工程化外泌体递送ANGPTL6 mRNA对小鼠肝纤维化的改善作用

doi:10.13481/j.1671-587X.20250602

摘要/Abstract

摘要：

目的探讨血管生成素样蛋白6（ANGPTL6）在肝脏纤维化中的作用，分析工程化外泌体（Exo）递送ANGPTL6 mRNA对肝脏纤维化的改善作用。方法将12只C57BL/6小鼠随机分为橄榄油组（OIL组）（腹腔注射橄榄油）和四氯化碳（CCl₄）组（腹腔注射橄榄油与CCl₄混合液），每组各6只；另12只C57BL/6小鼠，随机分为对照组（喂食含蛋氨酸胆碱对照饲料）和蛋氨酸胆碱缺乏（MCD）组（喂食MCD饲料），构建2种小鼠肝脏纤维化模型，采用实时荧光定量PCR（RT-qPCR）和Western blotting法检测各组小鼠肝组织中ANGPTL6 mRNA和蛋白表达水平。将30只小鼠随机分为橄榄油+磷酸盐缓冲液（PBS）组（OIL+PBS组）（腹腔注射橄榄油，每周2次，8周后，尾静脉注射PBS缓冲液6周，每周2次）、CCl₄+Exo-绿色荧光蛋白（GFP） mRNA组（腹腔注射CCl₄混合液制备肝纤维化模型后，尾静脉注射装载GFP mRNA的工程化Exo 6周）和CCl₄+Exo-ANGPTL6 mRNA组（腹腔注射CCl₄混合液制备肝纤维化模型后，尾静脉注射装载ANGPTL6 mRNA的工程化Exo 6周），每组10只。CCl₄+Exo-GFP mRNA组CCl₄+Exo-ANGPTL6 mRNA组小鼠每周注射工程化Exo 2次，每次每只小鼠注射20 μg（体积100 μL）。酶联免疫吸附试验（ELISA）法检测各组小鼠血清中丙氨酸氨基转移酶（ALT）和天冬氨酸氨基转移酶（AST）活性，Masson染色和天狼猩红染色观察各组小鼠肝脏胶原沉积情况，免疫组织化学法检测各组小鼠肝组织中α-平滑肌肌动蛋白（α-SMA）表达水平，RT-qPCR法检测各组小鼠肝组织中α-SMA、Ⅰ型胶原α1链（Col1a1）、转化生长因子β1（TGF-β1）和金属蛋白酶组织抑制剂1 （TIMP-1） mRNA表达水平。结果生物信息学分析，ANGPTL6在活化肝星状细胞（aHSC）中表达明显下调。超声检查，OIL组小鼠肝脏表面细腻光滑；与OIL组比较，CCl₄组小鼠肝脏切面粗糙，表面凹凸不平。RT-qPCR和Western blotting法检测，与OIL组比较，CCl₄组小鼠肝组织中ANGPTL6 mRNA和蛋白表达水平均明显降低（P<0.05）。从HEK293T细胞上清液中提取的工程化Exo结构完整，经尾静脉注射后能够在纤维化肝脏中大量富集，GFP蛋白在肝脏中大量表达。ELISA法检测，与OIL+PBS组比较，CCl₄+Exo-GFP mRNA组小鼠血清中ALT和AST活性均明显升高（P<0.05）；与CCl₄+Exo-ANGPTL6 mRNA组比较，CCl₄+Exo-GFP mRNA组小鼠血清中ALT和AST活性均明显降低（P<0.05）。Masson染色和天狼猩红染色观察，与OIL+PBS组比较，CCl₄+Exo-GFP mRNA组小鼠肝脏胶原沉积明显增加，相对胶原面积增加（P<0.05）；与CCl₄+Exo-GFP mRNA组比较，CCl₄+Exo-ANGPTL6 mRNA组小鼠肝脏胶原沉积明显减少，相对胶原面积减少（P<0.05）。免疫组织化学法检测，与OIL+PBS组比较，CCl₄+Exo-GFP mRNA组小鼠肝组织中α-SMA蛋白表达水平明显升高（P<0.05）；与CCl₄+Exo-GFPmRNA组比较，CCl₄+Exo-ANGPTL6mRNA组小鼠肝组织中α-SMA蛋白表达水平明显降低（P<0.05）。RT-qPCR法检测，与OIL+PBS组比较，CCl4+Exo-GFP mRNA组小鼠肝组织中Col1a1、α-SMA、TGF-β1和TIMP-1 mRNA表达水平明显升高（P<0.05）；与CCl₄+Exo-GFPmRNA组比较，CCl₄+Exo-ANGPTL6 mRNA组小鼠肝组织中Col1a1、α-SMA、TGF-β1和TIMP-1 mRNA表达水平明显降低（P<0.05）。结论小鼠尾静脉注射工程化Exo递送的ANGPTL6 mRNA主要富集在肝脏，工程化Exo递送ANGPTL6 mRNA对小鼠肝纤维化具有改善作用。

