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Treatment of hepatic fibrosis in rat treated with DMN by siRNA/HSP47 encapsulated in vitamin A conjugating liposome

维生素 A 脂质体包裹的 siRNA/HSP47 治疗 DMN 大鼠肝纤维化

基本信息

批准号：
17590664
负责人：
SATO Yasushi
金额：
$ 2.24万
依托单位：
Sapporo Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590664/
关键词：
HEPATIC FIBROSIS HSP47 siRNA retinol 肝線維化コラーゲン in vivo

项目摘要

To date, no modality has been approved for antifibrotic therapy of liver cirrhosis in humans, mainly due to insufficient specificity for particular target molecules or target cells. In the present study, we first confirmed substantial suppression of collagen secretion from normal rat kidney (NRK) fibroblasts by small interfering RNA (siRNA) for a 46-kDa collagen-binding glycoprotein-(gp46)(heat shock protein 47 of rat), a collagen-specific chaperon. Since hepatic stellate cells (HSC), which play a key role in fibrogenesis, are known to take up vitamin A (VA) through retinol-binding protein receptors (RBPRs), we then treated primary rat HSCs and a human HSC cell line (LI90) with carboxy-fluorescein (FAM)-labeled siRNA gp46 encapsulated in VA-coupled liposomes (VA-lip-siRNAgp46) in the presence of various concentrations of FBS, and affirmed that the fluorescence of these cells was appreciably suppressed by anti-RBP antibodies. Specific delivery of FAM-labeled VA-lip-siRNAgp46 to HSCs was also verified following i.v. injection in rats with liver cirrhosis induced by dimethylnitrosamine (DMN), since the fluorescence was mainly observed in HSC areas of the cirrhotic liver but scarcely in the liver parenchyma, lung, spleen or retina. When ^3H-VA-lip-siRNAgp46 was administered, radioactivity was seen mainly in the liver of DMN-treated rats, while in the normal rats, radioactivity in the liver was not prominent. Treatment of DMN rats with VA-lip-siRNAgp46 (i.v.) resulted in nearly complete histological resolution of liver cirrhosis, due to apoptosis of HSCs, and a 100% survival rate; in contrast, no untreated rats survived. This novel approach may be applicable not only to the therapy of liver cirrhosis, but also for treating fibrotic conditions in other organs, such as nephrosclerosis and chronic pancreatitis, in which stellate cells reportedly play an essential role.

迄今为止，还没有批准用于人类肝硬化的抗纤维化治疗的模式，主要是由于对特定靶分子或靶细胞的特异性不足。在本研究中，我们首先证实了实质性抑制正常大鼠肾（NRK）成纤维细胞的胶原蛋白分泌的小干扰RNA（siRNA）的46 kDa的胶原蛋白结合糖蛋白（gp 46）（热休克蛋白47的大鼠），胶原蛋白特异性伴侣。由于肝星状细胞（HSC）在纤维形成中起关键作用，已知其通过视黄醇结合蛋白受体（RBPR）摄取维生素A（VA），因此我们随后用封装在VA偶联脂质体中的羧基荧光素（FAM）标记的siRNA gp 46处理原代大鼠HSC和人HSC细胞系（LI 90（VA-lip-siRNAgp 46），并确认这些细胞的荧光被抗RBP抗体明显抑制。FAM标记的VA-lip-siRNAgp 46向HSC的特异性递送也在静脉内注射到由二甲基亚硝胺（DMN）诱导的肝硬化大鼠中后得到验证，因为荧光主要在硬化肝脏的HSC区域中观察到，而在肝实质、肺、脾或视网膜中几乎没有。当给予^3 H-VA-lip-siRNAgp 46时，在DMN处理的大鼠中主要在肝脏中观察到放射性，而在正常大鼠中，肝脏中的放射性并不显著。用VA-唇-siRNAgp 46（i. v.）由于HSC的凋亡，导致肝硬化的组织学几乎完全消退，并且100%的存活率;相反，未处理的大鼠没有存活。这种新的方法可能不仅适用于肝硬化的治疗，而且还适用于治疗其他器官的纤维化疾病，如肾硬化和慢性胰腺炎，据报道，星状细胞在其中发挥重要作用。