体外培养下可持续分泌人血管抑素和内皮抑素基因工程细胞对血管生成的抑制效应  

Anti-angiogenesis effects of human angiostatin and endostatin persistently secreted by in vitro genetically engineered cells

作　　者：潘静坤[1] 赵卉[2] 田磊[1] 崔忻[1] 高宇红[1] 薛毅珑[1] 

机构地区：[1]解放军总医院老年医学研究所细胞生物学研究室,北京市100853 [2]解放军总参谋部管理保障部中心门诊部,北京市100034

出　　处：《中国组织工程研究与临床康复》2009年第20期3886-3889,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:以往实验构建了可持续分泌人血管抑素和内皮抑素的基因工程细胞,即人血管抑素/293细胞和人内皮抑素293细胞,并已证明可持续分泌人血管抑素蛋白和人内皮抑素蛋白。但尚不了解其分泌物是否能发挥血管生成的抑制作用。目的:观察以基因工程技术构建的人血管抑素/293细胞和人内皮抑素/293细胞对鸡胚尿囊膜血管新生的抑制效应。设计、时间及地点:随机对照动物实验,于2006-06/2007-01在解放军总医院老年医学研究所细胞生物学研究室完成。材料:Hek/293细胞为人胚胎肾细胞,由解放军军事医学科学院提供。人血管抑素/293细胞和人内皮抑素/293细胞由课题组前期构建。SPF级鸡胚购自北京维通利华实验动物有限公司。方法:将鸡胚消毒后,置于37℃无菌恒温箱中孵育5d,在距胚头1cm两条卵黄静脉之间的卵壳投影部位磨切暴露尿囊膜,制成假气室,用无菌滤纸封闭窗口,制成鸡胚尿囊膜模型,备用。用灭菌蒸馏水配制体积分数为0.5%甲基纤维素溶液,制成甲基纤维素小杯。卵壳开窗后第3天,分别吸取浓缩的293细胞、人血管抑素/293细胞、人内皮抑素/293细胞培养上清液各20μL,或人血管抑素/293细胞和人内皮抑素/293细胞培养上清液各10μL,加于甲基纤维素小杯中,将其置于鸡胚尿囊膜的两条大血管之间的无血管区。主要观察指标:观察尿囊膜血管生成的变化。结果:共培养9d时,与单纯293细胞上清液组相比,人血管抑素/293细胞上清液或人内皮抑素/293细胞上清液组,甲基纤维素小杯内及邻近区的鸡胚尿囊膜血管明显发育不良,血管分布稀疏,数量明显减少,其一级血管数量虽未明显减少,但其管径较细,而二级血管和三级以下的血管数量明显减少、管径明显变细,伸入尿囊膜边缘区的血管数量减少,管径明显变细。与人血管抑素/293细胞组或人内皮抑素/293细胞组相比,人血管抑素/29BACKGROUND： The human angiostatin （hAS）/293 and human endostati （hES）/293 cells, which has been constructed previously could secrete human angiostatin and endostatin proteins persistently in vitro. However, it is still unclear whether the secreted cells can inhibit angiogenesis. OBJECTIVE： To observe the inhibitory effects of hAS/293 and hES/293 cells on angiogenesis in chick chorioallantoic membrane （CAM）. DESIGN, TIME AND SETTING： Randomized controlled animal experiment was conducted in Laboratory of Cell Biology, Institute of Geriatrics, General Hospital of PLA from June 2006 to January 2007. MATERIALS： Hek/293 cells were human embryonic kidney cells, which were supplied by the Academy of Military Medicine; hAS/293 and hES/293 cells were constructed previously. CAM was purchased from Merial Vital Rever Experimental Animal Co., Ltd. METHODS： Constructing the CAM model by exposing CAM on the shell subpoint between 2 vitelline veins and generating an air chamber after 5 days of incubated with aseptic incubator at 37 ℃. The methyl cellulose cup was made with 0.5% methyl cellulose solution. Putting 10 μL concentrated culture supernatant of hAS/293, hES/293 and 293 cells or 20 μL culture supernatant on the avascular area of CAM at the 3＇d day of shell exposed. MAIN OUTCOME MEASURES： Changes of CAM angiogenesis. RESULTS： At days 9 of culture, compared with control 293 group, there was apparent angiodysplasia around the cup area of hAS/293 and hES/293 groups, with sparse distribution, decreased quantity, thinner caliber of Grade 1 blood vessels, as well remarkably decreased number and caliber of Grade 2 blood vessel and Grade 3 below blood vessels. When applied combination supernatants of hAS/293 and hES/293, numbers of Grade 2 and Grade 3 below vessels decreased apparently, and blood vessel trees disappeared. CONCLUSION： The secreted hAS/293 and hES/293 cells have apparent inhibitory effect on angiogenesis on CAM, and this inhibitory effect is enhanced when applied jointly.

关 键 词：人血管抑素 人内皮抑素 基因工程细胞 鸡胚尿囊膜 血管新生 

分 类 号：R349.83[医药卫生—基础医学]