| 41 | 0 | 52 |
| 下载次数 | 被引频次 | 阅读次数 |
目的 基于规律间隔短回文重复序列/CRISPR相关蛋白9(CRISPR/Cas9)技术构建人源Serpin Family A Member1(SERPINA1)Q129无义突变敲入小鼠(SERPINA1 p.Q129*)并建立心肌梗死(MI)模型，探究其术后7 d、30 d心功能变化及表型差异。方法 设计靶向gRNA并体外转录，构建含同源臂的供体质粒，测序验证后，显微注射Cas9 mRNA与gRNA至C57BL/6J小鼠受精卵，获得F0代基因编辑小鼠并繁育至纯合子。采用PCR方法进行基因型鉴定，通过实时荧光定量聚合酶链反应(Quantitative real-time polymerase chain reaction, qRT-PCR)和蛋白免疫印迹(Western blot, WB)检测SERPINA1 mRNA表达水平及其编码的α1-抗胰蛋白酶(Alpha-1-antitrypsin, AAT)蛋白水平。建立小鼠MI模型，假手术组仅开胸不结扎。术后1 d氯化三苯基四氮唑(TTC)染色测心肌梗死面积；术后7 d、30 d,记录体重、心脏湿重、肺脏湿重和死亡率并超声检测心功能指标；术后30 d,苏木精-伊红(Hematoxylin-eosin, HE)染色法观察心脏病理学形态变化。结果 PCR凝胶电泳及mRNA表达结果证实成功获得SERPINA1 p.Q129*小鼠。术后1 d,与WT心肌梗死小鼠相比，SERPINA1 p.Q129*小鼠心肌梗死面积差异无统计学意义；术后7 d,仅体重降低(P<0.05),心功能、心重比、肺重比差异均无统计学意义；术后30 d,心功能显著恶化(LVEF、LVFS降低，均P<0.05),心重比、肺重比升高(P<0.05),存活率降低；HE染色提示SERPINA1 p.Q129*小鼠心肌梗死面积显著扩大及心腔扩张更明显。结论 SERPINA1 Q129无义突变在MI慢性期(30 d)加剧心功能衰退、心脏不良重塑及死亡风险，其病理效应具有时间依赖性，或为心肌梗死后心力衰竭潜在干预靶点。
Abstract：Objective A humanized serpin family A member 1(SERPINA1) Q129 nonsense mutation knock-in mouse model(SERPINA1 p.Q129*) was generated using CRISPR/Cas9 technology, followed by the establishment of a myocardial infarction(MI) model, to investigate the post-MI changes in cardiac function and phenotypic differences at 7 and 30 days. Methods The targeted gRNA was designed and transcribed in vitro. A donor plasmid containing the homologous arms was constructed. After sequencing verification, Cas9 mRNA and gRNA were microinjected into fertilized eggs of C57BL/6J mice to obtain F0 gene-edited mice that were then bred to homozygotes. Genotype identification was performed by PCR. The expression levels of SERPINA1 mRNA and its encoded α1-antitrypsin(AAT) protein were detected by quantitative real-time polymerase chain reaction(qRT-PCR) and Western blot(WB). An MI model was established in mice. The sham-operated group underwent thoracotomy without coronary artery ligation. 1 day after MI, infarct area was measured by triphenyltetrazolium chloride(TTC) staining; on postoperative days 7 and 30, body weight, heart weight, lung weight and mortality rate were recorded, and echocardiography was used to assess cardiac function. Pathological changes in the heart were observed using the hematoxylin-eosin(HE) staining at 30 days post-operation. Results The gel electrophoresis results of PCR and mRNA expression confirmed the successful generation of SERPINA1 p.Q129* mice. 1 day after myocardial infarction, there was no significant difference in myocardial infarct size between SERPINA1 p.Q129* mice and WT mice. 7 days after operation, only body weight decreased significantly(P<0.05), but there was no difference in heart function, heart weight ratio and lung weight ratio. But at 30 days after operation, cardiac function deteriorated significantly(LVEF and LVFS decreased, all P<0.05), heart weight ratio and lung weight ratio increased(P<0.05), and survival rate decreased significantly. HE staining indicated SERPINA1 p.Q129* mice showed significantly enlarged infarct area and more obvious ventricular dilation. Conclusion The SERPINA1 Q129 nonsense mutation aggravates cardiac function decline, maladaptive cardiac remodeling, and the risk of death during the chronic phase of MI(30 days). Its pathological effect is time-dependent, which may be a potential intervention target for heart failure after MI.
[1] TSAO C W,ADAY A W,ALMARZOOQ Z I,et al.Heart disease and stroke statistics-2023 update:a report from the American heart association [J].Circulation,2023,147(8):E93-E621.
[2] XIANG S Y,YANG L,HE Y,et al.Alpha-1 antitrypsin as a regulatory protease inhibitor modulating inflammation and shaping the tumor microenvironment in cancer[J].Cells,2025,14(2):17.
