TLR4干扰RNA腺病毒的构建及其对肺泡巨噬细胞内毒素反应的影响

Construction of the adenovirus expressing rats expressing TLR4 siRNA and its effect on alveolar macrophage reaction to LPS

目的构建表达大鼠TLR4siRNA的腺病毒,并观察对内毒素诱导的大鼠肺泡巨噬细胞TLR4的抑制和TNF-α、IL-10释放的影响。方法合成双链寡核苷酸并克隆入pShuttleH1载体,pShuttleH1-TLR4线性化后电转化到含pAdEasy-1的BJ5183感受态细菌中获取重组质粒,重组质粒用脂质体转染293细胞获取重组腺病毒Ad-siTLR4;扩增、纯化腺病毒,TCID50法测定病毒滴度。原代培养大鼠肺泡巨噬细胞,腺病毒Ad-siTLR4感染后加入内毒素,采用RT-PCR和Western blot观察TLR4蛋白及mRNA的表达,ELISA检测TNF-α、IL-10的释放情况。结果目的基因序列正确并成功克隆入腺病毒,腺病毒Ad-siTLR4滴度为5.72×109pfu/mL。转染腺病毒后的内毒素诱导的肺泡巨噬细胞TLR4蛋白和mRNA水平下降,TNF-α、IL-10的表达均明显减弱。结论本研究成功构建了表达大鼠TLR4 siRNA的腺病毒,具有良好的抑制TLR4和TNF-α、IL-10的作用,为下一步动物体内实验基因治疗急性肺损伤奠定良好的基础。

Objective To construct the incompetent -replication adenovirus expressing rats TLR4 siRNA and to identify its effect on alveolar macrophage reaction to LPS （ Lipopolysaccharide ） in vitro. Methods The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was cloned into the pShuttleH1 vector. Linearized pShuttleH1 - siTLR4 was transformed into E. coli BJ5183 cells containing backbone plasmid pAdEasy- 1 by electroporation. The recombinant plasmid was transfected into 293 cells to package the adenovirus Ad - siTLR4. The titers of adenovirus were determined using the specific 50% tissue culture infection dosage （TCID50） method. After virus infected the cultured alveolar macrophage, the effect on LPS - induced TLR4 protein and its mRNA expression were observed by western blot and RT- PCR. TNF -α, IL- 10 expression were also detected by ELISA. Resuits It was identified that the sequence of gene was correctly inserted into the genome of virus. The titer of recombinant adenovirus was 5.72 × 10^9pfu/mL. TLR4 protein and its mRNA expression were greatly reduced after virus infection. TNF - α, IL - 10 expression were also decreased. Conclusion The recombinant adenovirus expressing rats TLR4 siRNA were successfully constructed,which probably can be further used in treatment of acute lung injury in vivo.