PD-1对脓毒症小鼠肝脏枯否细胞功能的影响与机制研究

发布时间：2018-04-17 08:03

本文选题：脓毒症 + 盲肠结扎穿孔　；参考：《第二军医大学》2013年博士论文

【摘要】：【研究目的】脓毒症是感染引起的全身炎症反应综合征，死亡率一直居高不下。免疫抑制是脓毒症后期常见的免疫异常状态。巨噬细胞功能障碍是这种免疫抑制的一个重要原因，但是其具体发生机制目前尚不明了。程序性死亡分子1（programmed cell death1, PD-1）是近年来发现的共抑制受体，参与外周组织免疫耐受和慢性病毒感染。PD-1基因敲除可以显著提高脓毒症小鼠的生存率，提示PD-1在脓毒症免疫抑制中发挥着重要的作用。本课题拟首先检测PD-1在肝脏枯否细胞的表达，进而应用PD-1基因敲除小鼠，观察PD-1在脓毒症肝脏枯否细胞功能障碍的作用，并初步探索PD-1在枯否细胞发挥作用的分子机制及PD-1参与脓毒症多器官功能障碍的分子机制。【研究方法】 1、复制盲肠结扎穿孔（cecal ligation and puncture, CLP）致脓毒症小鼠模型。将16只野生型C57BL/6小鼠随机分为假手术组（Sham, n=8）和CLP组（CLP, n=8）。术后24h时取肝脏，分离肝脏非实质细胞，应用流式细胞仪检测枯否细胞（F4/80+）表面PD-1的表达水平。 2、将10只野生型C57BL/6小鼠及10只PD-1基因敲除小鼠随机分为野生型假手术组（WT Sham, n=4），PD-1基因敲除假手术组（PD-1-/-Sham, n=4），野生型CLP组（WT CLP, n=6）和PD-1基因敲除CLP组（PD-1-/-CLP, n=6）。术后24h时，分离肝脏非实质细胞，应用流式细胞仪检测枯否细胞表面MHCΠ、CD69、CD80和CD86的表达水平。 3、将8只野生型C57BL/6小鼠及8只PD-1基因敲除小鼠随机分为野生型假手术组（WT Sham, n=4），PD-1基因敲除假手术组（PD-1-/-Sham, n=4），野生型CLP组（WT CLP, n=4）和PD-1基因敲除CLP组（PD-1-/-CLP, n=4）。术后24h时，分离肝脏非实质细胞，贴壁法纯化枯否细胞。加入分子探针pHrodoTME. coli BioParticlesConjugate共培养1h，应用流式细胞仪检测枯否细胞吞噬功能。 4、将10只野生型C57BL/6小鼠及10只PD-1基因敲除小鼠随机分为野生型假手术组（WT Sham, n=4），PD-1基因敲除假手术组（PD-1-/-Sham, n=4），野生型CLP组（WT CLP, n=6）和PD-1基因敲除CLP组（PD-1-/-CLP, n=6）。术后24h时，分离肝脏非实质细胞，贴壁法纯化枯否细胞。LPS（1μg/ml）刺激或不刺激24h后收集培养上清及细胞，ELISA法检测细胞因子TNF-α、IL-1β、IL-6、IL-10、IL-12p40和MCP-1水平。 5、将10只野生型C57BL/6小鼠及10只PD-1基因敲除小鼠随机分为野生型假手术组（WT Sham, n=4），PD-1基因敲除假手术组（PD-1-/-Sham, n=4），野生型CLP组（WT CLP, n=6）和PD-1基因敲除CLP组（PD-1-/-CLP, n=6）。术后24h时，分离肝脏非实质细胞，贴壁法纯化枯否细胞。LPS（1μg/ml）刺激24h后收集细胞，Westernblot法检测cleaved caspase-3的水平。 6、将10只野生型C57BL/6小鼠及10只PD-1基因敲除小鼠随机分为野生型假手术组（WT Sham, n=4），PD-1基因敲除假手术组（PD-1-/-Sham, n=4），野生型CLP组（WT CLP, n=6）和PD-1基因敲除CLP组（PD-1-/-CLP, n=6）。术后24h时，分离肝脏非实质细胞，贴壁法纯化枯否细胞。LPS（1μg/ml）刺激24h后收集细胞，TUNEL法检测凋亡情况。 7、将10只野生型C57BL/6小鼠及10只PD-1基因敲除小鼠随机分为野生型假手术组（WT Sham, n=4），PD-1基因敲除假手术组（PD-1-/-Sham, n=4），野生型CLP组（WT CLP, n=6）和PD-1基因敲除CLP组（PD-1-/-CLP, n=6）。术后24h时，分离肝脏非实质细胞，贴壁法纯化枯否细胞。LPS（1μg/ml）刺激10min后收集细胞，Western blot法检测磷酸化Akt及总Akt水平。 