课题背景：课题为广东省自然科学基金资助项目(06301417)；广东省卫生厅资助项目(A2006726)。本实验拟通过基因工程与组织工程的有机结合来获得新生骨组织。实验结果表明转染人骨形态发生蛋白2较未转染在体内形成新骨能力明显增强，人骨形态发生蛋白2基因修饰的自体细胞复合支架材料，改善成骨环境促进骨愈合，加速了组织工程化骨的形成。

同行评价：联合应用基因工程和组织工程技术研制具有较强生物诱导活性的组织工程骨是近5年来引人注目的研究方向。文章采用转染人骨形态发生蛋白2基因的组织工程化骨修复骨质疏松症下颌骨缺损，具有一定创新性，对相关领域研究具有一定指导意义和参考价值。

偏倚或不足：实验中虽然人骨形态发生蛋白2基因修饰的组织工程化骨植入绝经后骨质疏松症大鼠下颌骨骨缺损区，增强了其骨再生能力，但8周时的结果显示仍未达到完全的骨性愈合。究竟是在体内相关细胞外环境的改变，还是脂质体介导的基因转移效率不高所致，尚待深入观察。

摘要

目的：桡动脉作为血管桥材料在冠状动脉旁路移植术中具有重要作用，但血管桥痉挛一直是困扰桡动脉移植的难题。实验通过腺病毒载体介导的人内皮型一氧化氮合成酶基因（AdCMVeNOS）转染人桡动脉，体外培养后检测人内皮型一氧化氮合成酶基因的表达，观察其对内膜增生的影响。
方法：①实验于2005-03/04在解放军沈阳军区总医院完成。实验用Enos-Ad由该院麻醉科张铁铮主任惠赠。桡动脉来自15例冠状动脉旁路移植手术患者剩余桡动脉。将桡动脉横切成5 mm血管环，采用随机数字表法分为3组，对照组、空载腺病毒液感染组、内皮型一氧化氮合酶基因转染组，每组8份。空载腺病毒液感染组、内皮型一氧化氮合酶基因转染组桡动脉血管环分别置于空载腺病毒液和AdCMVeNOS溶液中1 h，取出后体外培养14 d。应用多聚寡核苷酸探针和高敏感标记技术检测外源性基因内皮型一氧化氮合酶mRNA的表达；免疫组织化学染色方法检测内皮型一氧化氮合酶蛋白的表达；苏木精-伊红染色及弹性纤维染色后，应用计算机图像分析系统检测桡动脉内膜、中膜增生情况。
结果：①3组培养的桡动脉在14 d后有新内膜形成和显著的中膜增厚，内皮型一氧化氮合酶基因转染组内膜厚度及内膜/中膜厚度比值低于对照组 (P < 0.01)，空载腺病毒液感染组与对照组差异无显著性 (P > 0.05)。②培养14 d后，内皮型一氧化氮合酶基因转染组内膜和中膜可检测到内皮型一氧化氮合酶mRNA和蛋白质表达，对照组及空载腺病毒液感染组未见明显表达。
结论：腺病毒载体介导的人内皮型一氧化氮合成酶基因成功转染体外培养桡动脉中并有效表达，内皮型一氧化氮合酶基因具有防治体外培养桡动脉内膜增生的作用。
关键词：桡动脉；一氧化氮合酶；腺病毒科；组织构建

陶登顺，王辉山，汪曾炜，朱洪玉，姜志明. 人内皮型一氧化氮合成酶基因转染桡动脉的表达[J].中国组织工程研究与临床康复，2008，12(18):3461-3464 [www.zglckf.com/zglckf/ejournal/upfiles/08-18/18k-3461(ps).pdf]

Expression of transfected human endothelial nitric oxide synthase gene in radial artery

Abstract

AIM: As blood vessel bridge material, radial artery plays an important role in coronary artery bypass graft (CABG), but blood vessel bridge spasm limits the application of radial artery grafting. This study measured the expression of adenoviral-mediated human endothelial nitric oxide synthase gene (AdCMVeNOS) transfected in radial artery in vivo and explored its effect on intimal hyperplasy.
METHODS: The experiment was performed at General Hospital of Shenyang Military Area Command of Chinese PLA from March to April 2005. Enos-Ad used in this study was gifted by Zhang. Radial arteries from 15 CABG patients were transected into 5 mm blood vessel rings and randomly divided into 3 groups (n=8): control group, adenoviral infection group, and eNOS gene transfection group. The radial artery rings in the latter two groups were sunk into adenoviral solution and AdCMVeNOS solution for 1 hour, respectively, and then cultured in vivo for 14 days. Expressions of eNOS mRNA were measured by in situ hybridization, and high sensitive marking technology, and expressions of eNOS protein by immunohistochemistry. Intimal and medial hyperplasy of radial artery were stained by HE and elastic fiber, and analyzed by computer graph-analyzing system.
RESULTS: ①Neointima formed and media proliferated obviously in radial artery cultured for 14 days. Intimal thickness and ratio of intimal/medial thickness was significantly lower in eNOS gene transfection group than in control group (P < 0.01), but there was no significant difference between adenoviral infection group and control group (P > 0.05). ②Expressions of eNOS mRNA and protein were found in intima and media of eNOS gene transfested radial artery after culture for 14 days. But no obvious expression was found in the other groups.
CONCLUSION: AdCMVeNOS gene is transfected into in vitro cultured radial artery successfully and expressed effectively. eNOS gene could inhibit intimal hyperplasia in cultured radial artery in vitro.

Tao DS, Wang HS, Wang ZW, Zhu HY, Jiang ZM. Expression of transfected human endothelial nitric oxide synthase gene transfection in radial artery.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(18):3461-3464(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-18/18k-3461 (ps).pdf]