Toll样受体4下游炎性因子在四氯化碳急性肝损伤和修复中的作用

发布时间：2018-04-20 05:05

  本文选题：Toll样受体4 + 四氯化碳　； 参考：《安徽医科大学》2014年硕士论文

【摘要】：背景和目的肝脏疾病严重危害机体健康,急性肝损伤是慢性肝病的发病基础,阐明急性肝损伤的发病机理对急慢性肝病的防治具有重要意义。Toll样受体(TLR)4是介导先天性免疫反应的重要信号通路,其下游炎性相关因子通过介导肝脏炎症参与肝脏的损伤和修复过程,在肝脏疾病中发挥重要作用；另一方面,维生素D(Vit D)可下调TLR4介导的信号通路起到免疫调节作用,其可能通过影响TLR4下游炎性相关因子表达参与调控炎症反应。本课题拟通过研究(1)TLR4在四氯化碳(CCl4)急性肝损伤和修复中的作用,(2)Vit D缺乏对CCl4急性肝损伤过程肝脏炎症的影响,阐明TLR4及下游炎性因子在CCl4急性肝损伤和修复中的作用以及Vit D缺乏调控TLR4下游炎性因子表达对CCl4急性肝损伤的影响。 方法本课题包括二个部分：(1)为探讨TLR4在CCl4急性肝损伤和修复中的作用,将TLR4野生型(C3H/HeN,TLR4+/+)和突变型(C3H/HeJ,TLR4-/-)小鼠各24只分别经腹腔注射给予单剂量CCl4(按体质量0.3ml/kg)处理,在CCl4处理后不同时点(0、24、48、72h)每组分别剖杀6只小鼠。所有小鼠在剖杀前均禁食12h。所有小鼠在剖杀前均称量体重,剖杀后收集血液和肝脏组织样本,称量肝脏重量。分离的血清用于ALT水平的检测；用HE染色检测肝脏组织病理学损害；用TUNEL法检测肝脏细胞凋亡；用免疫组织化学技术检测增殖细胞核抗原(PCNA)在肝脏细胞中的分布与定位；用Western blot检测小鼠肝脏组织核蛋白PCNA水平；用RT-qPCR检测肝脏细胞增殖相关基因细胞周期素(Cyclin)D1及肝脏吞噬相关基因(CD36、Gpmb、Cd81和Cd51)、促炎细胞因子(TNF-α、IL-1β和IL-6)、趋化因子(KC、MCP、1和MIP-1α)、抗炎细胞因子(IL-4和IL-10)和促纤维化因子TGF-β1mRNA水平。(2)为探讨维生素D缺乏对CCl4急性肝损伤过程肝脏炎症的影响,将36只成年健康雄性ICR小鼠随机分为维生素D缺乏(LVD)组和标准饲料(STD)组。LVD组喂养缺乏维生素D的饲料；STD组喂养标准饲料。所有小鼠在喂养1W后经腹腔注射给予CC14(0.3ml/kg)处理,于不同时点(0、24、72h)剖杀小鼠并收集血清和肝组织。检测血清丙氨酸转氨酶(ALT)水平；HE染色分析肝脏病理学改变；RT-qPCR技术检测肝脏KC、MCP-1、MIP-1α、NF-α、TGF-β1和α-SMA mRNA水平。 结果(1)TLR4+/+小鼠血清ALT水平和肝脏坏死面积在24h明显升高,72h则基本恢复至正常；TLR4"/-小鼠血清ALT水平的变化类似于TLR4+/+小鼠,但同时点的坏死面积均比TLIR4+/+小鼠显著,损伤恢复速度慢于TLR4+/+小鼠。用免疫组织化学技术检测发现,CCl4处理后72h肝组织切片中有细胞核染色呈棕黄色,且TLR4+/+小鼠的PCNA阳性细胞数显著高于TLR4-/-小鼠。用Western Blotting和RT-qPCR技术检测发现,TLR4+/+小鼠肝脏组织核蛋白PCNA水平和Cyclin D1mRNA表达明显高于TLR4-/-小鼠。并且,RT-qPCR检测结果显示,与TLR4+/+小鼠相比,同时点TLR4-/-小鼠肝脏趋化因子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水平均下调；与吞噬作用相关的基因包括CD36、Gpnmb、Cd81和Cd51mRNA水平也均低于TLR4+/+小鼠；CCl4处理显著上调TLR4+/+小鼠肝脏TGF-β1mRNA水平,尽管TLR4-/-小鼠肝脏的TGF-β1mRNA基础水平显著高于TLR4+/+小鼠,但CCl4对TLR4-/-小鼠肝脏的TGF-β1mRNA无明显影响。(2)与STD组小鼠比较,VD组小鼠各时点ALT水平和肝脏组织病理学改变均无显著差异；RT-qPCR检测结果显示,与STD组同时点相比,LVD组小鼠肝脏趋化因子KC和MCP-1mRNA水平上调更明显,促炎因子TNF-α以及促纤维化因子TGF-β1和α-SMA mRNA水平升高更显著。 结论(1)TLR4通过诱导下游促炎细胞因子和趋化因子,参与CCl4急性肝损伤引起的炎症反应,显著上调吞噬相关基因、增殖标志基因和促纤维化因子在调控肝脏修复和肝再生反应中发挥重要作用。(2)Vit D缺乏上调TLR4下游肝脏趋化因子、促炎细胞因子和促纤维化相关细胞因子表达,加重CCl4急性肝损伤引起的炎症反应和促纤维化反应。
[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伪 , NF - 伪 , TGF - 尾1 and 伪 - 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�浣滅敤鐩稿叧鐨勫熀鍥犲寘鎷珻D36,Gpnmb,Cd81鍜孋d51mRNA姘村钩涔熷潎浣庝簬TLR4+/+灏忛紶锛，

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