本文關(guān)鍵詞：NALP3炎癥小體參與小鼠肝臟缺血再灌注損傷的作用及其機(jī)制　出處：《華中科技大學(xué)》2011年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： NALP3 肝臟缺血再灌注 IL-lβ IL-18 HMGB1

【摘要】：肝臟缺血再灌注損傷(LIRI)是一個級聯(lián)炎癥反應(yīng)過程,發(fā)病機(jī)制復(fù)雜,涉及多種因素的參與及相互作用,包括：肝臟微血管紊舌L Kupffer細(xì)胞激活、活性氧簇(ROS)增多、炎性細(xì)胞因子[如腫瘤壞死因子(TNF-α)、白細(xì)胞介素1β(IL-1β)、白細(xì)胞介素18(IL-18)、白細(xì)胞介素6(IL-6)]及高遷移率組蛋白B1(HMGB1)等。 炎癥小體(inflammasome)是由多種蛋白組成的復(fù)合體,能激活炎性caspase復(fù)合物,caspase-1能被NALP3及NALP1炎癥小體激活�；罨腸aspase-1能將proIL-1β和proIL-18分別剪切為IL-1β和IL-18,也有文獻(xiàn)報道其能將proIL-33剪切為IL-33。NALP3炎癥小體能識別許多內(nèi)源性和外源性危險信號,包括細(xì)菌RNA、ATP、尿酸(urid acid, UA)、鋁、石棉、硅石等。另外,胞漿內(nèi)低濃度鉀離子(鉀離子外流)和ROS升高能激活NALP3炎癥小體。 近期研究表明,多種以發(fā)熱、貧血及急性反應(yīng)蛋白增高為特征的系統(tǒng)性炎癥與IL-1β大量釋放相關(guān)。缺血再灌注過程可釋放多種激活炎癥小體的啟動因子,如鈣聚集、鉀離子外流、ROS及多種危險信號(如HMGB1、DNA及RNA)等,故推測炎癥小體可能參與缺血再灌注損傷。文獻(xiàn)已報道：炎癥小體激活所釋放IL-1β和IL-18在小鼠LIRI中是重要的啟動因子,某些針對IL-1β和IL-18的治療策略顯示較好療效；IL-1β在缺血再灌注損傷中發(fā)揮關(guān)鍵作用,IL-1β基因敲除小鼠能明顯減緩缺血再灌注損傷；IL-1受體拮抗基因敲除小鼠缺血再灌注損傷明顯加重。迄今,有關(guān)NALP3炎癥小體是否參與肝臟缺血再灌注損傷,以及干預(yù)NALP3對小鼠肝臟缺血再灌注損傷的影響,均尚未見報道。 本課題建立小鼠LIRI動物模型,并構(gòu)建針對小鼠NALP3的干擾質(zhì)粒,觀察NALP3炎癥小體參與LIRI的作用,并初步探討其機(jī)制。結(jié)果發(fā)現(xiàn)：NALP3炎癥小體在小鼠肝臟缺血再灌注所致無菌性炎癥及組織損傷中起重要作用,其機(jī)制為：NALP3炎癥小體參與IL-1β和IL-18等細(xì)胞因子成熟、釋放；NALP3炎癥小體參與肝臟細(xì)胞凋亡及炎性細(xì)胞浸潤。本課題所獲研究成果為臨床上探索防治LIRI的策略提供了新的實(shí)驗(yàn)依據(jù)。一.NALP3炎癥小體參與小鼠LIRI 建立雄性C57/BL小鼠LIRI模型,同時設(shè)立假手術(shù)對照組。 1.小鼠LIR后血清ALT/AST變化及肝臟形態(tài)學(xué)改變 小鼠肝臟缺血1h再灌注6h后收集血清檢測谷丙轉(zhuǎn)氨酶(ALT)及谷草轉(zhuǎn)氨酶(AST)變化,同時制作肝臟石蠟切片,HE染色觀察形態(tài)學(xué)改變。結(jié)果顯示：模型組ALT/AST明顯高于假手術(shù)組,形態(tài)學(xué)觀察發(fā)現(xiàn)模型組出現(xiàn)大量肝細(xì)胞變性、凋亡、壞死及炎性細(xì)胞浸潤。提示小鼠肝臟缺血再灌注損傷模型制作成功。 2.小鼠LIR后不同時間點(diǎn)IL-1β水平 分別采集小鼠肝臟缺血1h再灌注1h、3h、6h及24h后血樣,ELISA檢測IL-1β水平。結(jié)果顯示：與假手術(shù)對照組相比,血清IL-1β在1h后開始升高,6h達(dá)高峰,24h后回落至1h水平(p0.05)。 3.小鼠LIR后不同時間點(diǎn)NALP3蛋白表達(dá) 小鼠肝臟缺血1h再灌注6h、12h及24h后分別取小鼠肝臟組織,免疫印記法檢測NALP3蛋白水平。結(jié)果發(fā)現(xiàn)：與假手術(shù)組對照,小鼠缺血1h再灌注后NALP3蛋白開始上調(diào),持續(xù)到12h,以6h最為明顯(p0.05)。 4.小鼠肝臟缺血再灌注損傷后ROS變化 小鼠肝臟缺血1h再灌注6h后分離非實(shí)質(zhì)細(xì)胞(NPC), DCF染色,流式細(xì)胞術(shù)檢測ROS含量。結(jié)果顯示,與假手術(shù)對照組相比,缺血lh再灌注6h后ROS明顯增高。 以上結(jié)果提示,NALP3炎癥小體參與小鼠LIRI過程。二、NALP3基因干擾載體的構(gòu)建及鑒定 1.針對小鼠NALP3基因干擾序列的篩選 利用siRNA設(shè)計軟件篩選針對小鼠NALP3基因的3條干擾序列及1條無關(guān)序列,分別合成并命名為siRNA1(S1), siRNA2(S2), siRNA3(S3)及scramble siRNA(SS),利用脂質(zhì)體2000分別轉(zhuǎn)染CHO細(xì)胞,Western-blot檢測目的蛋白NALP3沉默效果,發(fā)現(xiàn)siRNA3沉默效果最佳,scramble siRNA無明顯影響。故選擇siRNA3序列為NALP3有效干擾序列。 2.