酸敏感离子通道1a对PDGF诱导的肝星状细胞活化的影响及其可能机制

发布时间：2018-04-05 14:09

  本文选题：肝星状细胞　切入点：酸敏感离子通道　出处：《安徽医科大学》2014年硕士论文

【摘要】：酸敏感离子通道(ASICs)是一类配体门控性离子通道，能被胞外H+激活，开放的通道对Na+、Ca2+具有通透性。研究表明，ASICs参与一系列以组织酸化为特征的疾病，如炎症、脑缺血、肿瘤等。前期实验结果研究表明在大鼠肝纤维化的肝星状细胞上存在ASIC1a的高表达，并参与肝星状细胞的活化过程，但具体机制尚不清楚。为此，本研究选用大鼠肝星状细胞为研究对象，采用胞外血小板衍生因子(Platelet derived growth factor, PDGF)刺激肝星状细胞活化体外模型，探讨ASIC1a在PDGF诱导的肝星状细胞活化中发挥的作用及其可能的分子机制。目的：本研究旨在观察ASIC1a对血小板衍生生长因子(Platelet derived growth factor,PDGF)诱导肝星状细胞活化的影响，并探讨其作用的分子机制。 内容： 1.观察PDGF刺激HSCs过程中，ASIC1a和CaMKII的表达变化； 2.利用ASIC1a siRNA建立体外ASIC1a的表达沉默模型并验证其沉默效率； 3.观察沉默或阻滞ASIC1a后，肝星状细胞活化指标的改变； 4.探讨ASIC1a在PDGF诱导肝星状细胞活化过程中作用的分子机制； 方法： 1. PDGF(0、5、10、20ng/mL)，(0、12、24、48h)作用于大鼠肝星状细胞后，通过Western-blot法检测ASIC1a和CaMKII的表达。 2.采用特异性ASIC1a siRNA建立体外ASIC1a的表达沉默，采用荧光倒置显微镜、RT-PCR、实时荧光定量PCR(q-RT-PCR)及Western-Blot法检测特异性siRNA对ASIC1a的沉默效率及其对ASIC1a mRNA和蛋白的抑制作用； 3.选择HSC-T6细胞株为实验对象，设正常组、PDGF模型组(10ng/mL)、电压门控性Ca2+通道阻滞剂组(5M nimodipine,3M ω-conotoxin MVIIC)、PcTx1组（100ng/mL）、特异性ASIC1a SiRNA沉默组、ASIC1a SiRNA沉默的阴性对照组，激光共聚焦技术检测胞内[Ca2+]i水平；MTT和Transwell法检测细胞增殖和趋化能力；实时荧光定量PCR及Western-Blot法检测肝星状细胞活化相关基因-SMA,TGF-, Col-1, MMP-13, TIMP-1的含量，比较其差异性。 4.将PDGF作用于肝星状细胞24h后，采用Western-blot法检测各组细胞p38、ERK1/2、JNK的磷酸化水平。 结果： 1. PDGF诱导肝星状活化过程中，ASIC1a和CaMKII蛋白表达呈时间和剂量依赖性，且均在24h,10ng/mL时二者表达量达到最大水平； 2.化学合成的特异性ASIC1a siRNA可成功转染入大鼠肝星状细胞中，且明显下调ASIC1a mRNA和蛋白表达量； 3.基因水平干预ASIC1a表达或阻断ASIC1a可导致肝星状细胞胞内Ca2+浓度降低，，CaMKII的表达降低，且可抑制PDGF诱导的大鼠肝星状细胞活化，具体表现为：细胞增殖和趋化能力降低，炎症因子的表达量下降，胶原沉积减少，基质金属蛋白酶及其抑制剂的比值升高； 4.沉默或阻断ASIC1a可抑制PDGF诱导的ERK1/2及JNK磷酸化水平的升高。 结论： 1. ASIC1a和CaMKII参与PDGF诱导肝星状细胞活化过程； 2. ASIC1a siRNA转染可成功构建大鼠肝星状细胞ASIC1a表达沉默模型； 3.沉默或抑制ASIC1a抑制PDGF诱导Ca2+超载，延缓肝星状细胞的活化； 4. ASIC1a通过调节ERK1/2及JNK信号通路发挥抑制PDGF诱导的大鼠肝星状细胞活化的作用；
[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 .

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.2

【参考文献】

相关期刊论文 前10条

1 遇玲;李名扬;郭余龙;;RNA干扰机理与应用[J];安徽农业科学;2009年07期

2 王慧;陈真;;PDGF及TGF信号转导通路与肝星状细胞活化增殖的研究进展[J];安徽医药;2011年07期

3 唐文;周德江;;c-Jun氨基端激酶信号传导通路及其在肝纤维化中的作用研究进展[J];西南军医;2008年04期

4 李婧;蒋炜;;肝星状细胞的免疫学特性[J];复旦学报(医学版);2010年03期

5 刘兰;白玛多吉;;血小板衍生生长因子PDGF与肿瘤血管生成的研究进展[J];西藏大学学报(自然科学版);2011年02期

6 袁凤来;陈飞虎;李霞;雄志刚;黄学应;刘永靖;吴繁荣;姚莹;张晓明;;大鼠关节软骨酸敏感离子通道的表达[J];安徽医科大学学报;2007年05期

7 罗波;官阳;袁静萍;杨木兰;徐惠;石玉香;;罗格列酮对癌性条件下肝星状细胞表达PDGF-B、α-SMA的影响[J];肿瘤防治研究;2011年02期

8 杨清高;刘绍能;;P38MAPK信号通路与肝纤维化[J];世界华人消化杂志;2012年24期

9 刘立民;张兴霞;赵广圣;司叶俊;林国强;张彦明;何广胜;吴德沛;;原发免疫性血小板减少症患者外周血Th22细胞水平的变化及意义[J];细胞与分子免疫学杂志;2012年12期

10 姜辉;夏伦祝;李颖;李翔;吴健;;三七总皂苷对肝纤维化大鼠基质金属蛋白酶-13及其抑制因子-1表达的影响[J];中国中药杂志;2013年08期

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