用载维生素 A 的脂质体包裹针对胶原特异性伴侣的 siRNA 治疗胰腺纤维化。

Treatment of pancreatic fibrosis with siRNA against a collagen-specific chaperone in vitamin A-coupled liposomes.

机构信息

Fourth Department of Internal Medicine, Sapporo Medical University, Sapporo, Japan.

出版信息

Gut. 2013 Sep;62(9):1328-39. doi: 10.1136/gutjnl-2011-301746. Epub 2012 Nov 20.

DOI:10.1136/gutjnl-2011-301746

PMID:23172890

Abstract

BACKGROUND AND OBJECTIVE

Fibrosis associated with chronic pancreatitis is an irreversible lesion that can disrupt pancreatic exocrine and endocrine function. Currently, there are no approved treatments for this disease. We previously showed that siRNA against collagen-specific chaperone protein gp46, encapsulated in vitamin A-coupled liposomes (VA-lip-siRNAgp46), resolved fibrosis in a model of liver cirrhosis. This treatment was investigated for pancreatic fibrosis induced by dibutyltin dichloride (DBTC) and cerulein in rats.

METHODS

Specific uptake of VA-lip-siRNAgp46, conjugated with 6'-carboxyfluorescein (FAM) by activated pancreatic stellate cells (aPSCs), was analysed by fluorescence activated cell sorting (FACS). Intracellular distribution of VA-lip-siRNAgp46-FAM was examined by fluorescent microscopy. Suppression of gp46 expression by VA-lip-siRNAgp46 was assessed by immunoblotting. Collagen synthesis in aPSCs was assayed by dye-binding. Specific delivery of VA-lip-siRNAgp46 to aPSCs in DBTC rats was verified following intravenous VA-lip-siRNA-FAM and (3)H-VA-lip-siRNAgp46. The effect of VA-lip-siRNA on pancreatic histology in DBTC- and cerulein-treated rats was determined by Azan-Mallory staining and hydroxyproline content.

RESULTS

FACS analysis revealed specific uptake of VA-lip-siRNAgp46-FAM through the retinol binding protein receptor by aPSCs in vitro. Immunoblotting and collagen assay verified knockdown of gp46 and suppression of collagen secretion, respectively, by aPSCs after transduction of VA-lip-siRNAgp46. Specific delivery of VA-lip-siRNAgp46 to aPSCs in fibrotic areas in DBTC rats was confirmed by fluorescence and radioactivity 24 h after the final injection. 10 systemic VA-lip-siRNAgp46 treatments resolved pancreatic fibrosis, and suppressed tissue hydroxyproline levels in DBTC- and cerulein-treated rats.

CONCLUSION

These data suggest the therapeutic potential of the present approach for reversing pancreatic fibrosis.

摘要

背景与目的

慢性胰腺炎相关的纤维化是一种不可逆的病变，可破坏胰腺外分泌和内分泌功能。目前，尚无针对这种疾病的批准治疗方法。我们之前曾表明，包裹在维生素 A 偶联脂质体（VA-脂质-siRNAgp46）中的胶原蛋白特异性伴侣蛋白 gp46 的 siRNA 可解决肝硬化模型中的纤维化。该治疗方法已在二丁基锡二氯化物（DBTC）和 cerulein 诱导的大鼠胰腺纤维化中进行了研究。

方法

通过荧光激活细胞分选（FACS）分析激活的胰腺星状细胞（aPSC）对结合 6'-羧基荧光素（FAM）的 VA-脂质-siRNAgp46 的特异性摄取。通过荧光显微镜检查 VA-脂质-siRNAgp46-FAM 的细胞内分布。通过免疫印迹评估 VA-脂质-siRNAgp46 对 gp46 表达的抑制作用。通过染料结合测定法测定 aPSC 中的胶原合成。通过静脉内 VA-脂质-siRNA-FAM 和（3）H-VA-脂质-siRNAgp46 验证 VA-脂质-siRNAgp46 在 DBTC 大鼠中 aPSC 的特异性递送。通过 Azan-Mallory 染色和羟脯氨酸含量确定 VA-脂质-siRNA 对 DBTC 和 cerulein 处理大鼠胰腺组织学的影响。

结果

FACS 分析显示，体外 aPSC 通过视黄醇结合蛋白受体特异性摄取 VA-脂质-siRNAgp46-FAM。免疫印迹和胶原测定分别验证了 gp46 的敲低和 VA-脂质-siRNAgp46 转导后 aPSC 中胶原分泌的抑制。DBTC 大鼠纤维化区域内 aPSC 的特异性递送通过荧光和放射性 24 小时后最后一次注射得到证实。10 次全身 VA-脂质-siRNAgp46 治疗可解决胰腺纤维化，并抑制 DBTC 和 cerulein 处理大鼠的组织羟脯氨酸水平。