tTG反义寡核苷酸对小梁细胞tTG表达的抑制作用

Inhibiting effect of tTG antisense oligodeoxynucleotides on its expression of trabecular meshwork cells

目的通过计算机程序模拟组织型谷氨酰胺转移酶(tissue transglutaminase,tTG)基因的mRNA二级结构,优化硫代反义寡核苷酸(antisense oligodeoxynucleotide,A-SODN)药物设计,为有效抑制小梁细胞表达tTG提供新的途径。方法根据牛tTG mRNA的二级结构,选择环与茎结合区(280～300bp)和茎区(2720～2740bp)作为反义药物作用的靶点,分别合成ASODN1、ASODN2,用脂质体包埋后转染牛眼小梁细胞。通过半定量RT-PCR法检测转染后tTG mRNA表达的变化来评价tTG-ASODN的生物学效应。结果ASODN1、ASODN2对tTG mRNA的表达均有抑制作用,与空白对照组相比差异有显著性(P<0.05),且tTG-A-SODN1抑制作用明显强于tTG-ASODN2(P<0.05)。结论选择环与茎连接的部位作为反义作用的靶点,其反义作用明显强于茎区。从机制上讲,可能是因为环与茎连接的部位的形成需要吸收能量,是整个tTG mRNA序列中最不稳定的区域,ASODN容易接近而发挥作用,提示通过tTG mRNA二级结构的模拟而选择ASODN是设计反义药物的新途径。

Objective According to the secondary structure of tissue transglutaminase （tTG）, design and select the best antisense oligodeoxynucleotide （ASODN） and to reduce the expression of tTG in cultured bovine trabecular meshwork cells. Methods We selected two bands which were complementary to the combined position of ring and caudex （ASODN1, 280-300bp） and the caudex region （A- SODN2, 2720-2740bp）. Both were designed, synthesized, pbosphorothioated and embedded in Lipofectamine to transfect bovine trabocular meshwork cells. Through measuring the expression of tTG in mRNA level by semi-Quantitative RT-PCR, we estimate the inhibiting effect of tTG-ASODN. Results The tTG-ASODN were significantly decreased the expression of tTG, but the inhibiting effect of tTG-ASODN1 was stronger than tTG-ASODN2. Moreover, there had significantly difference. Conclusion The ASODN1 which was located on the combined position of ring and candex is better than the ASODN2. The reason is that that position was very unstable because of its wasting energy for maintaining the ring structure, inducing the band to approximate mRNA of tTG easily. So this study may be exploring a new way for designing anti-tTG through the secondary structure of tTG.