特異性pNALP3shRNA干擾載體的構(gòu)建及鑒定 分別將siRNA3特異性干擾序列及scramble siRNA無關(guān)序列構(gòu)建入干擾載體pNALP3shRNA及pshRNANC中,經(jīng)酶切鑒定及測序驗(yàn)證其正確性。利用小鼠巨噬細(xì)胞核轉(zhuǎn)試劑盒分別轉(zhuǎn)染pNALP3shRNA禾(?)pshRNANC至巨噬細(xì)胞,設(shè)立：nock組對照。熒光實(shí)時定量PCR檢測NALP3 mRNA水平,Western blot檢測其蛋白水平。結(jié)果發(fā)現(xiàn),pNALP3shRNA干擾載體能特異性沉默NALP3基因(p0.05),而對其他相關(guān)基因(NALP1b、NLRC4等)無影響。提示pNALP3shRNA干擾載體具有沉默特異性。 3. pNALP3shRNA干擾載體對巨噬細(xì)胞分泌IL-1β的影響 利用小鼠巨噬細(xì)胞核轉(zhuǎn)試劑盒轉(zhuǎn)染pNALP3shRNA和pshRNANC至巨噬細(xì)胞,設(shè)mock組對照。48h后繼續(xù)給予LPS及ATP刺激,檢測上清IL-11β含量。結(jié)果發(fā)現(xiàn),轉(zhuǎn)染pNALP3shRNA組IL-1β含量明顯降低(p0.05)。三、沉默NALP3基因?qū)π∈驦IRI的影響及其機(jī)制 1.體內(nèi)干擾NALP3基因?qū)π∈驦IRI的保護(hù)作用 (1) pNALP3shRNA對小鼠肝臟的沉默效果 尾靜脈高壓注射pNALP3shRNA質(zhì)粒后48h,制作小鼠缺血1h再灌注6h動脈模型,實(shí)時定量PCR、Western blot、免疫組化及流式細(xì)胞術(shù)檢測肝組織NALP3表達(dá)。結(jié)果表明：與生理鹽水及pshRNANC組相比,pNALP3shRNA組NALP3表達(dá)明顯下降；免疫組化顯示NALP3主要表達(dá)于肝臟Kupffer細(xì)胞和肝竇內(nèi)皮細(xì)胞。流式細(xì)胞術(shù)檢測發(fā)現(xiàn),Kupffer細(xì)胞和肝竇狀內(nèi)皮細(xì)胞NALP3表達(dá)均被抑制。 (2) pNALP3shRNA預(yù)處理對小鼠缺血再灌注損傷后肝功能的影響 小鼠肝臟缺血1h再灌注6h、12h及24h后收集血清及肝組織制作切片。檢測ALT/AST水平及HE染色。結(jié)果表明：與生理鹽水及pshRNANC對照組相比,pNALP3shRNA組血清ALT/AST.水(?)平明顯降低(p0.01),HE染色顯示肝細(xì)胞變性壞死及炎性細(xì)胞浸潤明顯減少。提示pNALP3shRNA預(yù)處理小鼠能明顯減輕肝臟缺血再灌注損傷。 2.沉默NALP3基因?qū)π∈驦IRI保護(hù)作用的可能機(jī)制 (1) pNALP3shRNA預(yù)處理對小鼠LIRI后血清IL-1β、IL-18水平的影響 小鼠缺血1h再灌注6h后,血清IL-1β和IL-18水平均升高；pNALP3shRNA預(yù)處理能明顯降低血清IL-1β和IL-18水平(p0.05),、Vestern blot檢測發(fā)現(xiàn)pNALP3shRNA預(yù)處理能降低活化的Caspase-1水平。體外實(shí)驗(yàn)證實(shí),pNALP3-shRNA能抑制巨噬細(xì)胞分泌IL-1β(p0.05)。 (2) pNALP3shRNA對小鼠血清細(xì)胞因子及HMGB1水平的影響 與生理鹽水及pshRNANC對照組相比,pNALP3shRNA預(yù)處理組小鼠缺血1h再灌注6h后血清TNF-α和IL-6水平明顯減低,同時肝組織HMGB1表達(dá)明顯降低(p0.05)。再灌注后1h提取肝臟核蛋白,凝膠遷移率(EMSA)實(shí)驗(yàn)檢測NF-κB的DNA結(jié)合活性,結(jié)果顯示,pNALP3shRNA組NF-κB活性明顯減低(p0.05)。 (3) pNALP3shRNA對小鼠肝細(xì)胞凋亡的影響 小鼠缺血1h再灌注6h后,切取肝組織,4%多聚甲醛固定,制作石蠟切片,TUNEL法檢測肝細(xì)胞凋亡。結(jié)果發(fā)現(xiàn),與生理鹽水組及pshRNANC組對比,pNALP3shRNA預(yù)處理能明顯減輕小鼠缺血1h再灌注6h后肝細(xì)胞凋亡(p0.05)。 (4) pNALP3shRNA對小鼠肝臟巨噬細(xì)胞及中性粒細(xì)胞浸潤的影響 小鼠缺血1h再灌注6h后,切取肝組織,4%多聚甲醛固定,制作石蠟切片,免疫組化檢測巨噬細(xì)胞(F4/80)和中性粒細(xì)胞(Gr-1)浸澗。結(jié)果發(fā)現(xiàn), pNALP3sh-RNA預(yù)處理能明顯降低巨噬細(xì)胞和中性粒細(xì)胞浸潤(p0.05)。四、結(jié)論 1. NALP3炎癥小體參與小鼠LIRI發(fā)生、發(fā)展。 2. pNALP3shRNA預(yù)處理能保護(hù)小鼠肝臟缺血再灌注損傷。 3.上述保護(hù)作用的可能機(jī)制為：抑制炎性因子IL-1β、IL-18、TNF-α,、IL-6及HMGB1釋放；抑制NF-κB活性；減少肝細(xì)胞凋亡；減少炎性細(xì)胞浸潤。
