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表皮生长因子受体(epidermal growth factor receptor， EGFR)是介导细胞增殖、分化与修复的关键受体，其异常表达与多种肿瘤定量方法，以猫EGFR重组质粒为标准品构建标准曲线，用于猫源EGFR拷贝数测定，并在此基础上对猫与鼠多组织EGFR mRNA丰度进行比较。结果显示：引物扩增特异性良好，标准曲线相关系数R2=0.996，扩增效率约99.7%，定量线性范围为发生发展密切相关。为获得伴侣动物正常组织中EGFR的定量基线，本研究建立了基于SYBR Green I的实时荧光定量PCR(qPCR)绝对5.2×108–5.2×101 copies/μL，最低检测限约为5.2×101 copies。对重复孔中偏离均值较大的数据点剔除后，健康猫13种组织中EGFR含量为1.64×102–2.02×103 copies/μg total RNA，其中小肠最高(2024.178)，其次为结膜(778.595)和心脏(724.542)，脾脏最低(164.276)。健康鼠12种组织中EGFR含量为1.97×102–2.38×103 copies/μg total RNA，以肾脏最高(2382.941)，肝脏(922.530)与脾脏(858.902)次之。跨物种比较显示，小肠与结膜在猫中高于鼠(分别约6.67倍与3.32倍)，而肾脏与肝脏在鼠中更高(分别约5.70倍与2.59倍)。综上，本研究建立的猫源EGFR绝对定量qPCR方法可获得跨组织可比的拷贝数结果，并为猫与鼠正常组织EGFR表达分布提供了定量基线，可为比较研究、疾病标志物筛选及靶向药物安全性评估提供参考。
Abstract：Epidermal growth factor receptor (EGFR) is a key regulator of cell proliferation， differentiation， and tissue repair， and its dysregulation is closely associated with the pathogenesis of multiple tumors. To establish a quantitative baseline of EGFR expression in normal tissues of companion animals， an absolute quantitative real-time PCR (qPCR) assay based on SYBR Green I was developed in this study. A recombinant feline EGFR plasmid was used as the standard to generate a calibration curve for copy number determination， and EGFR mRNA abundance in multiple tissues from cats and mice was subsequently compared. The assay showed high amplification specificity， with a correlation coefficient of R = 0.996， an amplification efficiency of 99.7%， a linear dynamic range of 5.2 × 10^8 to 5.2 × 10^1 copies/μL， and a detection limit of approximately 5.2 × 10^1 copies. After excluding replicate outliers that deviated substantially from the mean， EGFR expression in 13 tissues from healthy cats ranged from 1.64 × 10^2 to 2.02 × 10^3 copies/μg total RNA， with the highest level detected in the small intestine (2024.178 copies/μg total RNA)， followed by the conjunctiva (778.595) and heart (724.542)， whereas the spleen showed the lowest level (164.276). In 12 tissues from healthy mice， EGFR expression ranged from 1.97 × 10^2 to 2.38 × 10^3 copies/μg total RNA， with the highest level in the kidney (2382.941)， followed by the liver (922.530) and spleen (858.902). Cross-species comparison revealed that EGFR expression in the small intestine and conjunctiva was higher in cats than in mice， by approximately 6.67-fold and 3.32-fold， respectively， whereas expression in the kidney and liver was higher in mice， by approximately 5.70-fold and 2.59-fold， respectively. Taken together， the absolute quantitative qPCR assay established in this study provides comparable cross-tissue copy number measurements and defines a quantitative baseline for EGFR expression in normal feline and murine tissues， thereby offering useful reference data for comparative studies， biomarker discovery， and safety evaluation of EGFR-targeted therapeutics.
[1] 刘宝全， 范圣第. EGFR信号转导机制及靶向治疗[J]. 大连民族学院学报， 2008， 10(1): 13-16.
[2] Yarden Y， Sliwkowski MX. Untangling the ErbB signalling network[J]. Nat Rev Mol Cell Biol， 2001， 2(2): 127-137.
[3] Normanno N， De Luca A， Bianco C， et al. The epidermal growth factor receptor (EGFR) signaling network in cancer[J]. J Cell Physiol， 2006， 194(1): 13-19.
