靶向敲减KLF4基因的重组腺相关病毒载体的构建及其在肺动脉高压动物模型中的应用

目的 构建靶向敲减KLF4基因的重组腺相关病毒载体，并应用于肺血管疾病，为后期研究肺血管KLF4基因敲减在肺动脉高压大鼠中的作用及相关分子机制奠定基础。方法 根据大鼠KLF4基因序列，设计针对大鼠特异性的siRNA序列，以pHBAAV-U6-MCS-CMV-EGFP作为空白载体，通过酶切技术构建带有GFP荧光标记的KLF4干扰腺病毒载体pHBAAV-r-KLF4 shRNA-GFP，行测序分析，并进行病毒扩增、纯化及滴度测定。本研究对长时间（3个月）接触香烟烟雾的大鼠进行气道注入AAV1-KLF4-shRNA的干预治疗，观察大鼠右心室收缩压、平均右心室压等血流动力学指标改变。结果 经测序检测显示，大鼠AAV1-KLF4-shRNA腺相关病毒载体构建成功。健康对照组、生理盐水模型组、对照病毒模型组、治疗干预组的右心室收缩压和平均右心室压比较，差异均有统计学意义（P <0.05）。结论 对长时间接触香烟烟雾的肺动脉高压模型大鼠应用AAV1-KLF4-shRNA腺相关病毒载体，能有效改善右心室收缩压和平均右心室压，该载体在相关疾病动物模型中应用有效，为后期顺利开展AAV1-KLF4-s...

Construction and identification of recombinant adeno-associated virus vector targeting KLF4 gene

Objective The recombinant adeno-associated virus vector with KLF4 gene knockdown was constructed and applied to pulmonary vascular diseases, which laid a foundation for the later study of the role and related molecular mechanism of pulmonary vascular KLF4 gene knockdown in rat pulmonary hypertension.Methods According to the sequence of the rat KLF4 gene, a rat-specific siRNA sequence was designed. Using pHBAAV-U6-MCS-CMV-EGFP as the blank vector, the GFP labeled KLF4 interference adenovirus vector pHBAAV-r-KLF4 shRNA-GFP was constructed by enzyme digestion. The virus was amplified, purified, and titrated. We injected AAV1-KLF4-shRNA into the airway of rats exposed to cigarette smoke for 3 months, and observed the changes in hemodynamic indexes such as right ventricular systolic pressure and mean right ventricular pressure.Results The AAV1-KLF4-shRNA adeno-associated virus vector was constructed successfully. The application of adeno-associated virus vector in pulmonary hypertension rats modeled by cigarette smoke can effectively ameliorate hemodynamic indexes such as right ventricular systolic pressure and mean right ventricular pressure (P < 0.05).Conclusion AAV1-KLF4-shRNA adeno-associated virus vector can be successfully constructed, which plays an important role in the treatment of pulmonary hypertension animal model in the study.