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口服肝靶向制剂介导受体ASBT和ASGPR在肝泡型包虫病大鼠模型体内的表达分析
DOI: 10.3969/j.issn.1001-5256.2021.04.024
利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。
作者贡献声明：高瑞雪、胡春晖、张发斌负责课题设计，撰写论文，修改论文；高瑞雪、高攀、甘雪辉、张耀刚、姜博璠参与收集数据及数据分析；高瑞雪、胡春晖、张发斌负责拟定写作思路，指导撰写文章并最后定稿。
Expression of ASBT and ASGPR mediated receptors for oral liver-targeting preparations in a rat model of hepatic alveolar echinococcosis
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摘要:
目的 通过测定顶端钠依赖性胆盐转运体(ASBT)和去唾液酸糖蛋白受体(ASGPR)的表达研究，探讨其在肝泡型包虫病(HAE)药物治疗方面口服肝靶向制剂设计的可行性。 方法 18只雄性SD大鼠，10只大鼠造模，造模成功的8只大鼠为HAE组，正常组大鼠8只作为对照。采用免疫荧光、Western Blot、实时荧光定量PCR对ASBT在HAE模型大鼠及正常大鼠的回肠组织的表达分布、蛋白及基因表达水平进行检测分析；采用同样的检测方法分析ASGPR在HAE大鼠模型的非患病肝组织、肝组织病灶边缘带及正常大鼠肝组织的表达水平。符合正态分布的计量资料2组间比较采用t检验，3组间比较采用单因素方差分析，两两比较采用LSD-t检验。 结果 免疫荧光、Western Blot、实时荧光定量PCR结果均显示ASBT在HAE组回肠组织的表达较正常组表达上调(t值分别为5.309、4.110、28.060，P值均 < 0.05)。免疫荧光、Western Blot、实时荧光定量PCR结果均显示，ASGPR在正常对照组大鼠肝组织、HAE组大鼠非患病肝组织和HAE组大鼠肝织病灶边缘带中表达差异有统计学意义(F值分别为110.666、128.201、143.879，P值均 < 0.001)，其中HAE组较正常组表达水平高，且越靠近病灶组织表达越高。 结论 ASBT和ASGPR可作为HAE潜在的口服肝靶向制剂的介导受体，为设计用于治疗HAE的口服肝靶向制剂提供理论基础。 -
关键词:
- 棘球蚴病, 肝 /
- 分子靶向治疗 /
- 顶端钠依赖性胆盐转运体 /
- 去唾液酸糖蛋白受体 /
- 大鼠, Sprague-Dawley
Abstract:Objective To investigate the feasibility of apical sodium-dependent bile salt transporter (ASBT) and asialoglycoprotein receptor (ASGPR) in the design of oral liver-targeting preparations for the treatment of hepatic alveolar echinococcosis (HAE) by measuring the expression of ASBT and ASGPR. Methods A total of 18 male Sprague-Dawley rats were selected, among which 10 were used to establish a model of HAE (HAE group) and 8 were used as controls (normal group). Immunofluorescence assay, Western blotting, and quantitative real-time PCR were used to measure the expression distribution, protein expression level, and mRNA expression level of ASBT in the ileal tissue of HAE model rats and normal rats; the same methods were used to measure the expression level of ASGPR in the non-diseased liver tissue and the marginal zone of liver tissue lesion of HAE model rats and the liver tissue of normal rats. The 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 three groups, and the least significant difference t-test was used for comparison between two groups. Results The results of immunofluorescence assay, Western blotting, and quantitative real-time PCR showed that compared with the normal group, the HAE group had significantly upregulated expression of ASBT in the ileal tissue (t=5.309, 4.110, and 28.060, all P < 0.05) and a significantly higher expression level of ASGPR (the closer to the lesion, the higher the expression) (F=110.666, 128.201, and 143.879, all P < 0.001). Conclusion ASBT and ASGPR can be used as potential mediated receptors for oral liver-targeting preparations for HAE, which provides a theoretical basis for the design of oral liver-targeting preparations for the treatment of HAE. -
图 3 ASBT在正常大鼠及HAE大鼠回肠组织的表达分布(免疫荧光染色，×200)
注: DAPI，蓝色荧光(细胞核)；CY3，红色荧光(ASGPR阳性表达)；MERGE，叠加。
图 5 ASGPR在正常大鼠及HAE大鼠肝组织的表达分布(免疫荧光染色，×200)
注: DAPI，蓝色荧光(细胞核)；CY3，红色荧光(ASGPR阳性表达)；MERGE，叠加。
表 1 实时荧光定量PCR引物
靶基因 引物序列 ASBT Forward: 5′-TGCCGCCCTCCCCACAAC-3′ Reverse: 5′-GGACTGACCCTGGACTCTGACC-3′ ASGPR Forward: 5′-TGGCTGAGAGTCTGGCTTCCTG-3′ Reverse: 5′-AGTCATCTGGCTGCCCTGGTC-3′ β-actin Forward: 5′-CCTGGCACCCAGCACAAT-3′ Reverse: 5′-GCCGATCCACACGGAGTACT-3′ 
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表 2 ASBT在正常大鼠及HAE大鼠的表达比较
组别 大鼠数(只) 免疫荧光面密度值(%) ASBT/β-actin mRNA相对表达量(2-△△CT) 正常对照组 8 1.286±0.261 0.331±0.025 0.988±0.031 HAE组 8 2.603±0.490 0.946±0.054 2.528±0.708 t值 5.309 4.110 28.060 P值 0.006 0.015 < 0.001
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表 3 ASGPR在正常大鼠及HAE大鼠的表达比较
组别 大鼠数(只) 免疫荧光面密度值(%) ASBT/β-actin mRNA相对表达量(2-△△CT) 正常对照组大鼠肝组织 8 0.162±0.004 0.540±0.035 1.051±0.124 HAE组大鼠非患病肝组织 8 0.532±0.0691) 0.866±0.0831) 2.208±0.2331) HAE组大鼠肝组织病灶边缘带 8 1.275±0.1422) 1.024±0.0572) 3.808±0.5002) F值 110.666 128.201 143.879 P值 < 0.001 < 0.001 < 0.001 注：与正常大鼠肝组织相比, 1)P < 0.001；与HAE大鼠非患病肝组织相比, 2)P < 0.001。
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