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

背景：平滑肌细胞增殖移行和血小板激活导致血栓形成是移植皿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可以抑制种植平滑肌细胞的人工血管内膜增生还未得到证实。 目的：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。 设计、时间及地点：重复观察测量，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。 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．随机分成3组，正常对照组植入未转染的人工血管．LacZ转染组植入种植转染lacZ的平滑肌细胞的人工血管，内皮型一氧化氮合酶转染组植入种植内皮型一氧化氮合酶的平滑肌细胞的人工血管。6只/组。 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。 主要观察指标：瓜氨酸法检测细胞培养上清中一氧化氮含量。移植血管内膜X-gal染色观察，种植的平滑肌细胞为蓝色，而内源性细胞为红色。显微镜下测量血管内膜增生厚度。 结果：18只兔均进入结果分析。内皮型一氧化氮合酶转染组一氧化氮含量明显高于正常对照组（P〈0．05）。转染lacZ基因的平滑肌细胞经X-gal染色呈蓝色。血管平滑肌细胞植入30d，各组内膜厚度基本相似（P〉0．05）：植入100d．正常对照组与内皮型一氧化氮合酶转染组内膜厚度基本相似（P〉0．05），两组明显均低于lacZ转染组（P〈0．05）。 结论：内皮型一氧化氮合酶基因转染可抑制种植平滑肌细胞的人工血管内膜增生。

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

BACKGROUND: 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 neointimal hyperplasia in graft seeded with SMCs remains uncertain.OBJECTIVE: This study was designed to further investigate the effect of eNOS gene transfection on neointimal hyperplasia in the grafts seeded with SMCs.DESIGN, TIME AND SETTING: This study, a repeated observation and measurement 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.MATERIALS: One 1-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 control group,the vessel graft with no SMCs were transplanted; In SMC/lacZ group, the vessel grafts with SMCs transfected with lacZ were transplanted;In SMC/eNOS group,the vessel grafts with SMCs seeded with eNOS were transplanted.METHODS: Rabbit SMCs were transduced with pseudotyped retroviral vectors, Murine leukemia virus/vesicular stomatitis virus G glycoprotein, carrying genes coding for eNOS or lacZ gene. The SMCs then were seeded on the vessel grafts and implanted into the rabbit abdominal aorta using vessel bypass transplantation.MAIN OUTCOME MEASURES: Nitric oxide (NO) content in the supernatant of cells transfected with eNOS and lacZ gene was detected by citrulline method. The grafts were stained with X-gal to visualize the seeded cells: the seeded SMCs were stained blue,while eNOS were stained red. The thickness of the neointima on a graft was measured with a microscope.RESULTS: Eighteen rabbits were all included in the final analysis. NO content in the SMC/eNOS group was significantly higher than that in the normal control group (P<0.05). The SMCs transfected with lacZ gene showed blue after X-gal staining under the inverted microscope. Thirty 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 days after implantation,the neointimal thickness on grafts seeded with eNOS transduced SMCs was similar to that of unseeded grafts (P>0.05 ), but was significantly thinner than that on grafts seeded with SMCs transduced with only lacZ gene (P<0.05).CONCLUSION: eNOS gene transfection inhibits nenintimal hyperplasia in the vessel graft seeded with SMCs.