Abstract:

Objective The research aimed to investigate whether KLF4⁃shRNA carried by adeno⁃associated virus type 1（AAV1）injected into the airway of rats can effectively construct an animal model of pulmonary vascu⁃ lar gene knockdownand its application in pulmonary hypertension. Methods According to the rats′ KLF4 gene sequence，the rat⁃specific siRNA sequence was designed and verified by cell count and cell migration expriments and then the AAV1⁃KLF4⁃shRNA adeno⁃associated virus vector was constructed.A total of 44 rats were randomly assigned to four groups：sham group，saline group，AAV1⁃control vector group and AAV1⁃KLF4⁃shRNA group. The last three groups of rats were injected with normal saline，AAV1⁃carried control vector，and AAV1⁃KLF4⁃shR⁃ NA in the trachea，respectively. After 1 month，the latter 3 groups were whole body exposed to CS for 4 months of smoke. After each group of rats was anesthetized，hemodynamic testing was carried out，the expression of viral vectors in pulmonary blood vessels was observed by immunofluorescence assays，the level of pulmonary vascular remodeling was evaluated by HE staining，and the KLF4 level in pulmonary arteries was measured by PCR method. Then we harvested rats′ heart to calculate right heart hypertrophy index. Results Cell counting and cell migration experiments confirmed that the selected KLF4 ⁃ siRNA strands were effective. Immunofluorescence experiments showed that adeno⁃associated virus vector type 1 injected through the airway of rats was distributed along the pulmo⁃ nary blood vessels. Fluorescence quantitative PCR experiments showed that the mRNA expression of KLF4 in thepulmonary arterioles of the AAV1⁃KLF4⁃shRNA group was significantly lower than other two model groups. Hemo⁃ dynamic test results showed that the right ventricular systolic blood pressure and mean right ventricular pressure in the two groups of pulmonary hypertension models were significantly higher than those in the healthy control group， while the right ventricular systolic blood pressure and mean right ventricular pressure in rats undergoing knockdown of the pulmonary vascular KLF4 gene were significantly lower than those in both model groups. The HE staining and the right heart hypertrophy index results showed that the degree of pulmonary small vessel remodeling and the ventricular remodeling in the two groups of pulmonary hypertension models were significantly higher than those in the healthy control group，while the degree of pulmonary small vessel remodeling and the ventricular remodeling in rats undergoing knockdown of the pulmonary vascular KLF4 gene were significantly lower than those in both model groups. Conclusion The successful construction of a pulmonary vascular ⁃ specific KLF4 gene knockdown model and the successful use in pulmonary hypertension provide a platform and experimental basis for further in ⁃ depth study of the pathogenesis of pulmonary hypertension.

Key words:

pulmonary hypertension, gene knockdown, adeno?associated virus 1, KLF4