發(fā)布時(shí)間：2018-04-20 05:05

  本文選題：Toll樣受體4 + 四氯化碳��； 參考：《安徽醫(yī)科大學(xué)》2014年碩士論文

【摘要】：背景和目的肝臟疾病嚴(yán)重危害機(jī)體健康,急性肝損傷是慢性肝病的發(fā)病基礎(chǔ),闡明急性肝損傷的發(fā)病機(jī)理對(duì)急慢性肝病的防治具有重要意義。Toll樣受體(TLR)4是介導(dǎo)先天性免疫反應(yīng)的重要信號(hào)通路,其下游炎性相關(guān)因子通過(guò)介導(dǎo)肝臟炎癥參與肝臟的損傷和修復(fù)過(guò)程,在肝臟疾病中發(fā)揮重要作用；另一方面,維生素D(Vit D)可下調(diào)TLR4介導(dǎo)的信號(hào)通路起到免疫調(diào)節(jié)作用,其可能通過(guò)影響TLR4下游炎性相關(guān)因子表達(dá)參與調(diào)控炎癥反應(yīng)。本課題擬通過(guò)研究(1)TLR4在四氯化碳(CCl4)急性肝損傷和修復(fù)中的作用,(2)Vit D缺乏對(duì)CCl4急性肝損傷過(guò)程肝臟炎癥的影響,闡明TLR4及下游炎性因子在CCl4急性肝損傷和修復(fù)中的作用以及Vit D缺乏調(diào)控TLR4下游炎性因子表達(dá)對(duì)CCl4急性肝損傷的影響。 方法本課題包括二個(gè)部分：(1)為探討TLR4在CCl4急性肝損傷和修復(fù)中的作用,將TLR4野生型(C3H/HeN,TLR4+/+)和突變型(C3H/HeJ,TLR4-/-)小鼠各24只分別經(jīng)腹腔注射給予單劑量CCl4(按體質(zhì)量0.3ml/kg)處理,在CCl4處理后不同時(shí)點(diǎn)(0、24、48、72h)每組分別剖殺6只小鼠。所有小鼠在剖殺前均禁食12h。所有小鼠在剖殺前均稱量體重,剖殺后收集血液和肝臟組織樣本,稱量肝臟重量。分離的血清用于ALT水平的檢測(cè)；用HE染色檢測(cè)肝臟組織病理學(xué)損害；用TUNEL法檢測(cè)肝臟細(xì)胞凋亡；用免疫組織化學(xué)技術(shù)檢測(cè)增殖細(xì)胞核抗原(PCNA)在肝臟細(xì)胞中的分布與定位；用Western blot檢測(cè)小鼠肝臟組織核蛋白PCNA水平；用RT-qPCR檢測(cè)肝臟細(xì)胞增殖相關(guān)基因細(xì)胞周期素(Cyclin)D1及肝臟吞噬相關(guān)基因(CD36、Gpmb、Cd81和Cd51)、促炎細(xì)胞因子(TNF-α、IL-1β和IL-6)、趨化因子(KC、MCP、1和MIP-1α)、抗炎細(xì)胞因子(IL-4和IL-10)和促纖維化因子TGF-β1mRNA水平。(2)為探討維生素D缺乏對(duì)CCl4急性肝損傷過(guò)程肝臟炎癥的影響,將36只成年健康雄性ICR小鼠隨機(jī)分為維生素D缺乏(LVD)組和標(biāo)準(zhǔn)飼料(STD)組。LVD組喂養(yǎng)缺乏維生素D的飼料；STD組喂養(yǎng)標(biāo)準(zhǔn)飼料。所有小鼠在喂養(yǎng)1W后經(jīng)腹腔注射給予CC14(0.3ml/kg)處理,于不同時(shí)點(diǎn)(0、24、72h)剖殺小鼠并收集血清和肝組織。檢測(cè)血清丙氨酸轉(zhuǎn)氨酶(ALT)水平；HE染色分析肝臟病理學(xué)改變；RT-qPCR技術(shù)檢測(cè)肝臟KC、MCP-1、MIP-1α、NF-α、TGF-β1和α-SMA mRNA水平。 結(jié)果(1)TLR4+/+小鼠血清ALT水平和肝臟壞死面積在24h明顯升高,72h則基本恢復(fù)至正常；TLR4"/-小鼠血清ALT水平的變化類似于TLR4+/+小鼠,但同時(shí)點(diǎn)的壞死面積均比TLIR4+/+小鼠顯著,損傷恢復(fù)速度慢于TLR4+/+小鼠。用免疫組織化學(xué)技術(shù)檢測(cè)發(fā)現(xiàn),CCl4處理后72h肝組織切片中有細(xì)胞核染色呈棕黃色,且TLR4+/+小鼠的PCNA陽(yáng)性細(xì)胞數(shù)顯著高于TLR4-/-小鼠。用Western Blotting和RT-qPCR技術(shù)檢測(cè)發(fā)現(xiàn),TLR4+/+小鼠肝臟組織核蛋白PCNA水平和Cyclin D1mRNA表達(dá)明顯高于TLR4-/-小鼠。并且,RT-qPCR檢測(cè)結(jié)果顯示,與TLR4+/+小鼠相比,同時(shí)點(diǎn)TLR4-/-小鼠肝臟趨化因子KC、MCP-1和MIP-1α mRNA,促炎因子TNF-α、IL-1β和IL-6mRNA以及抗炎因子IL-4和IL-10mRNA水平均下調(diào)；與吞噬作用相關(guān)的基因包括CD36、Gpnmb、Cd81和Cd51mRNA水平也均低于TLR4+/+小鼠；CCl4處理顯著上調(diào)TLR4+/+小鼠肝臟TGF-β1mRNA水平,盡管TLR4-/-小鼠肝臟的TGF-β1mRNA基礎(chǔ)水平顯著高于TLR4+/+小鼠,但CCl4對(duì)TLR4-/-小鼠肝臟的TGF-β1mRNA無(wú)明顯影響。(2)與STD組小鼠比較,VD組小鼠各時(shí)點(diǎn)ALT水平和肝臟組織病理學(xué)改變均無(wú)顯著差異；RT-qPCR檢測(cè)結(jié)果顯示,與STD組同時(shí)點(diǎn)相比,LVD組小鼠肝臟趨化因子KC和MCP-1mRNA水平上調(diào)更明顯,促炎因子TNF-α以及促纖維化因子TGF-β1和α-SMA mRNA水平升高更顯著。 結(jié)論(1)TLR4通過(guò)誘導(dǎo)下游促炎細(xì)胞因子和趨化因子,參與CCl4急性肝損傷引起的炎癥反應(yīng),顯著上調(diào)吞噬相關(guān)基因、增殖標(biāo)志基因和促纖維化因子在調(diào)控肝臟修復(fù)和肝再生反應(yīng)中發(fā)揮重要作用。(2)Vit D缺乏上調(diào)TLR4下游肝臟趨化因子、促炎細(xì)胞因子和促纖維化相關(guān)細(xì)胞因子表達(dá),加重CCl4急性肝損傷引起的炎癥反應(yīng)和促纖維化反應(yīng)。
[Abstract]:Background and Objective The pathogenesis of acute liver injury is the basis of chronic liver disease . Toll - like receptor ( TLR ) 4 is an important signal pathway mediating the innate immune response . Toll - like receptor ( TLR ) 4 plays an important role in liver disease . Toll - like receptor ( TLR ) 4 plays an important role in liver disease . Toll - like receptor ( TLR ) 4 is an important signal pathway mediating innate immune response .
On the other hand , vitamin D ( vitamin D ) can down - regulate the signaling pathway mediated by TL4 to play an immunoregulatory role , which may be involved in the regulation of inflammatory response by affecting the expression of the downstream inflammatory - related factors .