8、将10只野生型C57BL/6小鼠及10只PD-1基因敲除小鼠随机分为野生型假手术组（WT Sham, n=4），PD-1基因敲除假手术组（PD-1-/-Sham, n=4），野生型CLP组（WT CLP, n=6）和PD-1基因敲除CLP组（PD-1-/-CLP, n=6）。术后24h时，分离肝脏非实质细胞，贴壁法纯化枯否细胞。LPS（1μg/ml）刺激10min后收集细胞，Western blot法检测磷酸化p38及总p38水平。 9、将10只野生型C57BL/6小鼠及10只PD-1基因敲除小鼠随机分为野生型假手术组（WT Sham, n=4），PD-1基因敲除假手术组（PD-1-/-Sham, n=4），野生型CLP组（WT CLP, n=6）和PD-1基因敲除CLP组（PD-1-/-CLP, n=6）。术后24h时，取心脏、肝脏和肾脏。组织匀浆，Western blot法检测磷酸化Akt、总Akt、磷酸化STAT3和总STAT3水平。【结果】 1、术后24h时，脓毒症小鼠肝脏枯否细胞表面PD-1表达显著高于假手术组（P0.001）. 2、术后24h时，野生型脓毒症小鼠肝脏枯否细胞表面MHCΠ、CD86表达均显著低于野生型假手术组（P 0.001），CD80表达显著高于野生型假手术组（P 0.001）。PD-1基因敲除可部分但显著恢复枯否细胞表面MHCΠ、CD80和CD86表达（P 0.001）。各组CD69表达无显著改变。 3、术后24h时，野生型脓毒症小鼠肝脏枯否细胞吞噬功能显著下降（P 0.01），PD-1基因敲除脓毒症小鼠可显著增强枯否细胞功能（P 0.001）。 4、术后24h分离肝脏枯否细胞，体外LPS继续刺激或不刺激24h时，野生型脓毒症小鼠肝脏枯否细胞分泌TNF-α、IL-1β、IL-6、IL-10、IL-12p40和MCP-1能力显著下降（P 0.05）。PD-1基因敲除可部分但显著恢复枯否细胞分泌上述细胞因子能力。 5、术后24h分离肝脏枯否细胞，体外LPS继续刺激24h时，野生型脓毒症小鼠肝脏枯否细胞cleaved caspase-3水平显著升高（P 0.001），PD-1基因敲除可显著减少枯否细胞cleaved caspase-3水平（P 0.001）。 6、术后24h分离肝脏枯否细胞，体外LPS继续刺激24h时，野生型脓毒症小鼠肝脏枯否细胞TUNEL染色阳性比例显著升高，PD-1基因敲除可显著减少枯否细胞TUNEL染色阳性比例。 7、术后24h分离肝脏枯否细胞，体外LPS继续刺激10min时，，野生型脓毒症小鼠肝脏枯否细胞磷酸化Akt/总Akt比例显著降低（P 0.001），PD-1基因敲除可显著增加枯否细胞磷酸化Akt/总Akt比例（P 0.001）。 8、术后24h分离肝脏枯否细胞，体外LPS继续刺激10min时，野生型脓毒症小鼠肝脏枯否细胞磷酸化p38/总p38比例显著升高（P 0.001），PD-1基因敲除可显著减少枯否细胞磷酸化p38/总p38比例（P 0.001）。 9、术后24h时，野生型脓毒症小鼠心脏、肝脏和肾脏磷酸化Akt/总Akt比例显著降低，PD-1基因敲除可显著增加肝脏和肾脏磷酸化Akt/总Akt比例，但心脏磷酸化Akt/总Akt比例无显著变化。野生型脓毒症小鼠心脏、肝脏和肾脏磷酸化STAT3/总STAT3比例显著升高，PD-1基因敲除可显著减少心脏和肾脏磷酸化STAT3/总STAT3比例，但是肝脏STAT3/总STAT3比例无显著变化。【结论】 PD-1参与了脓毒症导致的肝脏枯否细胞功能障碍，可能通过影响枯否细胞Akt磷酸化和p38磷酸化发挥作用，还可能通过影响Akt磷酸化和STAT3磷酸化参与脓毒症导致的多器官功能障碍。
[Abstract]:[purpose]
Sepsis is a systemic inflammatory reaction caused by infection syndrome, mortality is high. The immunosuppressive state is immune abnormalities common sepsis later. Macrophage dysfunction is an important reason for this immune suppression, but the specific mechanism is unclear.
