端粒酶基因治疗对压力超负荷心力衰竭小鼠的影响

doi:10.3969/j.issn.1674-8115.2025.08.002

摘要/Abstract

摘要：

目的·探讨端粒酶基因治疗（JV001）对主动脉弓缩窄（transverse aortic constriction，TAC）小鼠心力衰竭的治疗效果。方法·将雄性C57BL/6J小鼠随机分为假手术组（Sham组）、模型组（TAC组）和JV001治疗组（TAC+JV001组）。采用腺相关病毒9（adeno-associated virus 9，AAV9）载体携带催化失活（D868A）和核输出磷酸化（Y707F）突变的人端粒酶反转录酶（human telomerase reverse transcriptase，hTERT）基因（AAV9-modhTERT，命名为JV001），以4×10¹² gc/kg（genomic copy per kilogram）剂量单次尾静脉注射至TAC+JV001组小鼠模型中。Sham组和TAC组小鼠尾静脉注射等量的生理盐水。在给药后第6周，通过实时荧光定量PCR（real-time quantitative PCR，RT-qPCR）明确JV001在心脏中的表达。通过超声心动图检测各组小鼠心功能。记录各组小鼠生理指标并计算心脏质量/体质量比值（heart weight/body weight ratio，HW/BW）和心脏质量/胫骨长度比值（heart weight/tibial length ratio，HW/TL）。通过苏木精-伊红（hematoxylin and eosin，H-E）染色和小麦胚芽凝集素（wheat germ agglutinin，WGA）染色分析心肌肥大情况。通过马松（Masson）染色观察心脏组织胶原沉积情况。通过RT-qPCR检测心肌肥大相关基因（Nppa、Nppb、Myh7、Myh6）和心肌纤维化相关基因（Col1a1、Col3a1、Ctgf）的表达水平。结果·给药后第6周，小鼠心脏中表达高水平的modhTERT mRNA。与Sham组相比，TAC组小鼠表现出左心室射血分数（left ventricular ejection fraction，LVEF）和左心室缩短分数（left ventricular fractional shortening，LVFS）下降，JV001治疗后小鼠的LVEF和LVFS较TAC组显著升高。相比TAC组，JV001治疗后TAC小鼠的HW/BW和HW/TL降低，心肌细胞平均横截面积减小，心肌肥大相关基因（Nppa、Nppb、Myh7、Myh6）的表达水平改善。JV001治疗改善了TAC诱导的小鼠心肌间质纤维化增加和纤维化相关基因（Col1a1、Col3a1、Ctgf）的表达水平升高。结论·AAV9-modhTERT可改善TAC所致小鼠心脏功能损害，减轻心肌肥厚和心肌纤维化。

关键词: 心力衰竭, 心肌纤维化, 腺相关病毒, 基因治疗, 端粒酶

Abstract:

Objective ·To investigate the therapeutic effect of telomerase gene therapy (JV001) on heart failure induced by transverse aortic constriction (TAC) in mice. Methods ·Male C57BL/6J mice were randomly divided into sham operation group (Sham group) , model group (TAC group), and JV001 treatment group (TAC+JV001 group). An adeno-associated virus 9 (AAV9) vector containing the catalytic inactivation (D868A) and nuclear export phosphorylation (Y707F) mutations in the human telomerase reverse transcriptase (hTERT) gene (AAV9-modhTERT, named JV001) was intravenously administered to TAC animals at a single dose of 4×10¹² gc/kg. The Sham group and the TAC group received equivalent volumes of saline via tail vein injection. Six weeks after administration, the expression of JV001 in the heart was determined by real-time quantitative PCR (RT-qPCR). Murine cardiac functions were assessed using echocardiography. Physiological indexes of mice were recorded for calculating heart weight/body weight ratio (HW/BW) and heart weight/tibial length ratio (HW/TL). Cardiac hypertrophy was assessed using hematoxylin-eosin (HE) staining and wheat germ agglutinin (WGA) staining. Cardiac collagen deposition was observed by Masson staining. The expressions of myocardial hypertrophy-related genes (Nppa, Nppb, Myh7, Myh6) and myocardial fibrosis-related genes (Col1a1, Col3a1, Ctgf) were detected by RT-qPCR. Results ·High levels of modhTERT mRNA were expressed in the hearts of mice at 6 weeks post-injection. Compared with sham-operated mice, TAC mice exhibited significantly reduced left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS). Compared to TAC mice, TAC+JV001 mice exhibited a significant improvement in LVEF and LVFS. Concurrently, there was a downregulation in the HW/BW and HW/TL in TAC+JV001 mice compared to TAC mice. Furthermore, JV001 treatment reduced the mean cardiomyocyte cross-sectional area and improved the expression levels of myocardial hypertrophy-related genes, including Nppa, Nppb, Myh7 and Myh6. Additionally, JV001 treatment ameliorated the TAC-induced increase in myocardial interstitial fibrosis and reduced the elevated expression levels of myocardial fibrosis-related genes, including Col1a1, Col3a1, and Ctgf. Conclusion ·AAV9-modhTERT treatment can alleviate TAC-induced cardiac dysfunction, cardiac hypertrophy, and fibrosis in mice.

