课题背景：课题为国家自然科学基金资助项目（30470456），主要进行组织工程人工血管的研究。目前，课题组已将人工血管内皮化，并在内皮细胞种植前先铺种平滑肌细胞，使血管结构更接近生理结构。为了提高血管的抗血栓能力和抑制内膜增生，分别在内皮细胞和平滑肌细胞中转入内皮型一氧化氮合酶和组织型纤溶酶原激活物基因。将转染了基因的细胞作为种子细胞，联合种植在人工血管上，提高人工血管长期通畅率已取得初步的成功。

应用要点：实验采用假型反转录病毒载体进行基因转染，使细胞内基因的转染率更高，持续时间更长，使应用基因修饰人工血管技术最终应用于临床成为可能。在平滑肌细胞中导入内皮型一氧化氮合酶基因，调控平滑肌细胞的增殖，防止内膜增生，从而提高人工血管的长期通畅率。

相关链接：理想的人工血管应该有良好的组织相容性、抗血栓性、物理稳定性、抗感染性和易使用性。为了提高人工血管的血液和组织相容性，人们采用具有很好微孔效应的人造材料，将人造血管内皮化，在人工血管上预衬纤维粘连蛋白或种植平滑肌细胞提高内皮细胞黏附率，利用组织型纤溶酶原激活物、内皮型一氧化氮合酶、lacZ等基因修饰人工血管，增强人工血管的抗血栓和抗内膜增生能力。提高人工血管临床治疗效果的手段多种多样，无论从形态还是功能上，越接近生理血管的人工血管临床效果就越好，未来的研究发展中也得从这个方向出发。

摘要
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30，100 d 后X-gal染色及苏木精-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30 d，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）；100 d后，内皮型一氧化氮合酶转染组内膜厚度与正常细胞组无明显差异，与LacZ转染组相比较，差异显著（P < 0.05）。
结论：内皮型一氧化氮合酶基因转染抑制了种植平滑肌细胞的人工血管内膜增生。
关键词：人工血管；平滑肌细胞；转染；内皮型一氧化氮合酶基因；内膜增生；组织工程化血管；组织构建

裴斐，李俊彦，张莉，何蕊. 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[J].中国组织工程研究与临床康复，2008，12(7):1225-1229 [www.zglckf.com/zglckf/ejournal/upfiles/08-7/7k-1225(ps).pdf]

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

Abstract

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 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 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 days after implantation, the neointimal thickness on grafts seeded with eNOS transduced SMCs was similar to that of unseeded grafts, but was significantly thinner than those on grafts seeded with SMCs transduced with only lacZ gene (P < 0.05).
CONCLUSION: eNOS gene in the seeded cells inhibits neointimal hyperplasia in the vessel graft seeded with SMCs.

Pei F, Li JY, Zhang L, He R. Neointimal hyperplasia in the vessel grafts transfected with endothelial nitric oxide synthase gene.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(7):1225-1229(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-7/7k-1225(ps).pdf]