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平足蛋白（PDPN）在肝星状细胞活化以及肝纤维化中的作用分析
DOI: 10.12449/JCH240316
伦理学声明：临床研究于2020年8月12日经由上海中医药大学附属普陀医院伦理委员会审批，批号：PTEC-2020-32（Y）-1，符合临床研究伦理规范。动物实验于2022年6月30日由上海中医药大学附属普陀医院实验动物伦理委员会审批，批号：QWEC-A-202206008，符合实验室动物管理与使用准则。
利益冲突声明：本文不存在任何利益冲突。
作者贡献声明：王知意、杨广越、张玮、赵鑫负责实验操作及资料分析，撰写论文；马文婷、刘旭凌、吴柳、浦娅琼参与收集数据，修改论文；刘成、陶乐负责课题设计，指导撰写文章并最后定稿。
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摘要:
目的 探究平足蛋白（PDPN）在肝星状细胞活化以及肝纤维化中的作用，并对其机制进行初步探讨。 方法 收集2019年9月—2022年6月首次就诊于上海中医药大学附属普陀医院感染科的75例慢性乙型肝炎患者肝活检组织样本，实时定量PCR（RT-PCR）和免疫组化检测PDPN在不同肝纤维化分期患者肝组织中的表达。12只雄性C57/BL6小鼠随机分为正常组和模型组，每组各6只。模型组腹腔注射含10% CCl4，对照组注射等体积橄榄油共6周，HE染色、天狼星红染色观察肝组织病理学变化，分离小鼠肝脏原代细胞，检测PDPN mRNA在各类型细胞中表达；使用PDPN蛋白处理小鼠原代肝星状细胞，并使用NF-κB抑制剂BAY11-7082处理，分析PDPN诱导肝星状细胞炎症因子表达情况。计量资料2组间比较采用成组t检验，多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。两组数据相关性分析采用Spearman相关性分析法。 结果 慢性乙型肝炎患者肝活检样本中，PDPN mRNA在正常肝脏中表达较低，S3、S4期肝组织中表达显著升高，与正常肝组织比较差异均有统计学意义（P值均<0.001）；免疫组化染色结果显示，PDPN阳性表达主要集中在纤维间隔及肝窦，S4期肝组织PDPN阳性面积比S0期表达显著升高（t=8.892，P=0.001）。正常组小鼠中PDPN主要在肝窦表达少许，在CCl4模型小鼠中PDPN表达显著升高（t=0.95，P<0.001），主要在纤维间隔表达；RT-PCR结果显示，PDPN mRNA在CCl4模型小鼠中显著升高（t=11.25，P=0.002）。与肝细胞、肝星状细胞、Kupffer细胞和肝内胆管细胞相比，PDPN在肝窦内皮细胞中明显高表达（F=20.56，P<0.001）；PDPN蛋白处理小鼠原代肝星状细胞15 min，炎症相关因子TNF-α、CCL3、CXCL1和CXCR1的mRNA表达，与对照组相比均显著升高（P值均<0.05），BAY11-7082（NF-κB信号抑制剂）处理后，上述炎症指标水平均明显下降（P值均<0.05）。 结论 在肝纤维化中主要表达在肝窦内皮细胞的PDPN增加，其通过NF-κB信号调节肝星状细胞活化，促进肝纤维化进展 。 Abstract:Objective To investigate the role and mechanism of podoplanin （PDPN） in hepatic stellate cell （HSC） activation and liver fibrosis. Methods Liver biopsy samples were collected from 75 patients with chronic hepatitis B who attended Department of Infectious Diseases， Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine， for the first time from September 2019 to June 2022， and RT-PCR and immunohistochemistry were used to measure the expression of PDPN in liver tissue of patients in different stages of liver fibrosis. A total of 12 male C57/BL6 mice were randomly divided into control group and model group. The mice in the model group were given intraperitoneal injection of 10% CCl4， and those in the control group were injected with an equal volume of olive oil， for 6 weeks. HE staining and Sirius Red staining were used to observe liver histopathological changes； primary mouse liver cells were separated to measure the mRNA expression of PDPN in various types of cells； primary mouse HSCs were treated with PDPN protein， followed by treatment with the NF-κB inhibitor BAY11-708， to measure the expression of inflammatory factors in HSCs induced by PDPN. The independent-samples t test was used for comparison of normally distributed continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. The Spearman correlation analysis was used to investigate data correlation. Results As for the liver biopsy samples， there was a relatively low mRNA expression level of PDPN in normal liver， and there was a significant increase in the mRNA expression level of PDPN in liver tissue of stage S3 or S4 fibrosis （all P<0.001）. Immunohistochemical staining showed that PDPN was mainly expressed in the fibrous septum and the hepatic sinusoid， and the PDPN-positive