Treatment of pancreatic fibrosis with siRNA against a collagen-specific chaperone in vitamin A-coupled liposomes
Treatment of pancreatic fibrosis with siRNA against a collagen-specific chaperone in vitamin A-coupled liposomes
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DOI：
10.1136/gutjnl-2011-301746
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发表时间：
2013-09-01
期刊：
影响因子：
24.5
通讯作者：
Niitsu, Yoshiro
中科院分区：
文献类型：
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作者：
Ishiwatari, Hirotoshi;Sato, Yasushi;Niitsu, Yoshiro
背景和目的慢性胰腺炎相关的纤维化是一种不可逆的损害，可扰乱胰腺的外分泌和内分泌功能。目前，还没有批准的治疗这种疾病的方法。我们先前发现，针对胶原特异性伴侣蛋白gp46的siRNA被包裹在维生素A偶联脂质体(VA-Lip-siRNAgp46)中，可以解决肝硬变模型中的纤维化。采用二丁基二氯化锡(DBTC)和雨蛙素诱导的大鼠胰腺纤维化模型。方法用荧光激活细胞分选(FACS)法检测活化的胰腺星状细胞(APSCs)对VA-Lip-siRNAgp46的特异性摄取。荧光显微镜观察VA-LIP-siRNAgp46-FAM在细胞内的分布。免疫印迹法检测VA-Lip-siRNAgp46对gp46表达的抑制作用。用染料结合法检测aPSCs的胶原合成。静脉注射VA-LIP-siRNA-FAM和H-3-VA-LIP-siRNAgp46后，证实了VA-LIP-siRNAgp46特异性地输送到DBTC大鼠的aPSCs。通过Azan-Mallory染色和羟脯氨酸含量检测VA-Lip-siRNA对DBTC和雨蛋白处理大鼠胰腺组织学的影响。结果流式细胞仪分析显示，aPSCs通过视黄醇结合蛋白受体特异性摄取VA-Lip-siRNAgp46-FAM。免疫印迹和胶原检测分别证实了转导VA-Lip-siRNAgp46后aPSCs对gp46的抑制和胶原分泌的抑制。最后一次注射后24小时，荧光和放射性检测证实VA-Lip-siRNAgp46被特异性地输送到DBTC大鼠纤维化区域的aPSCs。10次全身性VA-Lip-siRNAgp46治疗可缓解胰腺纤维化，并抑制DBTC和雨蛙素治疗大鼠的组织羟脯氨酸水平。结论这些数据表明本方法具有逆转胰腺纤维化的治疗潜力。
Background and objectiveFibrosis 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.MethodsSpecific 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 H-3-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.ResultsFACS 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 24h after the final injection. 10 systemic VA-lip-siRNAgp46 treatments resolved pancreatic fibrosis, and suppressed tissue hydroxyproline levels in DBTC- and cerulein-treated rats.ConclusionThese data suggest the therapeutic potential of the present approach for reversing pancreatic fibrosis.