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FAK靶向RNAi重组体的构建及其抗多细胞球体形成效应

, PP. 1633-1636

Keywords: 黏着斑激酶,RNA干扰,构建,多细胞球

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Abstract:

目的构建RNA干扰(RNAi)重组体抑制黏着斑激酶(FAK)表达,并探讨其对结肠癌多细胞球体(MCSs)形成的影响。方法针对FAKcDNA序列设计并合成1对特异性的含有短发卡的寡核苷酸序列及其对照序列,经退火后插入pGenesil-1中构建重组体。经双酶切鉴定和DNA测序后,用脂质体2000将其转染结肠癌HT29细胞,并用G418稳定筛选,采用Real-timePCR和Westernblot检测干扰前后FAK在HT29内的表达变化,并作细胞悬浮培养观测靶向干扰FAK对MCSs形成的影响。结果双酶切和测序鉴定证实插入序列完全正确;靶向干扰FAK后,其mRNA和蛋白表达分别显著下调(79.20±2.97)%和(78.47±4.39)%,且细胞不易聚集形成MCSs。结论成功构建FAK靶向RNAi重组载体,显著地抑制FAK表达,并能有效地抑制MCSs的形成。

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