[Abstract]:Hepatic ischemia - reperfusion injury ( LIRI ) is a cascade of inflammatory reaction processes , pathogenesis is complex , involves the participation and interaction of various factors , including : the activation of the liver microvessels , the increase of reactive oxygen species ( ROS ) , the inflammatory cytokines , such as tumor necrosis factor ( TNF - 偽 ) , interleukin - 1尾 ( IL - 1尾 ) , interleukin - 18 ( IL - 18 ) , interleukin - 6 ( IL - 6 ) and high - mobility group protein , B1 , and so on . Inflammatory caspase - 1 can be activated by NALP3 and NALP1 . The activated caspase - 1 can cut proIL - 1尾and proIL - 18 into IL - 1尾 and IL - 18 respectively . It is also reported that it is capable of cutting proIL - 1尾and proIL - 18 into IL - 1尾 and IL - 18 . It is also reported that it can identify many endogenous and exogenous risk signals including bacterial RNA , ATP , uric acid ( UA ) , aluminum , asbestos , silica , etc . In addition , low concentration of potassium ( potassium ion efflux ) and ROS increase in cytoplasm can activate NALP3 inflammatory small body . IL - 1尾 and IL - 18 play a key role in ischemia - reperfusion injury . IL - 1尾 and IL - 18 play a key role in ischemia - reperfusion injury . IL - 1尾 and IL - 18 play a key role in ischemia - reperfusion injury . In this study , we established an animal model of mouse LIRI , and constructed an interfering plasmid against NALP3 in mice , and observed the role of NALP3 inflammatory small body in LIRI . It was found that NALP3 inflammation was involved in the maturation and release of cytokines such as IL - 1尾 and IL - 18 . The results showed that NALP3 - inflammatory small body was involved in the apoptosis of liver cells and inflammatory cell infiltration . A male C57 / BL mouse LIRI model was established and sham operation control group was established . 1 . Serum ALT / AST changes and hepatic morphological changes after LIR in mice The results showed that ALT / AST in model group was significantly higher than that in sham operation group . The results showed that ALT / AST in model group was significantly higher than that in sham operation group . The results showed that ALT / AST in model group was significantly higher than that in sham operation group . 2 . IL - 1尾 levels at different time points after LIR in mice The levels of IL - 1尾 and IL - 1尾 in liver of mice were measured at 1h , 3h , 6h and 24h after reperfusion . The results showed that the serum IL - 1尾 increased after 1 h , peaked at 6 h , and returned to 1 hour after 24 h ( p < 0.05 ) . 