RNA干扰技术抑制人内皮细胞非肌肉肌球蛋白重链ⅡA（MYH9）表达.docVIP

RNA干扰技术抑制人内皮细胞非肌肉肌球蛋白重链ⅡA（MYH9）表达 　 作者：黄娅琳, 寇俊萍, 宋佳希, 余伯阳 【摘要】 　　目的: 构建人MYH9（非肌肉肌球蛋白重链ⅡA）的siRNA 表达体系, 抑制原代人脐静脉内皮细胞（HUVEC）MYH9 基因的表达。方法: 针对人MYH9 基因序列, 构建siRNA 表达质粒, 通过酶切和测序鉴定确定其序列正确性。分别转染重组质粒pGCsiMYH91/2/3 至HUVEC 细胞, 通过半定量RTPCR 和蛋白质印迹, 分别检测在转染后24 h、 48 h、 72 h MYH9 在mRNA 转录水平及蛋白表达的变化。结果: 在构建的3种重组质粒中, pGCsiMYH92、 pGCsiMYH93在转染后24 h 均表现出一定的干扰作用, 转染后48 h干扰作用最强, 转染后72 h 干扰作用减弱; pGCsiMYH93的干扰作用最明显, 转染后48 h, 能明显下调HUVEC 的MYH9 蛋白表达, 抑制率平均可达71.2%, 此时MYH9 基因mRNA 转录明显减少, 抑制率为86.5%。结论: 在转染后48 h pGCsiMYH93 可明显抑制HUVEC 细胞内源MYH9 的表达, 为进一步研究MYH9在相关疾病中的功能奠定基础。 　　AIM: To construct the expression vector of small hairpin RNA (shRNA) and to investigate its efficacy in silencing nonmuscle myosin heavy chain ⅡA (MYH9)gene. METHODS: According to the MYH9 cDNA sequence in GenBank, three shRNA expression vectors targeting MYH9 gene were constructed and were confirmed by digestion with restriction enzymes and DNA sequencing. The recombinant plasmids were transfected into HUVEC cells. The expression of MYH9 was determined by semiquantitative RTPCR at mRNA level and by western blotting at protein level at 24 h, 48 h and 72 h after transfection. RESULTS: The decrease of MYH9 expression at mRNA and protein levels gradually became more evident from 24 h to 48 h, and achieved the maximal degree at 48 h, then became weakened and restored at 72 h. The efficiency of pGCsiMYH93 was the best among these three plasmids. The introduction of pGCsiMYH93 was showed to efficiently and specifically inhibit the expression of MYH9 with inhibitory rate at 71.2% according to results of Western blot. RTPCR results showed that mRNA transcription of MYH9 gene was reduced by 86.5% at 48 h after introduction of pGCsiMYH93. CONCLUSION: Plasmid pGCsiMYH93 could inhibit MYH9 expression in HUVEC cells successfully and effectively, which promised its further application in related function analysis of MYH9. 【关键词】 RNA 干扰; 人脐静脉内皮细胞（HUVEC）; 非肌肉肌球蛋白重链ⅡA（MYH9） 　　非肌肉肌球蛋白重链ⅡA（nonmuscle myosin heavy