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基于流体动力学的肝转染技术使用短发夹 RNA 质粒在肝硬化大鼠中实现 CB1 的基因沉默。

Hydrodynamics-based liver transfection achieves gene silencing of CB1 using short hairpin RNA plasmid in cirrhotic rats.

机构信息

Institute of Molecular Biology in Medicine, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara, Mexico.

Tecnologico de Monterrey, Campus Guadalajara, Guadalajara, Mexico.

出版信息

PLoS One. 2020 Feb 13;15(2):e0228729. doi: 10.1371/journal.pone.0228729. eCollection 2020.

DOI:10.1371/journal.pone.0228729
PMID:32053633
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7018086/
Abstract

BACKGROUND

There is a correlation between the endocannabinoid system and hepatic fibrosis based on the activation of CB1 and CB2 receptors; where CB1 has profibrogenic effects. Gene therapy with a plasmid carrying a shRNA for CB1 delivered by hydrodynamic injection has the advantage of hepatic tropism, avoiding possible undesirable effects of CB1 pharmacological inhibition.

OBJECTIVE

To evaluate hydrodynamics-based liver transfection in an experimental model of liver cirrhosis of a plasmid with the sequence of a shRNA for CB1 and its antifibrogenic effects.

METHODS

Three shRNA (21pb) were designed for blocking CB1 mRNA at positions 877, 1232 and 1501 (pshCB1-A, B, C). Sequences were cloned in the pENTR™/U6. Safety was evaluated monitoring CB1 expression in brain tissue. The silencing effect was determined in rat HSC primary culture and CCl4 cirrhosis model. Hydrodynamic injection in cirrhotic liver was through iliac vein and with a dose of 3mg/kg plasmid. Serum levels of liver enzymes, mRNA levels of TGF-β1, Col IA1 and α-SMA and the percentage of fibrotic tissue were analyzed.

RESULTS

Hydrodynamic injection allows efficient CB1 silencing in cirrhotic livers and pshCB1-B (position 1232) demonstrated the main CB1-silencing. Using this plasmid, mRNA level of fibrogenic molecules and fibrotic tissue considerably decrease in cirrhotic animals. Brain expression of CB1 remained unaltered.

CONCLUSION

Hydrodynamics allows a hepatotropic and secure transfection in cirrhotic animals. The sequence of the shCB1-B carried in a plasmid or any other vector has the potential to be used as therapeutic strategy for liver fibrosis.

摘要

背景

内源性大麻素系统与肝纤维化之间存在相关性，这是基于 CB1 和 CB2 受体的激活；其中 CB1 具有促纤维化作用。通过水动力注射携带 CB1 的短发夹 RNA (shRNA) 的质粒基因治疗具有肝靶向性的优势，可避免 CB1 药理学抑制的可能不良影响。

目的

在肝纤维化的实验模型中，评估基于水动力的肝转染对携带 CB1 shRNA 序列的质粒的疗效及其抗纤维化作用。

方法

设计了三个针对 CB1 mRNA 的 21bp shRNA（位置 877、1232 和 1501）（pshCB1-A、B、C）。序列被克隆到 pENTR™/U6 中。通过监测脑组织中 CB1 的表达来评估安全性。在大鼠 HSC 原代培养物和 CCl4 肝硬化模型中测定沉默效果。通过髂静脉以 3mg/kg 质粒的剂量进行肝内水动力注射。分析血清肝酶水平、TGF-β1、ColIA1 和α-SMA 的 mRNA 水平以及纤维化组织的百分比。

结果

水动力注射可有效沉默肝硬化肝脏中的 CB1，pshCB1-B（位置 1232）显示出主要的 CB1 沉默效果。使用该质粒，肝硬化动物的纤维生成分子和纤维组织的 mRNA 水平显著降低。大脑中 CB1 的表达保持不变。

结论

水动力法可在肝硬化动物中实现肝靶向和安全转染。携带 shCB1-B 序列的质粒或任何其他载体都有可能成为治疗肝纤维化的策略。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b8a5/7018086/5573c73d71cb/pone.0228729.g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b8a5/7018086/02a3f65906ed/pone.0228729.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b8a5/7018086/97a3b64e317c/pone.0228729.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b8a5/7018086/a21ff9f46572/pone.0228729.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b8a5/7018086/9eb1cf4e8c19/pone.0228729.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b8a5/7018086/2e65075ba3fc/pone.0228729.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b8a5/7018086/66484ba79adb/pone.0228729.g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b8a5/7018086/5573c73d71cb/pone.0228729.g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b8a5/7018086/02a3f65906ed/pone.0228729.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b8a5/7018086/97a3b64e317c/pone.0228729.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b8a5/7018086/a21ff9f46572/pone.0228729.g003.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b8a5/7018086/2e65075ba3fc/pone.0228729.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b8a5/7018086/66484ba79adb/pone.0228729.g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b8a5/7018086/5573c73d71cb/pone.0228729.g007.jpg

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