摘要
目的探讨AP-1和Smad3双功能诱骗寡核苷酸对L929成纤维细胞增殖及纤维化介质表达的影响。方法设计并合成AP-1和Smad3双功能诱骗寡核苷酸、AP-1,Smad3阳性对照诱骗寡核苷酸及随机诱骗寡核苷酸,然后通过脂质体分别转染进入L929成纤维细胞内。分别通过ELISA法、细胞计数、ATP生物荧光法及免疫印迹法检测合成的诱骗寡核苷酸及其对L929细胞增殖及纤维化介质表达的影响。结果与随机诱骗核酸对照相比,双功能诱骗核苷酸既能明显抑制AP-1的DNA结合活性,又能抑制Smad3的DNA结合活性(P<0.01)。与随机诱骗寡核苷酸相比,AP-1和Smad3双功能诱骗寡核苷酸、AP-1,Smad3阳性对照诱骗核酸均能抑制TGF-β诱导的L929细胞的增殖并且降低其表达FN、PAI-1、c-fos、Collα2、TGF-β1、CTGF等纤维化介质,其中以AP-1和Smad3双功能诱骗寡核苷酸的作用最为显著。结论设计并合成的AP-1和Smad3双功能诱骗寡核苷酸可以有效地抑制L929成纤维细胞的增殖和纤维化介质的表达。
Objective To explore the effect of the dual AP - 1 and Smad decoy oligodeoxynucleotide on the proliferation and expres- sion of fibrotic mediators in L929 fibroblasts. Methods The dual AP - 1 and Smad3 decoy oligonucleotide, AP - I and Smad3 positively controlled decoy oligonucleotide, and random decoy oligonucleotide were designed and synthesized. Then they were transfected into L929 fibroblasts via lipofectamine. The dual AP - 1 and Smad3 decoy oligonncleotides was detected by ELISA. The effect of decoy oligonucleoti- des on the proliferation of L929 cells was detected by cell counting, ATP bioluminescence assay, and the effect on the expression of fibrot- ic mediators was detected by Western blot. Results Compared with random decoy oligonucleotide, the dual AP - 1 and Smad3 decoy oli- gonucleotide could inhibit both the DNA binding activities of AP - 1 and Smad3. （ P 〈 0.01 ）. Compared with random decoy oligonucleoti- de, both the dual AP - 1 and Smad3 decoy oligonucleotide and the positive control of AP - 1 and Smad3 decoy oligonucleotide inhibited the proliferation of the L929 fibroblasts stimulated by TGF - β and inhibited the expression of fibrotic mediators such as FN,PAI - 1 ,c - los, Collct2,TGF - β1, CTGF. Such effects were stronger among the group of the dual AP - 1 and Smad3 decoy oligonucleotide, when com- pared with those of the positive control group of AP - 1 and Smad3 decoy oligonucleotide. Conclusion The dual AP - 1 and Smad decoy oligodeoxynucleotide was successfully designed and synthesized, which could effectively inhibit the proliferation of fibroblast and the ex- pression of fibrotic mediators.
出处
《医学研究杂志》
2014年第3期98-102,共5页
Journal of Medical Research
