摘要
目的:构建猪活化转录因子6-短发夹RNA(ATF6-shRNA)重组腺病毒干扰载体,观察其对猪瓣膜间质细胞(VIC)增殖与凋亡的影响。方法:针对猪ATF6序列设计3个shRNA干扰靶点,构建质粒,包装后进行病毒扩增,使用腺病毒转染VIC,通过定量PCR检测腺病毒对VIC中ATF6的干扰效率,通过Western blot法测定下调ATF6后VIC中胱天蛋白酶(caspase)-3的表达情况,并通过CCK-8法检测VIC增殖情况。结果:成功构建靶向ATF6的shRNA重组腺病毒载体。腺病毒转染VIC后,腺病毒对VIC中ATF6干扰效率提高;而 ATF6、 caspase-3表达显著降低;自第2天起VIC存活率升高,差异均有统计学意义( P 均<0.05)。结论:成功构建靶向ATF6的shRNA重组腺病毒载体,通过降低VIC中ATF6的表达水平,减少VIC凋亡并促进细胞存活。
Objective:To construct the short hairpin RNA (shRNA) recombinant adenoviral vector targeting the activating transcription factor 6 (ATF6),and explore its effect on the proliferation and apoptosis of porcine valve interstitial cells (VIC). Methods:We identified 3 shRNA target sites based on the porcine ATF6 gene sequences.Then the plasmid was constructed and packaged for virus amplification.The interference efficiency was detected by quantitative PCR after porcine VIC were transfected by adenovirus vector carrying ATF6-shRNA,and the expression of caspase-3 in VIC was measured by western blot when down-regulating the ATF6.Furthermore,CCK-8 method was used to measure proliferation of VIC. Results:The recombinant adenovirus vector targeting ATF6,with high interference efficiency was constructed successfully.The expression of ATF6 and caspase-3 significantly decreased,and the survival rate of VIC increased since day 2 with statistical significance (all P <0.05). Conclusions:The recombinant adenovirus vector targeting ATF6 could be constructed successfully,which significantly reduces the expression of ATF6 in VIC,and decreases the apoptosis of VIC to promote their survival rate.
作者
田军
刘晓红
韩林
TIAN Jun;LIU Xiaohong;HAN Lin(Department of Cardiothoracic Surgery,Changhai Hospital,Navy Military Medical University,Shanghai 200433,China)
出处
《国际心血管病杂志》
2019年第3期142-145,共4页
International Journal of Cardiovascular Disease
基金
国家自然科学基金(81770383,81570351)
关键词
短发夹RNA
活化转录因子6
重组腺病毒载体
瓣膜间质细胞
凋亡
Short hairpin RNA
Activated transcription factor 6
Recombinant adenovirus vector
Valve interstitial cells
Apoptosis