[3] AMBROSINO P,MARCUCCIO G,LOMBARDI C,et al.Cardiovascular risk associated with Alpha-1 antitrypsin deficiency (AATD) genotypes:a meta-analysis with meta-regressions[J].J Clin Med,2023,12(20):6490.
[4] FÄHNDRICH S,BIERTZ F,KARCH A,et al.Cardiovascular risk in patients with alpha-1-antitrypsin deficiency[J].Respir Res,2017,18(1):171.
[5] MAHTA A,YAGHI S,REZNIK M E,et al.Serum alpha-1 antitrypsin in acute ischemic stroke:A prospective pilot study[J].J Clin Neurosci,2020,76:20-24.
[6] ROTONDO J C,AQUILA G,OTON-GONZALEZ L,et al.Methylation of SERPINA1 gene promoter may predict chronic obstructive pulmonary disease in patients affected by acute coronary syndrome[J].Clin Epigenetics,2021,13(1):79.
[7] MALIK R,DAU T,GONIK M,et al.Common coding variant in SERPINA1 increases the risk for large artery stroke[J].Proc Natl Acad Sci U S A,2017,114(14):3613-3618.
[8] DASí F.Alpha-1 antitrypsin deficiency[J].Med Clin (Barc),2024,162(7):336-342.
[9] FOIL K E.Variants of SERPINA1 and the increasing complexity of testing for alpha-1 antitrypsin deficiency[J].Ther Adv Chronic Dis,2021,12_suppl:20406223211015954.
[10] LONG O S,BENSON J A,KWAK J H,et al.A C.elegans model of human α1-antitrypsin deficiency links components of the RNAi pathway to misfolded protein turnover[J].Hum Mol Genet,2014,23(19):5109-5122.
[11] RUDNICK D A,PERLMUTTER D H.Alpha-1-antitrypsin deficiency:a new paradigm for hepatocellular carcinoma in genetic liver disease[J].Hepatology,2005,42(3):514-521.
[12] YAMASAKI M,SENDALL T J,PEARCE M C,et al.Molecular basis of α1-antitrypsin deficiency revealed by the structure of a domain-swapped trimer[J].EMBO Rep,2011,12(10):1011-1017.
[13] LEE J H,BRANTLY M.Molecular mechanisms of alpha1-antitrypsin null alleles[J].Respir Med,2000,94 Suppl C:S7-11.
[14] CUMMINGS E E,O′REILLY L P,KING D E,et al.Deficient and null variants of SERPINA1 are proteotoxic in a caenorhabditis elegans model of α1-antitrypsin deficiency[J].PLoS One,2015,10(10):e0141542.
[15] LUO H,WU P,CHEN X,et al.Novel insights into the relationship between α-1 anti-trypsin with the pathological development of cardio-metabolic disorders[J].Int Immunopharmacol,2022,111:109077.
[16] LUBRANO V,BALZAN S.Role of oxidative stress-related biomarkers in heart failure:galectin 3,α1-antitrypsin and LOX-1:new therapeutic perspective?[J].Mol Cell Biochem,2020,464(1-2):143-152.
[17] MARCONDES A M,KAROOPONGSE E,LESNIKOVA M,et al.α-1-Antitrypsin (AAT)-modified donor cells suppress GVHD but enhance the GVL effect:a role for mitochondrial bioenergetics[J].Blood,2014,124(18):2881-2891.
[18] AGNÉ A,RICHTER K,TUMPARA S,et al.Does heart surgery change the capacity of α1-antitrypsin to inhibit the ATP-induced release of monocytic interleukin-1β A preliminary study[J].Int Immunopharmacol,2020,81:106297.
[19] JANCIAUSKIENE S M,NITA I M,STEVENS T.Alpha1-antitrypsin,old dog,new tricks.Alpha1-antitrypsin exerts in vitro anti-inflammatory activity in human monocytes by elevating cAMP[J].J Biol Chem,2007,282(12):8573-8582.
[20] FRASS O M,BÜHLING F,TÄGER M,et al.Antioxidant and antiprotease status in peripheral blood and BAL fluid after cardiopulmonary bypass [J].Chest,2001,120(5):1599-1608.
[21] SHOEIBI S,GOU W,YEUNG T,et al.AAT-MSC-EVs:Novel implications for suppressing ferroptosis,fibrosis and pain associated with chronic pancreatitis[J].Mol Ther,2025,33(7):3321-3338.
基本信息:
中图分类号:R542.22
引用信息:
[1]陈红霞,马丽娟,李嫣晓,等.SERPINA1 Q129无义突变加重心肌梗死后心脏重塑的机制研究[J].新疆医科大学学报,2026,49(02):182-189.
基金信息:
“新疆地区高发疾病研究”教育部重点实验室开放课题项目(2024B02); 新疆维吾尔自治区杰出青年科学基金项目(2022D01E70)
2026-02-15
2026-02-15