Abstract: BACKGROUND: Vascular endothelial growth factor receptor 2 is mainly expressed in vascular endothelial cells and plays a crucial role in angiogenesis, tissue repair, and the occurrence and development of diseases. Adeno-associated virus, due to its unique advantages, has been widely applied in mechanism research, disease modeling, and gene therapy fields.
OBJECTIVE: To construct an adeno-associated viral vector for vascular endothelial growth factor receptor 2 gene knockdown in rat muscle tissue, determine the knockdown efficiency of vascular endothelial growth factor receptor 2 gene-knockdown adeno-associated virus in the rat sternocleidomastoid muscle and assess its effects on muscle and blood vessels.
METHODS: The vector was constructed, and packaged into adeno-associated virus. Following target screening experiments in Sprague-Dawley rats, immunofluorescence assays were conducted to assess viral infection efficiency and vascular endothelial growth factor receptor 2 protein expression. Quantitative real-time PCR was used to measure vascular endothelial growth factor receptor 2 mRNA expression. Ultimately, the optimal shRNA sequences were determined to be Y29478 and the control sequence Y9957. Twelve Sprague-Dawley rats were randomly divided into an adeno-associated virus group and a control adeno-associated virus group for functional validation. Adeno-associated virus was injected into the rat splenius capitis muscle. Twenty weeks later, fluorescent quantitative PCR and western blot were used to detect vascular endothelial growth factor receptor 2 mRNA and protein expression in the neck muscles. Hematoxylin-eosin staining was used to measure the cross-sectional area of muscle fibers in the neck muscles, and CD31 immunohistochemistry was used to detect the number of microvessels in the neck muscles.
RESULTS AND CONCLUSION: (1) The shRNA sequence and adeno-associated virus dose that could knock down the expression of vascular endothelial growth factor receptor 2 were successfully screened, and the vascular endothelial growth factor receptor 2 knockdown efficiency of rat muscle tissue after in-situ injection of adeno-associated virus with vascular endothelial growth factor receptor 2 reached 60%. (2) Compared with the control adeno-associated virus group, the mRNA and protein expression of vascular endothelial growth factor receptor 2, the muscle fiber area and the number of microvessels in the adeno-associated virus group decreased. (3) A rat model of vascular endothelial growth factor receptor 2 knockdown was successfully established. Knockdown of vascular endothelial growth factor receptor 2 in muscle tissue resulted in reduced muscle fiber cross-sectional area and decreased microvessel number. 

Key words: vascular endothelial growth factor receptor 2, adeno-associated virus, gene knockdown, in-situ injection, angiogenesis, muscle fiber area, muscle vessel