3 . NALP3 protein expression at different time points after LIR in mice The results showed that the NALP3 protein was up - regulated after 1 h of ischemia in mice and 12 h after reperfusion . The results showed that the NALP3 protein was up - regulated after 1 h of ischemia - reperfusion in mice and was the most obvious in 6 h ( p < 0.05 ) . 4 . ROS changes after liver ischemia / reperfusion injury in mice The content of ROS was detected after reperfusion for 6 hours after reperfusion in the liver of the mice , and the ROS content was detected by flow cytometry . The results showed that ROS increased significantly after 6 hours of ischemia - reperfusion compared with the sham - operated control group . The results suggested that NALP3 was involved in the process of LIRI in mice . The construction and identification of NALP3 gene interference vector 1 . Screening of Mouse NALP3 Gene Interference Sequence Three interfering sequences and 1 unrelated sequences of NALP3 gene of mouse were screened by siRNA design software . siRNA1 ( S1 ) , siRNA2 ( S2 ) , siRNA3 ( S3 ) and siRNA ( SS ) were synthesized and named respectively . Construction and identification of specific pNALP3shRNA interference vector pNALP3shRNA and pshRNANC were transfected into pNALP3shRNA and pshRNANC respectively . The results showed that pNALP3shRNA interfering vector could specifically silence NALP3 mRNA level and Western blot was used to detect NALP3 mRNA level . 3 . Effect of pNALP3shRNA interfering vector on IL - 1尾 secretion in macrophages The results showed that the content of IL - 1尾 in pNALP3shRNA group was significantly lower than that of pNALP3shRNA and pshRNANC to macrophages ( p < 0.05 ) . The effect of silencing NALP3 gene on LIRI in mice and its mechanism were found . 1 . Protective effect of NALP3 gene on LIRI in mice ( 1 ) Effect of pNALP3shRNA on Mouse Liver The expression of NALP3 in liver tissue was detected by RT - PCR , Western blot , immunohistochemistry and flow cytometry . The results showed that the expression of NALP3 in the pNALP3shRNA group was significantly decreased compared with normal saline and pshRNANC group . The expression of NALP3 was inhibited by flow cytometry . The expression of NALP3 was inhibited by flow cytometry . ( 2 ) Effect of pretreatment of pNALP3shRNA on liver function after ischemia / reperfusion injury in mice The levels of ALT / AST and water ( ? ) were significantly decreased in the pNALP3shRNA group ( P0.01 ) . The results showed that the serum ALT / AST and water ( ? ) level of pNALP3shRNA group were significantly decreased ( P0.01 ) compared with normal saline and pshRNANC control group . 