磷酸化修饰的反义TGF-β1寡核苷酸抑制血管损伤后内膜增殖

 目的：探讨反义转化生长因子β1（TGF-β1)寡核苷酸对血管损伤后血管平滑肌细胞（VSMCs）表型转变的影响及对内膜增殖的作用。方法：在跨过TGF-β1 cDNA序列的起始密码区ATG范围内，设计含15个碱基、无修饰或硫代磷酸化修饰的反义TGF-β1寡核苷酸（AS TGF-β1）及对照的正义（与反义寡核苷酸互补）寡核苷酸（S TGF-β1）。球囊导管损伤SD大鼠颈总动脉，术后7 d取出正常或损伤的血管进行VSMCs的培养,RT-PCR及蛋白印迹分别检测VSMCs生物学标志平滑肌22α蛋白（SM22α）、基质Gla蛋白和骨桥蛋白，以及TGF-β1和纤维连结蛋白（fibronectin，FN）mRNA和蛋白质的表达。采用ALZET 泵皮下注射硫代磷酸化修饰AS TGF-β1及S TGF-β1（90 μg·kg-1·d-1），连续给药28 d后测定血管内膜与中膜的面积比（I/M）。结果：（1）AS TGF-β1对血管损伤后VSMCs TGF-β1 mRNA表达并无明显作用，但呈浓度依赖性抑制TGF-β1蛋白质的表达，而S TGF-β1不影响TGF-β1mRNA和蛋白质的表达。（2）AS TGF-β1呈浓度依赖性抑制血管损伤后VSMCs DNA的合成，而不管是AS TGF-β1还是S TGF-β1对正常VSMCs DNA的合成均无明显的量效作用；同时AS TGF-β1显著抑制了血管损伤后VSMCs FN的合成。（3）AS TGF-β1显著促进了VSMCs SM22α mRNA的表达，但抑制了基质Gla蛋白和骨桥蛋白mRNA的表达，这种相反的作用在001及01 μmol/L的水平最为明显。（4）硫代磷酸化修饰的AS TGF-β1治疗28 d后，显著抑制了颈动脉损伤后新生内膜的增殖，I/M比对照组下降了68%。结论：AS TGF-β1特异性抑制了VSMCs TGF-β1蛋白的表达，抑制血管损伤后VSMCs增殖及合成、分泌FN，减轻血管损伤后新生内膜的增殖。上述作用可能与逆转血管损伤后VSMCs的表型转变有关。

Phosphorothioate-modified antisense TGF-β1 oligodeoxynucleotide inhibits neointimal hyperplasia after vascular balloon injury in rats

AIM: To evaluate the effects  of antisense TGF-β1 oligodeoxynucleotide (AS TGF-β1) on the expression of TGF-β1, deposition of extracellular matrix (ECM) and the neointima formation in the arteries after balloon injury. METHODS: The unmodified and  phosphorothioate-modified AS TGF-β1 which containing 15 bases  and surrounding the initiation codon region (ATG) of rat TGF-β1 complementary DNA (cDNA) were designed. At the same time, sense TGF-β1 oligodeoxynucleotide (S TGF-β1) with the base sequence complement to AS TGF-β1 was synthesized as a control. The oligodeoxynucleotides were introduced into in vivo and in vitro experiments, respectively. RESULTS: The AS TGF-β1 significantly inhibited the protein expression of TGF-β1 in a concentration-dependent manner, and S TGF-β1 did not have the same effect. Furthermore, no effect of the AS TGF-β1 on the mRNA expression of TGF-β1 in injured VSMCs was observed. Moreover, for the injured VSMCs, AS TGF-β1 significantly and concentration-dependently inhibited the basal DNA synthesis. Both AS TGF-β1 and S TGF-β1 did not exhibit dose-dependent effects on DNA synthesis in uninjured VSMCs. Fibronectin (FN) mRNA expression in injured VSMCs was significantly decreased by AS TGF-β1 in a concentration (001~1 μmol/L)-dependent manner. AS TGF-β1 significantly increased the mRNA expression of contractile marker SM22α, and decreased the mRNA expression of synthetic markers osteopontin and matrix Gla, especially at the concentration of 001 μmol/L and 01 μmol/L. After treatment with AS TGF-β1 (90 μg·kg-1·d-1) for 28 d, the neointima formation was significantly inhibited, and the area ratio of intima/media was markedly decreased by 68% compared with untreated group, but S TGF-β1 had no effect on neointimal formation. CONCLUSION：The AS TGF-β1 specifically inhibits the protein expression of TGF-β1 in the VSMCs derived from injured arteries. Moreover, it significantly inhibits DNA synthesis and cell proliferation, and decreases the expression of FN. Therefore, AS TGF-β1 dramatically attenuates neointima formation after balloon njury. The effects of AS TGF-β1 on the injured VSMCs may be associated with its reverse effects on the alteration of VSMC phenotype after balloon injury.