Programmed cell death 1 (programmed cell, death1, PD-1) is a co inhibitory receptor found in recent years, in the knockout can significantly improve the survival rate of sepsis mice.PD-1 gene in peripheral tissues and immune tolerance in chronic viral infection, suggesting that PD-1 in sepsis immunosuppression plays an important role in this paper. The first detection of PD-1 in Kupffer cells and expression of PD-1 gene knockout mice, to observe the effect of PD-1 on sepsis Kupffer cell dysfunction, and to explore the molecular mechanism and molecular mechanism of PD-1 play the role of PD-1 involved in sepsis and multiple organ dysfunction in Kupffer cells.
[research methods]
1, copy the cecal ligation and perforation (cecal ligation and puncture, CLP) induced sepsis mice model. 16 male wild-type C57BL/6 mice were randomly divided into sham operation group (Sham, n=8) and CLP group (CLP, n=8). After 24h, the liver and isolated liver non parenchymal cells, detect Kupffer by flow cytometry (F4/80+) expression level of PD-1 on the surface.
2, 10 wild type C57BL/6 mice and 10 PD-1 knockout mice were randomly divided into sham operation group (wild type WT Sham, n=4), PD-1 knockout sham operation group (PD-1-/-Sham, n=4), wild type CLP group (WT CLP, n=6) and PD-1 knockout (PD-1-/-CLP CLP group n=6, 24h). After the operation, the separation of liver nonparenchymal cells, Kupffer cell surface MHC PI assay, flow cytometry was used to CD69, the expression level of CD80 and CD86.
3, 8 wild type C57BL/6 mice and 8 PD-1 knockout mice were randomly divided into sham operation group (wild type WT Sham, n=4), PD-1 knockout sham operation group (PD-1-/-Sham, n=4), wild type CLP group (WT CLP, n=4) and PD-1 knockout (PD-1-/-CLP CLP group n=4, 24h). After the operation, the separation of liver nonparenchymal cells, adhesion purified Kupffer cells. The molecular probe pHrodoTME. coli BioParticlesConjugate joined the co culture 1H, flow cytometer was used to detect the phagocytosis of Kupffer cells.
4, 10 wild type C57BL/6 mice and 10 PD-1 knockout mice were randomly divided into sham operation group (wild type WT Sham, n=4), PD-1 knockout sham operation group (PD-1-/-Sham, n=4), wild type CLP group (WT CLP, n=6) and PD-1 knockout (PD-1-/-CLP CLP group n=6, 24h). After the operation, the separation and purification of liver non parenchymal cells and Kupffer cells adherent.LPS (1 g/ml) stimulated or not after 24h stimulation and cell culture supernatants were collected, detection of cytokines TNF-, ELISA IL-1 IL-6, IL-10, beta, IL-12p40 and MCP-1 levels.
5, 10 wild type C57BL/6 mice and 10 PD-1 knockout mice were randomly divided into sham operation group (wild type WT Sham, n=4), PD-1 knockout sham operation group (PD-1-/-Sham, n=4), wild type CLP group (WT CLP, n=6) and PD-1 knockout (PD-1-/-CLP CLP group n=6, 24h). After the operation, the separation and purification of liver non parenchymal cells and Kupffer cells adherent.LPS (1 g/ml) stimulation of 24h cells were collected after detection of cleaved caspase-3 by Westernblot.
6, 10 wild type C57BL/6 mice and 10 PD-1 knockout mice were randomly divided into sham operation group (wild type WT Sham, n=4), PD-1 knockout sham operation group (PD-1-/-Sham, n=4), wild type CLP group (WT CLP, n=6) and PD-1 knockout (PD-1-/-CLP CLP group n=6, 24h). After the operation, the separation and purification of liver non parenchymal cells and Kupffer cells adherent.LPS (1 g/ml) after stimulation of 24h cells were collected to detect the apoptosis of TUNEL.
7, 10 wild type C57BL/6 mice and 10 PD-1 knockout mice were randomly divided into sham operation group (wild type WT Sham, n=4), PD-1 knockout sham operation group (PD-1-/-Sham, n=4), wild type CLP group (WT CLP, n=6) and PD-1 knockout (PD-1-/-CLP CLP group n=6, 24h). After the operation, the separation and purification of liver non parenchymal cells and Kupffer cells adherent.LPS (1 g/ml) stimulation of 10min cells were collected after Western blot detection of phosphorylated Akt and total Akt levels.