[4] Wee P， Wang Z. Epidermal growth factor receptor cell proliferation signaling pathways in cancer[J]. Cancers (Basel)， 2017， 9(5): 52.
[5] Ciardiello F， Tortora G. EGFR antagonists in cancer treatment[J]. N Engl J Med， 2008， 358(11): 1160-1174.
[6] Looper JS， Malarkey DE， Ruslander D， et al. Epidermal growth factor receptor expression in feline oral squamous cell carcinomas[J]. Vet Comp Oncol， 2006.
[7] Yoshikawa H， et al. Immunohistochemical characterization of feline oral squamous cell carcinoma[J]. Am J Vet Res， 2012， 73(11): 1801-1806.
[8] De Maria R， et al. Molecular targets for anticancer therapies in companion animals and humans[J]. Theranostics， 2021， 11: 3882-3907.
[9] Hanazono K， et al. Epidermal growth factor receptor expression in canine transitional cell carcinoma[J]. J Vet Med Sci， 2015， 77(1): 111-115.
[10] Mao Y， et al. Near infrared photoimmunotherapy targeting bladder cancer with a canine anti-EGFR antibody[J]. Oncotarget， 2018.
[11] 王鹏飞， 吴柯维， 郑莉， 等. EGFR靶向抗体类药物的研究进展[J]. 药学进展， 2024， 48(3): 176-190.
[12] 宋祥铭， 郭晓明， 江旭源. EGFR靶向治疗耐药机制及分子影像学研究进展[J]. 核医学进展， 2021， 42(3): 235-240.
[13] Altamura G， Borzacchiello G. Anti-EGFR monoclonal antibody Cetuximab displays potential anti-cancer activity against feline oral squamous cell carcinoma cell lines[J]. Front Vet Sci， 2022， 9: 1040552.
[14] 磨炼， 慕晓艳， 李和根. 中药改善非小细胞肺癌患者EGFR-TKIs获得性耐药研究现状及其中医病机探析[J]. 上海中医药杂志， 2023， 57(4): 24-28.
[15] Paoloni M， Khanna C. Translation of new cancer treatments from pet dogs to humans[J]. Nat Rev Cancer， 2008， 8(2): 147-156.
[16] Bustin SA， Benes V， Garson JA， et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments[J]. Clin Chem， 2009， 55(4): 611-622.
[17] Vandesompele J， De Preter K， Pattyn F， et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J]. Genome Biol， 2002， 3(7): research0034.
[18] Rutledge RG， C?té C. Mathematics of quantitative kinetic PCR and the application of standard curves[J]. Nucleic Acids Res， 2003， 31(16): e93.
[19] 李文涛， 王俊东， 杨利峰， 等. 实时荧光定量PCR技术及其应用[J]. 生物技术通讯， 2006(1): 112-114.
[20] 孙秀静， 李丽， 吴建刚， 等. SYBR Green I实时荧光定量PCR检测人C-MYC基因方法的建立[J]. 首都医科大学学报， 2009.
[21] Barrick CJ， Yu M， Chao HH， Threadgill DW. Chronic pharmacologic inhibition of EGFR leads to cardiac dysfunction in C57BL/6J mice. Toxicology and Applied Pharmacology， 2008， 228(3): 315–325..
[22] Guo S， et al. Epidermal growth factor receptor-dependent maintenance of adult cardiac function. Cardiovascular Research， 2022， 118(5): 1276–1290.
[23] Tao G， Chityala PK. Epidermal growth factor receptor inhibitor-induced diarrhea: clinical incidence， toxicological mechanism， and management. Toxicology Research， 2021， 10(3): 476–486.
[24] Borkar DS， Lacouture ME， Basti S. Spectrum of ocular toxicities from epidermal growth factor receptor inhibitors and their intermediate-term follow-up: a five-year review. Supportive Care in Cancer， 2013， 21(4): 1167–1174.
基本信息:
中图分类号:S854
引用信息:
[1]阿依提肯·帕热哈提,潘英,汤傲星,等.猫源EGFR基因绝对荧光定量检测方法的建立及其组织表达分析[J].经济动物学报().
基金信息:
国家重点研发计划项目（2023YFD1800705）
2026-05-25
2026-05-25
2026-05-25