Methods Two parts were studied : ( 1 ) To investigate the role of Toll - like receptor 4 in the acute liver injury and repair of CC14 . All the 24 mice were treated by intraperitoneal injection ( 0.3 ml / kg ) . All the mice were fasted for 12 hours before the killing . All the mice weighed body weight before killing . All the mice collected blood and liver tissue samples before killing . The weight of liver was weighed . The separated serum was used for the detection of ALT level .
The pathological damage of liver was detected by HE staining .
TUNEL was used to detect the apoptosis of liver cells .
The distribution and localization of proliferating cell nuclear antigen ( PCNA ) in liver cells was detected by immunohistochemistry .
The level of PCNA was detected by Western blot .
( 2 ) To investigate the effects of vitamin D deficiency on liver inflammation in rats with acute liver injury induced by CC14 , and 36 adult healthy male ICR mice were randomly divided into vitamin D deficiency ( LVD ) group and standard feed ( STD ) group .
All the mice were treated with CC14 ( 0.3 ml / kg ) by intraperitoneal injection after 1 W feeding , and the mice were killed at different time ( 0,24 , 72h ) and serum and liver tissues were collected . Serum alanine aminotransferase ( ALT ) levels were detected .
HE staining analysis of liver pathology changes ;
The mRNA levels of KC , MCP - 1 , MIP - 1偽 , NF - 偽 , TGF - 尾1 and 偽 - SMA were detected by RT - qPCR .

Results ( 1 ) The serum ALT level and the area of liver necrosis increased significantly at 24 h , and then recovered to normal after 72 h .
Compared with TLIR4 + / + mice , the level of PCNA - positive cells was significantly higher than that in TLIR4 + / + mice .
涓庡悶鍣綔鐢ㄧ浉鍏崇殑鍩哄洜鍖呮嫭CD36,Gpnmb,Cd81鍜孋d51mRNA姘村鉤涔熷潎浣庝簬TLR4+/+灝忛紶錛，

本文編號(hào)：1776346