【作者】 李俊；

【导师】 吴繁荣；

【作者基本信息】 安徽医科大学 ， 药学， 2020， 硕士

【摘要】 肝纤维化被认为是一种急慢性各种致病因子导致的肝细胞弥漫性损伤的病理过程,在其过程中细胞外基质（extracellμLar matrix,ECM）的过度积累是明显特征。静止的肝星状细胞（HSC）发生活化是肝纤维化过程中最重要的一步,分泌过多的α-SMA,是ECM的主要来源。酸敏感离子通道（acid-sensing ion channels,ASICs）是一类由细胞外阳离子激活的离子通道,当细胞外酸化时,酸敏感离子通道开放,与此同时对Na⁺和Ca²⁺通透性增高,从而引起细胞中生理病理的变化。课题组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,加速肝纤维化的发展进程。也有研究表明,ASIC1a与关节软骨细胞的焦亡密切相关,ASIC1a通道打开后,大鼠关节软骨细胞发生焦亡,加重风湿性关节炎的进程。焦亡是国内外学者关注的热点,其与肝纤维化也有密切联系,那么ASIC1a是否参与到活化的肝星状细胞焦亡过程中呢?为此,本次实验使用细胞外酸激活HSC-T6,探讨ASIC1a对其焦亡的影响极其可能的机制。本课题的研究内容包括以下4部分:1. 抑制、沉默和过表达肝星状细胞中的ASIC1a对肝星状细胞活性的影响建立酸诱导HSC-T6体外模型,采用ASIC1a特异性阻滞剂Pc TX1、转染ASIC1a-si RNA和ASIC1a过表达质粒干预,考察ASIC1a对HSC-T6细胞胞内细胞增殖及基质代谢的影响。MTT法观察ASIC1a阻滞剂Pc Tx-1预处理后对酸诱导HSC-T6的细胞增殖的影响,与p H6.0组相比,HSC-T6细胞生长明显被抑制。研究HSC-T6功能的影响,发现与p H7.4组相比,酸处理至p H6.0 HSC-T6细胞中α-SMA、Collagen-I表达增加,Pc Tx-1和ASIC1a-si RNA干预后α-SMA、Collagen-I表达降低,使用ASIC1a过表达质粒进行干预后,上述α-SMA、Collagen-I指标均增加。研究提示,ASIC1a参与HSCs功能和代谢过程,当酸刺激下,ASIC1a激活和通道开放,进而调节HSC-T6细胞生长和胶原分泌。2. 抑制、沉默和过表达ASIC1a对肝星状细胞中Ca²⁺内流的影响建立酸诱导HSC-T6体外模型,采用ASIC1a特异性阻滞剂Pc Tx-1、转染ASIC1a-si RNA和ASIC1a过表达质粒干预,考察ASIC1a对HSC-T6细胞胞内Ca²⁺浓度的影响。激光共聚焦显微镜、Ca²⁺成像研究HSC-T6胞内Ca²⁺的变化发现,p H6.0能明显促进Ca²⁺流入HSC-T6胞内,Pc Tx-1和ASIC1a-si RNA干预后内流程度降低,使用ASIC1a过表达质粒进行处理后,Ca²⁺内流明显均增加。研究提示,当酸刺激下,ASIC1a激活和通道开放,使HSC-T6细胞Ca²⁺内流增加。3. 抑制、沉默和过表达ASIC1a对活化的肝星状细胞焦亡的影响建立酸诱导HSC-T6体外模型,采用ASIC1a特异性阻滞剂Pc Tx-1、转染ASIC1a-si RNA和ASIC1a过表达质粒干预,考察ASIC1a对活化的HSC-T6焦亡的影响。研究结果显示,ASIC1a激活可影响HSC-T6焦亡相关指标ASC、caspase-1、NLRP3和GSDMD的表达。Pc Tx-1和ASIC1a-si RNA处理后,与模型组相比,焦亡相关指标的表达有所增加,ASIC1a过表达质粒干预后上述焦亡指标的表达有所降低。并且,LDH结果显示,抑制ASIC1a后,上清液中LDH含量较酸化组有所增高。此外,Elisa结果也表明,抑制或沉默ASIC1a后,培养基上清液中IL-1β和IL-18的含量有所升高。转染ASIC1a过表达预处理后上述焦亡相关指标则与上述叙述结果相反。研究提示,ASIC1a抑制细胞外酸诱导活化的肝星状细胞的焦亡。4. Ca²⁺对ASIC1a介导的肝星状细胞焦亡的影响建立酸诱导HSC-T6体外模型,采用无Ca²⁺培养基进行预处理,考察Ca²⁺对活化的HSC-T6焦亡的影响。研究结果显示,无Ca²⁺培养基进行预处理后,与模型组相比,焦亡相关指标的表达有所增加。研究提示,Ca²⁺抑制细胞外酸诱导活化的肝星状细胞的焦亡。综上所述,本次研究结果表明:在大鼠肝星状细胞中,酸诱导使其发生活化。在活化的肝星状细胞中,ASIC1a激活则抑制其焦亡,其可能机制是通过对Ca²⁺与炎症小体的调控作用的。更多还原

