腺病毒介导的重组Tum5基因对生理状态下脐静脉血管内皮细胞生物学行为的抑制作用

Inhibition of adenovirus-mediated recombinant Tum5 gene overexpression on human umbilical vein endothelial cells in physiological status

背景肿瘤抑素是迄今发现活性最强的内源性血管生成抑制因子，对病理性新生血管形成有明显抑制作用。Tum5片段为肿瘤抑素的抗血管生成活性片断。目的研究腺病毒介导的Tum5重组基因过表达对生理状态下人脐静脉内皮细胞（HUVECs）增生、迁移及管腔形成的影响。方法构建表达绿色荧光蛋白的空载体腺病毒（rAd—GFP）和携带重组几m5基因的腺病毒载体（rAd—Tum5）。将HUVECs分为正常对照组、空载体组（rAd-GFP组）和Tum5基因组（rAd-GFP—Tum5组）。将rAd—GFP和rAd—Tum5病毒颗粒（1×10^10／m1）各20μl分别加入rAd—GFP组和rAd—GFP—Tum5组培养液以感染培养的细胞48h，倒置荧光显微镜下观察各组细胞中GFP的表达，并计算病毒的感染效率；采用细胞计数试剂盒（CCK）-8检测各组细胞在波长为450nm处的吸光度（A）值并计算细胞增生率；采用Transwell小室实验测定各组的迁移细胞数目；采用基质胶（Matrigel）实验检测各组细胞的管腔形成数；采用人血管内皮生长因子（VEGF）ELISA试剂盒检测细胞感染后24、48和72h各组细胞培养上清液中VEGF质量浓度。结果倒置荧光显微镜下可见rAd—GFP组和rAd—GFP—Tum5组HUVECs中呈绿色荧光，rAd—GFP组和tad—GFP—Tum5组的感染效率分别为55．13％和50．31％。细胞感染后24h和48h，正常对照组、rAd—GFP组和rAd-GFP—Tum5组细胞增生率比较差异均无统计学意义（均P〉0．05）；细胞感染后72h，rAd-GFP—Tum5组细胞增生率明显低于正常对照组和rAd—GFP组，差异均有统计学意义（均P〈0．01）。细胞感染后48h，正常对照组、rAd—GFP组和rAd—GFP—Tum5组迁移细胞数分别为（2260．25±930．44）、（2370．00±441．06）和（723．75±363．80）个，总体比较差异有统计学意义（F=8．524，P=0．008），rAd—GFP—Tum5组迁移细胞数量较正常对照组和rAd—GFP组均明显减少，差异均有统计学意义（均P〈0．01）。正常对照组、rAd—GFP组和rAd-GFP—Tum5组平均每张图片中管腔形成数目分别为（95．67±5．86）、（88．00±4．58）和（20．67±3．51）个，总体比较差异有统计学意义（F=226．498，P〈0．01），其中rAd—GFP—Tum5组细胞管腔形成数量较正常对照组和rAd—GFP组明显减少，差异均有统计学意义（均P〈0．01）。细胞感染后24、48和72h，各组细胞培养上清液中VEGF蛋白质量浓度总体比较差异均有统计学意义（F分组 =73．260，P〈0．01；F时间=73．477，P〈0．01）；其中感染后48h和72h，rAd—GFP-Tum5组VEGF质量浓度均明显低于rAd—GFP组，差异均有统计学意义（均P〈0．01）。结论腺病毒介导的重组Tum5基因的过表达可抑制生理状态下HUVECs的增生、迁移及管腔形成，这可能与Tum5下调VEGF在细胞上清液中的含量有关。

Background Tumstatin is the most active endogenous angiogenesis inhibitor, which has a marked inhibitory effect on pathological neovascularization, and Tum5 is an angiogenesis inhibitors fragment of full- length tumstatin. Objective This study was to investigate the effects of adenovirus-mediated overexpression of recombinant Turn5 gene on the proliferation, migration and tube formation of human umbilical vein endothelial cells （HUVECs） in physiological status. Methods The empty adenoviral vector expressing green fluorescent protein （rAd-GFP） and the viral vector expressing recombinant Turn5 gene were constructed. The HUVECs cultured in RPMI1640 medium were divided into normal control group, empty vector group （rAd-GFP group） and Tum5 gene infection group （rAd-GFP-Tum5 group）. The rAd-GFP and rAd-GFP-Tum5 adenoviral particles at the density of 1 × 10^10/ml were added into the medium to infect the cells for 48 hours. The proliferation of the cells was assayed at 24, 48 and 72 hours by cell counting kit-8 （ CCK-8 ） to evaluate the proliferative rate; the migration number of the cells was detected at 48 hours after infection by Transwell chamber ; the tube formation number of the cells were detected by Matrigel method. The concentration of vascular endothelial growth factor （YEGF） in cell supernatants was assayed by ELISA at 24, 48, and 72 hours following adenoviral infection. Results The cultured ceils showed green fluorescence in the rAd-GFP group and rAd-GFP-Tum5 group under the inverted fluorescence microscope, and the infection efficiency of rAd-GFP and rAd-GFP-Tum5 was 55. 13 % and 50.31% , respectively. No significant difference was found in cell proliferative rate among normal control group,rAd-GFP group and rAd-GFP-Tum5 group both at 24 and 48 hours after infection （ both at P〉0.05） ,and the cell proliferative rate was significantly lower in the rAd-GFP- Tum5 group than that in the normal control group and rAd-GFP group at 72 hours after infection （ both at P〈0. 01 ）. The migration number of the cells at 48 hours after infection was 2 260.25±930.44,2 370.00±441.06 and 723.75± 363.80 in the normal control group, rAd-GFP group and rAd-GFP-Tum5 group, showing a significant difference among the groups （ F = 8. 524, P = 0. 008 ） , and the migrated cells were evidently decreased in the rAd-GFP-Tum5 group compared with the rAd-GFP group and the normal control group （ both at P〈 0. 01 ）. The tube number at 48 hours after infection was 95, 67±5.86,88.00±4.58 and 20. 67±3.51 in the normal control group ,rAd-GFP group and rAd- GFP-Tum5 group, showing a significant difference among the groups （F = 226. 498,P〈0.01 ） , and the tube number in the rAd-GFP-Tum5 group was significantly reduced in comparison with the normal control group and rAd-GFP group （both at P〈0.01 ）. The considerably differences in VEGF concentration in the cell supernatants were found in different groups and various time points （ Fsroup = 73. 260, P 〈 0.01 ; Ftime= 73. 477, P 〈 0. 01 ） , and VEGF concentration in the cell supernatants was significantly decreased in the rAd-GFP-Tum5 group compared with the rAd- GFP group at both 48 hours and 72 hours （ both at P〈0. 01 ）. Conclusions The overexpression of the recombinant Turn5 can inhibit the proliferation,migration and tube formation of the HUVECs in physiological status,which may be associated with Tum-5-mediated down-regulation of VEGF protein in the cell supernatant.