8, 10 wild type C57BL/6 mice and 10 PD-1 knockout mice were randomly divided into sham operation group (wild type WT Sham, n=4), PD-1 knockout sham operation group (PD-1-/-Sham, n=4), wild type CLP group (WT CLP, n=6) and PD-1 knockout (PD-1-/-CLP CLP group n=6, 24h). After the operation, the separation and purification of liver non parenchymal cells and Kupffer cells adherent.LPS (1 g/ml) stimulation of 10min cells were collected after Western blot detection of phosphorylated p38 and total p38 levels.
9, 10 wild type C57BL/6 mice and 10 PD-1 knockout mice were randomly divided into sham operation group (wild type WT Sham, n=4), PD-1 knockout sham operation group (PD-1-/-Sham, n=4), wild type CLP group (WT CLP, n=6) and PD-1 knockout (PD-1-/-CLP CLP group n=6, 24h). After the operation, take heart, liver and kidney homogenates. Detection of phosphorylated Akt, Western, blot total Akt, phosphorylated STAT3 and total STAT3 levels.
[results]
1, the expression of PD-1 on the surface of liver Kupffer cells in sepsis mice was significantly higher than that of the sham operation group (P0.001) at 24h after operation.
2, after 24h, the wild type of sepsis mice Kupffer cell surface MHC PI, the expression of CD86 was significantly lower than that of the wild type in sham operation group (P 0.001), the expression of CD80 was significantly higher than that of the wild type in sham operation group (P 0.001).PD-1 gene knockout partially but significantly restored Kupffer cell surface MHC Pi the expression of CD80 and CD86 (P, 0.001). The expression of CD69 in each group had no significant change.
3, after 24h, the phagocytic function of Kupffer cells in wild type sepsis mice was significantly decreased (P 0.01). PD-1 knockout mice could significantly enhance the function of Kupffer cells (P 0.001).
4, 24h after separation of Kupffer cells in vitro, LPS continues to stimulate or not to stimulate the 24h, wild type septic mice Kupffer cells secrete TNF- alpha, IL-1 beta, IL-6, IL-10, IL-12p40 and MCP-1 decreased significantly (P 0.05).PD-1 gene knockout partially but significantly restored the secretion of dry cytokines in Kupffer cells.
5, after 24h, the hepatic Kupffer cells were separated from the liver. When LPS continued to stimulate 24h in vitro, the cleaved caspase-3 level of Kupffer cells in wild type sepsis mice increased significantly (P 0.001). PD-1 knockout significantly reduced the cleaved caspase-3 level of Kupffer cells (P 0.001).
6, after 24h, the hepatic Kupffer cells were separated from the liver. When LPS continued to stimulate 24h in vitro, the positive rate of TUNEL staining in Kupffer cells of wild type sepsis mice increased significantly. PD-1 knockout significantly reduced the positive proportion of TUNEL staining in Kupffer cells.
7, after 24h, the hepatic Kupffer cells were separated from the liver. When LPS continued to stimulate 10min in vitro, the proportion of total Akt/ Akt in Kupffer cells in the wild type sepsis mice decreased significantly (P 0.001). PD-1 knockout significantly increased the percentage of Akt/ Akt in Kupffer cells (P 0.001).
8, after 24h, the liver Kupffer cells were separated from the liver. When LPS continued to stimulate 10min in vitro, the proportion of p38/ p38 in Kupffer cells of wild type sepsis mice increased significantly (P 0.001). PD-1 knockout significantly reduced the percentage of p38/ p38 in Kupffer cells (P 0.001).
9, after 24h, the wild type of sepsis mice heart, liver and kidney of phosphorylated Akt/ total Akt ratio was significantly decreased, PD-1 knockout significantly increased the phosphorylation of Akt/ in liver and kidney total Akt ratio, but the heart of the phosphorylation of Akt/ ratio of total Akt. No significant changes in wild type sepsis mouse heart the liver and kidney of phosphorylated STAT3/ and total STAT3 ratio was significantly increased, PD-1 knockout significantly reduced the phosphorylation of STAT3/ in heart and kidney total STAT3 ratio, but the liver STAT3/ ratio of total STAT3 showed no significant changes.
[Conclusion]
PD-1 participates in sepsis induced Kupffer cell dysfunction. It may play a role in the Akt phosphorylation and p38 phosphorylation of Kupffer cells. It may also play a role in multiple organ dysfunction caused by sepsis by affecting Akt phosphorylation and STAT3 phosphorylation.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R459.7

【共引文献】