Key words: heart failure, cardiac fibrosis, adeno-associated virus (AAV), gene therapy, telomerase

中图分类号:

R541

何苏荟, 赵银龙, 张家毓. 端粒酶基因治疗对压力超负荷心力衰竭小鼠的影响[J]. 上海交通大学学报（医学版）, 2025, 45(8): 949-956.

HE Suhui, ZHAO Yinlong, CHANG Alex Chia Yu. Effects of telomerase gene therapy on pressure overload-induced heart failure in mice[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(8): 949-956.

图/表 5

表1 RT-qPCR引物序列

Tab 1 Primer sequences for RT-qPCR

Species	Gene	Forward primer sequence (5′→3′)	Reverse primer sequence (5′→3′)
Mus musculus	Nppa	TTCGGGGGTAGGATTGACAG	CACACCACAAGGGCTTAGGA
Mus musculus	Nppb	AGTCCTTCGGTCTCAAGGCA	CCGATCCGGTCTATCTTGTGC
Mus musculus	Myh7	ACTGTCAACACTAAGAGGGTCA	TTGGATGATTTGATCTTCCAGGG
Mus musculus	Myh6	GCCCAGTACCTCCGAAAGTC	GCCTTAACATACTCCTCCTTGTC
Mus musculus	Col1a1	TGGTACATCAGCCCGAAC	GTCAGCTGGATAGCGACA
Mus musculus	Col3a1	CTGTAACATGGAAACTGGGGAAA	CCATAGCTGAACTGAAAACCACC
Mus musculus	Ctgf	GACCCAACTATGATGCGAGCC	CCCATCCCACAGGTCTTAGAAC
Mus musculus	Gapdh	CATCACTGCCACCCAGAAGACTG	ATGCCAGTGAGCTTCCCGTTCAG
Homo sapiens	TERT	GCGGAAGACAGTGGTGAACT	TTCAGGATGGAGTAGCAGAGG

图1 JV001的设计和给药后6周 modhTERT 在心脏中的表达情况Note： A. Design of AAV9-modhTERT. B. Relative mRNA level of modhTERT in heart tissue.

Fig 1 Design of JV001 and modhTERT expression in the hearts at 6 weeks post-administration

图2 JV001对小鼠心功能的作用Note： A. Representative M-mode echocardiographic images of the left ventricle in each group at 16 weeks (bar=2 mm). B. LVEF and LVFS at 6 weeks after JV001 treatment.

Fig 2 Effects of JV001 on cardiac function in mice

图3 各组小鼠心肌肥厚指标比较Note： A. Representative gross morphology of the hearts. B. Representative HE staining images of the hearts. C. HW/BW and HW/TL ratios at 16 weeks post-surgery. D. Representative images of WGA staining and quantification of cross-sectional area of cardiomyocytes. E. Relative mRNA levels of Nppa, Nppb, Myh7, and Myh6 in heart tissue.

Fig 3 Comparison of cardiac hypertrophy in each group of mice

图4 JV001对小鼠心脏纤维化的作用Note： A. Representative images of Masson staining and quantification of collagen in left ventricular cross-sections. B. Relative mRNA levels of Col1a1, Col3a1, and Ctgf in heart tissue.

Fig 4 Effects of JV001 on myocardial fibrosis in mice

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