【Abstract】 Hepatic fibrosis is considered to be a pathological process of diffuse damage to liver cells caused by various acute and chronic pathogenic factors,during which excessive accumulation of extracellular matrix（ECM）is a prominent feature.The activation of resting hepatic stellate cells（HSC）is the most important step in the process of liver fibrosis.Excessive secretion ofα-SMA is the main source of ECM.Acid-sensing ion channels（ASICs）are a type of ion channels activated by extracellular cations.When extracellular acidification occurs,acid-sensitive ion channels are opened,and at the same time,the permeability to Na⁺and Ca²⁺is increased,thereby causes physiological and pathological changes in cells.The previous experimental results of the research team showed that the high expression of ASIC1a exists in hepatic stellate cells of rat liver fibrosis,and participates in the activation process of hepatic stellate cells,accelerating the development of liver fibrosis.Studies have also shown that ASIC1a is closely related to the pyroptosis of articular chondrocytes.After the ASIC1a channel is opened,pyroptosis of the articular chondrocytes of rats will worsen the process of rheumatoid arthritis.Pyroptosis is a hot topic that scholars at home and abroad pay close attention to,and it is also closely related to liver fibrosis.So does ASIC1a participate in the process of activated hepatic stellate cells?To this end,this experiment uses extracellular acids to activate HSC-T6,and explores the most likely mechanism of ASIC1a’s effect on pyroptosis.The research content of this topic includes the following 4 parts:1. Inhibition,silencing and overexpression of ASIC1a in hepatic stellate cellsAn in vitro model of acid-induced HSC-T6 was established,and ASIC1a specific blocker Pc Tx-1,transfected with ASIC1a-si RNA and ASIC1a overexpression plasmids were used to investigate the effects of ASIC1a on intracellular proliferation and matrix metabolism of HSC-T6 cells.The effect of pretreatment of ASIC1a blocker Pc Tx-1 on the proliferation of HSC-T6 cells induced by acid was observed by MTT method.Compared with the p H 6.0 group,the growth of HSC-T6 cells was significantly inhibited.To study the effect of HSC-T6 function,it was found that compared with the p H 7.4 group,the expression ofα-SMA and Collagen-I increased in HSC-T6 cells treated with acid to p H 6.0,andα-SMA after Pc Tx-1 and ASIC1a-si RNA intervention The expression of Collagen-I decreased.After intervention with the ASIC1a overexpression plasmid,the above-mentioned indicators ofα-SMA and Collagen-I increased.Studies suggest that ASIC1a is involved in the function and metabolism of HSCs.When stimulated by acid,ASIC1a activates and channels are opened,which in turn regulates HSC-T6 cell growth and collagen secretion.2. The effects of inhibition,silencing and overexpression of ASIC1a on Ca²⁺influx in hepatic stellate cellsAn in vitro model of acid-induced HSC-T6 was established,and ASIC1a specific blocker Pc Tx-1,transfected with ASIC1a-si RNA and ASIC1a overexpression plasmid were used to investigate the effect of ASIC1a on intracellular Ca²⁺concentration in HSC-T6 cells.Laser confocal microscopy and Ca²⁺imaging studies of changes in Ca²⁺in HSC-T6 cells found that p H 6.0 significantly promoted Ca²⁺influx into HSC-T6cells.The degree of influx was reduced after Pc Tx-1 and ASIC1a-si RNA intervention.Overexpression of ASIC1a was used,after plasmid treatment,Ca²⁺influx increased significantly.Studies suggest that when acid stimulated,ASIC1a activation and channel opening increase Ca²⁺influx in HSC-T6 cells.3. The effects of inhibition,silencing and overexpression of ASIC1a on pyroptosising of activated hepatic stellate cellsAn in vitro model of acid-induced HSC-T6 was established,and ASIC1a-specific blocker Pc Tx-1,transfected with ASIC1a-si RNA and ASIC1a overexpression plasmid were used to investigate the effect of ASIC1a on activated HSC-T6 pyroptosis.The results show that ASIC1a activation can affect the expression of HSC-T6 pyroptosis related indicators ASC,caspase-1,NLRP3 and GSDMD.After treatment with Pc Tx-1and ASIC1a-si RNA,compared with the model group,the expression of pyroptosis death indicators increased,and the expression of the pyroptosis death indicators decreased after the intervention of ASIC1a overexpression plasmid.Moreover,the LDH results showed that after inhibiting ASIC1a,the LDH content in the supernatant was higher than that in the acidified group.In addition,Elisa results also showed that the levels of IL-1βand IL-18 in the culture supernatant increased after ASIC1a was inhibited or silenced.After transfection ASIC1a overexpression pretreatment,the above-mentioned indexes related to pyroptosis were contrary to the results described above.Studies suggest that ASIC1a inhibits extracellular acid-induced pyroptosising of activated hepatic stellate cells.4. Effect of Ca2+on ASIC1a-mediated focal death of hepatic stellate cellsAn in vitro model of acid-induced HSC-T6 was established and pre-treated with Ca²⁺-free medium to investigate the effects of Ca²⁺on activated HSC-T6 pyroptosis.The results showed that compared with the model group,the expression of pyroptosis-related indicators increased after pretreatment with Ca²⁺-free medium.Studies suggest that Ca²⁺inhibits extracellular acid-induced pyroptosising of activated hepatic stellate cells.To sum up,the results of this study show that:in rat hepatic stellate cells,acid induction activates them.In activated hepatic stellate cells,activation of ASIC1a inhibits its pyroptosising,and its possible mechanism is through the regulation of Ca²⁺and inflammatory bodies.更多还原