关键词: 血管生成素样蛋白6, 工程化外泌体, 肝脏纤维化, 肝脏损伤, 疾病模型，动物

Abstract:

Objective To discuss the role of angiopoietin-like protein 6 （ANGPTL6） in liver fibrosis， and to analyze the improving effect of engineered exosome（Exo）-delivered ANGPTL6 mRNA on liver fibrosis. Methods A total of 12 C57BL/6 mice were randomly divided into olive oil group （OIL group）（intraperitoneally injected with olive oil） and carbon tetrachloride （CCl₄） group （intraperitoneally injected with a mixture of olive oil and CCl?）， with 6 mice in each group； another 12 C57BL/6 mice were randomly divided into control group （fed a with methionine-choline sufficient diet） and methionine-choline deficient （MCD） group （fed a with MCD diet）， and two kinds of mouse liver fibrosis models were established. Real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting method were used to detect the ANGPTL6 mRNA and protein expression levels in liver tissue of the mice in various groups. A total of 30 mice were randomly divided into olive oil + phosphate buffered saline （PBS） group （OIL+PBS group）（intraperitoneally injected with olive oil twice a week for 8 weeks， then injected with PBS buffer by tail vein twice a week for 6 weeks）， CCl₄+Exo-green fluorescent protein （GFP） mRNA group （established liver fibrosis model by intraperitoneal injection of CCl₄ mixture and were injected by tail vein with engineered Exo loaded with GFP mRNA for 6 weeks）， and CCl?+Exo-ANGPTL6 mRNA group （established liver fibrosis model by intraperitoneal injection of CCl₄ mixture and were injected by tail vein with engineered Exo loaded with ANGPTL6 mRNA for 6 weeks）， with 10 mice in each group. The mice in CCl₄+Exo-GFP mRNA group and CCl₄+Exo-ANGPTL6 mRNA group were injected with engineered Exo twice a week， 20 μg per mouse each time （volume 100 μL）. ELISA method was used to detect the serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST） activities in the mice in various groups； Masson staining and Sirius red staining were used to observe the collagen deposition in liver tissue of the mice in various groups； immunohistochemistry method was used to detect the α-smooth muscle actin （α-SMA） expression levels in liver tissue of the mice in various groups； RT-qPCR method was used to detect the expression levels of α-SMA， collagen type Ⅰ alpha 1 chain （Col1a1）， transforming growth factor β1 （TGF-β1）， and tissue inhibitor of metalloproteinase 1 （TIMP-1） mRNA in liver tissue of the mice in various groups. Results The bioinformatics analysis results showed that ANGPTL6 expression was significantly down-regulated in activated hepatic stellate cell（aHSC）. The ultrasound examination results showed that the liver surface of the mice in OIL group was fine and smooth； compared with OIL group， the liver section of the mice in CCl? group was rough and uneven. The RT-qPCR and Western blotting results showed that compared with OIL group， the ANGPTL6 mRNA and protein expression levels in liver tissue of the mice in CCl? group were significantly decreased （P<0.05）. The engineered Exo extracted from the supernatant of HEK293T cells had intact structure and could be largely enriched in the fibrotic liver after tail vein injection， with GFP protein being largely expressed in the liver. The ELISA assay results showed that compared with OIL+PBS group， the ALT and AST activities in CCl₄+Exo-GFP mRNA group were significantly increased （P<0.05）； compared with CCl₄+Exo-ANGPTL6 mRNA group， the serum ALT and AST activities in CCl₄+Exo-GFP mRNA group were significantly decreased （P<0.05）. The Masson staining and Sirius red staining results showed that compared with OIL+PBS group， the collagen deposition in liver tissue of the mice in CCl?+Exo-GFP mRNA group was significantly increased， and the relative collagen area was increased （P<0.05）； compared with CCl₄+Exo-GFP mRNA group， the collagen deposition in tissue liver of the mice in CCl?+Exo-ANGPTL6 mRNA group was significantly decreased， and the relative collagen area was decreased （P<0.05）. The immunohistochemistry results showed that compared with OIL+PBS group， the α-SMA protein expression level in liver tissue of the mice in CCl?+ Exo-GFP mRNA group was significantly increased （P<0.05）； compared with CCl₄+Exo-GFP mRNA group， the α-SMA protein expression level in liver tissue of the mice in CCl?+Exo-ANGPTL6 mRNA group was significantly decreased （P<0.05）. The RT-qPCR results showed that compared with OIL+PBS group， the expression levels of Col1a1， α-SMA， TGF-β1， and TIMP-1 mRNA in liver tissue of the mice in CCl?+Exo-GFP mRNA group were significantly increased （P<0.05）； compared with CCl₄+Exo-GFP mRNA group， the expression levels of Col1a1， α-SMA， TGF-β1， and TIMP-1 mRNA in liver tissue of the mice in CCl?+Exo-ANGPTL6 mRNA group were significantly decreased （P<0.05）. Conclusion Engineered Exo-delivered ANGPTL6 mRNA injected via the tail vein in the mice is mainly enriched in the liver， and engineered Exo delivery of ANGPTL6 mRNA has an improving effect on liver fibrosis in the mice.

