前列腺环素合酶基因转移治疗兔颈总动脉球囊扩张后再狭窄实验研究

PROSTACYCLIN SYNTHASE GENE TRANSFER INHIBITS NEOINTIMAL FORMATION IN RABBIT CAROTID ARTERIES AFTER BALLOON INJURY

目的 本研究旨在探讨前列腺环素合酶 (PCS)克隆脱氧核糖核酸 (cDNA)转染对颈动脉经皮球囊扩张术 (PTA)后再狭窄的防治效果。方法 首先提取大鼠主动脉平滑肌细胞 (VSMC)总RNA ,再根据已知PCS序列通过逆转录合成PCScDNA ;然后制备脂质体复合物。在成功建立家兔颈动脉PTA模型的基础上 ,进行体内转染实验。将实验动物分为 4组 ,各 1 0只 :①空白对照组 ;②载体对照组 ,注入pCMV -LacZ 40 μg ;③pCMV -PCS - 5组 ,注入 pCMV -PCS 5μg ;④pCMV -PCS - 4 0组 ,注入 pCMV -PCS 40 μg。 2周后对新生血管内膜与血管中层比值进行形态学分析。结果 空白对照组、载体对照组、pCMV -PCS - 5组及 pCMV -PCS - 4 0组内膜与中层厚度比值 (I/M )分别为 :(1 .2 0± 0 .1 4 )、(1 .2 4± 0 .1 5)、(0 .6± 0 .1 2 )、(0 .1 4 0± 0 .0 5) ,转基因组与空白对照组、载体对照组比较 ,差异有高度显著性 (P <0 .0 1 ) ,中层厚度分别为 :(0 .1 2± 0 .0 7)mm、(0 .1 4± 0 .0 8)mm、(0 .1 3± 0 .0 9)mm、(0 .1 2± 0 .0 3)mm ,各组间差异无显著性。结论 本研究显示PCS基因转移能有效地抑制颈动脉球囊扩张血管成形术后再狭窄。其作用主要是抑制新生内膜 ,而对中层几乎没有影响。这种抑制作用与转基因量相关。

Objective To investigate the effects of local delivery of PCS gene on inhibition of neotimal formation after PTA in carotid artery. Methods Based on the obtained sequence of the rat PCS gene, total cellular RNA was extracted from rat aorta by the modified method of Chomczinski and Sacchi and subjected to reverse-transcription using a first-strand cDNA synthesis kit. The PCR product was extracted and cloned in the cytomegalovirus enhancer/promoter-directed vector. A plasmid carrying the LacZ gene substituted for the PCS gene was constructed as a control (pCMV-LacZ). Then 50μg pCMV-PCS or 40μg pCMV-PCS or 40μg pCMV-LacZ with 50μL lipofectoamin plus reagent in a total volume of 100μl were transfected into endothelium-denuded rabbit carotid arteries after arterial balloon injury and histological and morphometric analyses were performed. Results 15 days after the carotid baloon injury and transfection,the ratio of thickness of intimal and medium(I/M )of control?pCMV-LacZ?pCMV-PCS-5 and pCMV-PCS-40 was (1.20±0.14), (1.24±0.15),(0.6± 0.12),(0.140±0.05)(n=10,P<0.01 pCMV-PCS-5 or pCMV-PCS-40 vs control or pCMV-LacZ ),respectively; thickness of medium was (3.1±0.07)mm,(3.2±0.08)mm,(3.3±0.09)mm,(3.0±0.03)mm(n=10, no statistic significance among groups),respectively. Conclusion The results suggest that transfection of PCS gene may inhibit neointimal formation in vivo.