内皮型一氧化氮合酶基因转染对移植人工血管内膜增生的作用

目的：平滑肌细胞增殖移行和血小板激活导致血栓形成是移植血管再狭窄的主要原因，一氧化氮可以抑制上述生物反应，但内皮型一氧化氮合酶基因转染是否抑制种植了平滑肌细胞的人工血管内膜增生还未得到证实。实验拟进一步观察内皮型一氧化氮合酶基因转染对种植平滑肌细胞的人工血管内膜增生的影响。 方法：实验于2006-04／2007—05在西安交通大学医学院中心实验室及分子生物学实验室完成。①实验材料；1月龄新西兰大白兔1只，用来获取平滑肌细胞。成年新西兰大白兔18只，随机数字表法分成3组，每组6只。正常细胞组移植未种植细胞的人工血管；LacZ转染组移植种植转染lacZ的平滑肌细胞的人工血管，内皮型一氧化氮合酶转染组移植种植内皮型一氧化氮合酶的平滑肌细胞的人工血管。②实验方法：构建含有报道基因lacZ和内皮型一氧化氮合酶基因的假型反转录病毒载体小鼠白血病病毒，疱疹性口炎病毒G糖蛋白，并转染平滑肌细胞。将转染了基因的细胞种植在人工血管上，并用血管旁路移植的方法植入兔腹主动脉。③实验评估：测定转染内皮型一氧化氮合酶基因及LacZ基因细胞培养上清中一氧化氮含量。血管植入30，100d后X—gal染色及苏木精一伊红染色观察人工血管上的平滑肌细胞，同时显微镜下测量每段血管内膜增生的厚度。 结果：纳入成年新西兰大白兔18只，均进入结果分析。①内皮型一氧化氮合酶转染组一氧化氮含量明显高于未转染的正常细胞组（P〈0．05）。平滑肌细胞转染lacZ基因后经X—gal染色，倒置显微镜下可见转染了基因的细胞被染成蓝色。②血管植入30d，与正常细胞组比较，LacZ转染组和内皮型一氧化氮合酶转染组内膜厚度差异无显著性（P〉0．05）；100d后，内皮型一氧化氮合酶转染组内膜厚度与正常细胞组?

Neointimal hyperplasia in the vessel grafts transfected with endothelial nitric oxide synthase gene

AIM： Smooth muscle cells （SMCs） proliferation and transmigration and platelet activation cause thrombogenesis and lead to grafted vessel restenosis. Nitric oxide （NO） can inhibit the above-mentioned biological responses, but whether endothelial nitric oxide synthase （eNOS） gene transfection can inhibit the proliferation of neointimal hyperplasia with grafted SMCs is still uncertain. This study observed the effect of eNOS on neointimal hyperplasia seeded with SMCs.
METHODS： The experiment was performed at the Central Laboratory and Laboratory of Molecular Biology of Xi＇an Jiaotong University Medical College from April 2006 to May 2007. ①One l-month-old New Zealand rabbit was used to acquire SMCs. Another 18 adult New Zealand rabbits were randomly divided into 3 groups （n=6）. In normal cell group, the vascular prosthesis with no SMCs were transplanted; in SMC/lacZ group, the vascular prosthesis with SMCs transfected with lacZ were transplanted; in SMC/eNOS group, the vascular prosthesis with SMCs seeded with eNOS were transplanted, ②Rabbit SMCs were transduced with pseudotyped retroviral vectors carrying genes coding for eNOS or lacZ gene. The SMCs then were seeded on the vascular prosthesis and implanted into the rabbit abdominal aorta using vessel bypass transplantation. ③NO content in the supernatant of cells transfected with eNOS and lacZ gene. The grafts were stained with X-gal and HE to visualize the seeded cells after 30 and 100 days of implanted. The thickness of the neointima on a graft was measured with a microscope.
RESULTS： Eighteen rabbits were all included in the final analysis. ①NO content in the SMC/eNOS group was significantly higher than that in the normal cell group （P 〈 0.05）. The SMCs transfected with lacZ gene showed blue after X-gal staining under the inverted microscope. ②Thirty days after implantation, there was no difference in neointimal thickness between normal grafts and grafts seeded with eNOS or lacZ transduced SMCs （P 〉 0.05）. 100 day