CAJ | 학술논문

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（90μg· 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 SM22αmRNA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0.1μmol/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。
목적：탐토반의전화생장인자β1（TGF-β1）과핵감산대혈관손상후혈관평활기세포（VSMCs）표형전변적영향급대내막증식적작용。방법：재과과TGF-β1 cDNA서렬적기시밀마구ATG범위내，설계함15개감기、무수식혹류대린산화수식적반의TGF-β1과핵감산（ AS TGF-β1）급대조적정의（여반의과핵감산호보）과핵감산（ S TGF-β1）。구낭도관손상SD대서경총동맥，술후7 d취출정상혹손상적혈관진행VSMCs적배양， RT-PCR급단백인적분별검측VSMCs생물학표지평활기22α단백（ SM22α）、기질Gla단백화골교단백，이급TGF-β1화섬유련결단백（fibronectin，FN）mRNA화단백질적표체。채용ALZET 빙피하주사류대린산화수식AS TGF-β1급S TGF-β1（90μg· kg-1· d-1），련속급약28 d후측정혈관내막여중막적면적비（I/M）。결과：（1）AS TGF-β1대혈관손상후VSMCs TGF-β1 mRNA표체병무명현작용，단정농도의뢰성억제TGF-β1단백질적표체，이S TGF-β1불영향TGF-β1mRNA화단백질적표체。（2）AS TGF-β1정농도의뢰성억제혈관손상후VSMCs DNA적합성，이불관시AS TGF-β1환시S TGF-β1대정상VSMCs DNA적합성균무명현적량효작용；동시AS TGF-β1현저억제료혈관손상후VSMCs FN적합성。（3）AS TGF-β1현저촉진료VSMCs SM22αmRNA적표체，단억제료기질Gla단백화골교단백mRNA적표체，저충상반적작용재0.01급0.1μmol/L적수평최위명현。（4）류대린산화수식적AS TGF-β1치료28 d후，현저억제료경동맥손상후신생내막적증식，I/M비대조조하강료68％。결론：AS TGF-β1특이성억제료VSMCs TGF-β1단백적표체，억제혈관손상후VSMCs증식급합성、분비FN，감경혈관손상후신생내막적증식。상술작용가능여역전혈관손상후VSMCs적표형전변유관。
[ABSTRACT]AIM:ToevaluatetheeffectsofantisenseTGF-β1oligodeoxynucleotide(ASTGF-β1)ontheex-pression of TGF-β1 , deposition of extracellular matrix ( ECM) and the neointima formation in the arteries after balloon inju-ry.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 ob-served.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 SM 22α, and decreased the mRNA expression of synthetic markers osteopontin and matrix Gla , especially at the concentration of 0.01μmol/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 pro-tein 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 altera-tion of VSMC phenotype after balloon injury .