量PCR及Western-Blot法檢測肝星狀細胞活化相關(guān)基因-SMA,TGF-, Col-1, MMP-13, TIMP-1的含量，比較其差異性。 4.將PDGF作用于肝星狀細胞24h后，采用Western-blot法檢測各組細胞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蛋白表達呈時間和劑量依賴性，且均在24h,10ng/mL時二者表達量達到最大水平； 2.化學(xué)合成的特異性ASIC1a siRNA可成功轉(zhuǎn)染入大鼠肝星狀細胞中，且明顯下調(diào)ASIC1a mRNA和蛋白表達量； 3.基因水平干預(yù)ASIC1a表達或阻斷ASIC1a可導(dǎo)致肝星狀細胞胞內(nèi)Ca2+濃度降低，，CaMKII的表達降低，且可抑制PDGF誘導(dǎo)的大鼠肝星狀細胞活化，具體表現(xiàn)為：細胞增殖和趨化能力降低，炎癥因子的表達量下降，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)肝星狀細胞活化過程； 2. ASIC1a siRNA轉(zhuǎn)染可成功構(gòu)建大鼠肝星狀細胞ASIC1a表達沉默模型； 3.沉默或抑制ASIC1a抑制PDGF誘導(dǎo)Ca2+超載，延緩肝星狀細胞的活化； 4. ASIC1a通過調(diào)節(jié)ERK1/2及JNK信號通路發(fā)揮抑制PDGF誘導(dǎo)的大鼠肝星狀細胞活化的作用；
[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)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R575.2

【參考文獻】

相關(guān)期刊論文 前10條

1 遇玲;李名揚;郭余龍;;RNA干擾機理與應(yīng)用[J];安徽農(nóng)業(yè)科學(xué);2009年07期

2 王慧;陳真;;PDGF及TGF信號轉(zhuǎn)導(dǎo)通路與肝星狀細胞活化增殖的研究進展[J];安徽醫(yī)藥;2011年07期

3 唐文;周德江;;c-Jun氨基端激酶信號傳導(dǎo)通路及其在肝纖維化中的作用研究進展[J];西南軍醫(yī);2008年04期

4 李婧;蔣煒;;肝星狀細胞的免疫學(xué)特性[J];復(fù)旦學(xué)報(醫(yī)學(xué)版);2010年03期

5 劉蘭;白瑪多吉;;血小板衍生生長因子PDGF與腫瘤血管生成的研究進展[J];西藏大學(xué)學(xué)報(自然科學(xué)版);2011年02期

6 袁鳳來;陳飛虎;李霞;雄志剛;黃學(xué)應(yīng);劉永靖;吳繁榮;姚瑩;張曉明;;大鼠關(guān)節(jié)軟骨酸敏感離子通道的表達[J];安徽醫(yī)科大學(xué)學(xué)報;2007年05期

7 羅波;官陽;袁靜萍;楊木蘭;徐惠;石玉香;;羅格列酮對癌性條件下肝星狀細胞表達PDGF-B、α-SMA的影響[J];腫瘤防治研究;2011年02期

8 楊清高;劉紹能;;P38MAPK信號通路與肝纖維化[J];世界華人消化雜志;2012年24期

9 劉立民;張興霞;趙廣圣;司葉俊;林國強;張彥明;何廣勝;吳德沛;;原發(fā)免疫性血小板減少癥患者外周血Th22細胞水平的變化及意義[J];細胞與分子免疫學(xué)雜志;2012年12期

10 姜輝;夏倫祝;李穎;李翔;吳健;;三七總皂苷對肝纖維化大鼠基質(zhì)金屬蛋白酶-13及其抑制因子-1表達的影響[J];中國中藥雜志;2013年08期

本文編號：1715092