area in S4 liver tissue was significantly higher than that in S0 liver tissue （t=8.892， P=0.001）. In normal mice， PDPN was mainly expressed in the hepatic sinusoid， and there was a significant increase in the expression of PDPN in CCl4 model mice （t=0.95， P<0.001）， mainly in the fibrous septum. RT-PCR showed a significant increase in the mRNA expression of PDPN in the CCl4 model mice （t=11.25， P=0.002）. Compared with hepatocytes， HSCs， Kupffer cells， and bile duct endothelial cells， hepatic sinusoidal endothelial cells showed a significantly high expression level of PDPN （F=20.56， P<0.001）. Compared with the control group， the primary mouse HSCs treated by PDPN protein for 15 minutes showed significant increases in the mRNA expression levels of the inflammation-related factors TNFα， CCL3， CXCL1， and CXCR1 （all P<0.05）， and there were significant reductions in the levels of these indicators after treatment with BAY11-7082 （all P<0.05）. Conclusion There is an increase in the expression of PDPN mainly in hepatic sinusoidal endothelial cells during liver fibrosis， and PDPN regulates HSC activation and promotes the progression of liver fibrosis via the NF-κB signaling pathway. -
Key words:
- Hepatic Fibrosis /
- Hepatic Stellate Cell /
- Hepatic Sinus Endothelial Cell /
- Podoplanin
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图 1 PDPN在临床肝活检样本中的表达
注： a，肝活检HE染色和PDPN免疫组化染色；b，PDPN阳性面积统计；c、d，PDPN mRNA与肝纤维化分期的关系。
Figure 1. The expression of PDPN mRNA in liver biopsy tissue
图 2 两组小鼠肝组织病理染色结果
注： 红色箭头所指代表染色阳性位置。
Figure 2. The pathological staining of liver tissue in two groups
图 3 小鼠肝脏各类型细胞中PDPN mRNA相对表达
Figure 3. Relative expression of PDPN mRNA in various types of mouse liver cells
表 1 RT-PCR引物序列
Table 1. The RT-PCR primer sequences
名称 序列 18S rRNA 上游：5'-GTAACCCGTTGAACCCCATT-3' 下游：5'-CCATCCAATCGGTAGTAGCG-3' Human PDPN 上游：5'-AACCAGCGAAGACCGCTATAA-3' 下游：5'-CGAATGCCTGTTACACTGTTGA-3' Mouse PDPN 上游：5'-ACCGTGCCAGTGTTGTTCTG-3' 下游：5'-AGCACCTGTGGTTGTTATTTTGT-3' Moues TNFα 上游：5'-ATGAGAGGGAGGCCATTTG-3' 下游：5'-CAGCCTCTTCTCATTCCTGC-3' Moues CCL3 上游：5'-TGTACCATGACACTCTGCAAC-3' 下游：5'-CAACGATGAATTGGCGTGGAA-3' Moues CXCR1 上游：5'-ACTGCTGTAAGAGCCTTTGGG-3' 下游：5'-AGCACCAGAATCACTAGGACA-3' Moues CXCL1 上游：5'-ATCCGAAAATCTGTTGTGGC-3' 下游：5'-CTTTTGCTATCTTCCGCCAG-3' 
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表 2 CCl4模型中PDPN及其mRNA表达分析
Table 2. Statistics of PDPN protein and mRNA expression in two groups
指标 正常组（n=6） 模型组（n=6） t值 P值 PDPN mRNA 1.00±0.26 9.79±1.73 11.25 0.002 PDPN阳性面积（%） 0.86±0.15 2.26±0.23 0.95 <0.001 天狼星红染色面积（%） 0.57±0.13 3.76±0.39 3.04 <0.001
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表 3 小鼠原代HSC中炎症指标水平
Table 3. Expression of inflammatory in mouse primary HSC
组别 TNF-α mRNA CCL3 mRNA CXCL1 mRNA CXCR1 mRNA PBS+Veh组 1.00±0.09 1.00±0.04 1.00±0.02 1.00±0.05 PDPN+Veh组 1.42±0.061） 1.52±0.091） 2.03±0.111） 1.89±0.031） PBS+BAY11-7082组 0.97±0.13 0.94±0.12 1.03±0.15 0.82±0.09 PDPN+BAY11-7082组 0.86±0.052） 0.90±0.082） 0.99±0.022） 1.04±0.212） F值 24.46 31.74 85.93 49.96 P值 0.005 0.005 0.003 0.015 注：BAY11-7082，NF-κB信号抑制剂。与PBS+Veh组比较，1）P<0.05；与PDPN+Veh组比较，2）P<0.05。
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