Key words: Angiopoietin-like protein 6, Engineered exosome, Liver fibrosis, Liver injury, Disease model， animals

中图分类号:

唐小茜,温升聪,董振亚,陈静仪,曹煜,张云华. 工程化外泌体递送ANGPTL6 mRNA对小鼠肝纤维化的改善作用[J]. 吉林大学学报(医学版), 2025, 51(6): 1452-1463.

Xiaoqian TANG,Shengcong WEN,Zhenya DONG,Jingyi CHEN,Yu CAO,Yunhua ZHANG. Improvement effect of engineered exosomes delivering ANGPTL6 mRNA on liver fibrosis in mice[J]. Journal of Jilin University(Medicine Edition), 2025, 51(6): 1452-1463.

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Primer	Sequences (5'-3')	NCBI Refseq
ANGPTL6	Forward: CTGGGCCGTCGTGTAGTAG	NM_145154.2
ANGPTL6	Reverse: CAGTCCTCTAGGAGTATCAGCAG	NM_145154.2
α-SMA	Forward: GTCCCAGACATCAGGGAGTAA	NM_007392.3
α-SMA	Reverse: TCGGATACTTCAGCGTCAGGA	NM_007392.3
Col1a1	Forward: GCTCCTCTTAGGGGCCACT	NM_007742.4
Col1a1	Reverse: CCACGTCTCACCATTGGGG	NM_007742.4
TGF-β1	Forward: CTCCCGTGGCTTCTAGTGC	NM_011577.2
TGF-β1	Reverse: GCCTTAGTTTGGACAGGATCTG	NM_011577.2
TIMP-1	Forward: GCAACTCGGACCTGGTCATAA	NM_001044384.2
TIMP-1	Reverse: CGGCCCGTGATGAGAAACT	NM_001044384.2
β-actin	Forward: GGCTGTATTCCCCTCCATCG	NM_007393.5
β-actin	Reverse: CCAGTTGGTAACAATGCCATGT	NM_007393.5

Group	ALT	AST
OIL+PBS	27.42±10.02	25.35±6.31
CCl₄+Exo-GFP mRNA	262.80±17.07^*	189.20±25.15^*
CCl₄+Exo-ANGPTL6 mRNA	51.07±6.93^△	59.16±7.39^△

Group	Masson	Sirius red	α-SMA
OIL+PBS	0.72±0.06	0.16±0.02	0.46±0.08
CCl₄+Exo-GFP mRNA	5.16±0.78^*	1.21±0.09^*	1.87±0.13^*
CCl₄+Exo-ANGPTL6 mRNA	1.31±0.22^△	0.61±0.04^△	1.07±0.12^△