2 . Possible mechanism of silencing NALP3 gene on the protection of LIRI in mice ( 1 ) Effect of pretreatment of pNALP3shRNA on serum levels of IL - 1尾 and IL - 18 after LIRI in mice The levels of IL - 1尾 and IL - 18 in serum were increased after 1 hour reperfusion in mice . The pretreatment of pNALP3shRNA could significantly reduce the levels of IL - 1尾 and IL - 18 in serum ( P < 0.05 ) . The results showed that pNALP3 shRNA could inhibit the secretion of IL - 1尾 ( p . 05 ) . ( 2 ) Effect of pNALP3shRNA on serum cytokines and serum levels in mice Compared with normal saline and pshRNANC control group , the levels of serum TNF - 偽 and IL - 6 were significantly decreased after reperfusion for 6 h after reperfusion in the pretreatment group of pNALP3shRNA . The DNA binding activity of NF - 魏B was detected at 1h after reperfusion . The results showed that the activity of NF - 魏B in pNALP3shRNA group was significantly lower ( p < 0.05 ) . ( 3 ) Effect of pNALP3shRNA on Apoptosis of Mouse Hepatocytes The results showed that the pretreatment of pNALP3shRNA could significantly reduce the apoptosis of hepatocytes after 1 hour reperfusion ( p . 05 ) . ( 4 ) Effect of pNALP3shRNA on the infiltration of macrophages and neutrophils in mouse liver The results showed that pNALP3sh - RNA pretreatment significantly decreased the infiltration of macrophages and neutrophils ( p . 05 ) . Conclusion : The results showed that pNALP3sh - RNA pretreatment could significantly reduce the infiltration of macrophages and neutrophils ( p . 05 ) . 1 . NALP3 inflammation was involved in the development and development of LIRI in mice . 2 . The pretreatment of pNALP3shRNA can protect the liver ischemia - reperfusion injury in mice . 3 . The possible mechanism of the above protection is to inhibit the release of inflammatory cytokines such as IL - 1尾 , IL - 18 , TNF - 偽 , IL - 6 , and IL - 6 , inhibit NF - 魏B activity , reduce hepatocellular apoptosis , and reduce inflammatory cell infiltration .

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

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本文編號：1433799