3,4苯并芘对内皮祖细胞生物学功能的影响

Effects of 3,4 benzo(a)pyrene on the biological functions of endothelial progenitor cell

目的研究3,4苯并芘(BaP)对人脐血来源的内皮祖细胞(EPC)生物学功能的影响。方法密度梯度离心法分离获取人脐血单个核细胞,采用贴壁培养法培养MNC中的EPC,通过Dil标记的乙酰化低密度脂蛋白(Dil-ac-LDL)摄取实验和FITC标记的植物凝集素(FITC-UEA-I lectin)结合实验鉴定细胞。消化收集第3代细胞,分别采用细胞计数试剂盒(CCK-8)、黏附能力测定实验、Transwell小室法及Matrigel体外成血管试验观察BaP对EPC增殖能力、粘附能力、迁移能力及成血管能力的影响,并检测各组细胞培养上清液SOD含量。采用单因素方差分析及LSD-t检验进行统计学分析。结果采用贴壁培养法能成功培养出EPC;与正常对照组相比,BaP呈浓度依赖性降低EPC的增殖能力{正常对照组OD(1.02±0.04)显著高于BaP各组[BaP①组OD(0.66±0.04),BaP②组OD(0.55±0.04),BaP③组OD(0.35±0.05),均P<0.01],BaP染毒组间增殖能力差异亦有统计学意义(两两比较,均P<0.01)};与正常对照组比较,BaP呈浓度依赖性降低EPC的黏附能力{正常对照组[(117.50±17.16)个/200倍镜]显著高于BaP各组[BaP①组(80.00±14.46)个/200倍镜,BaP②组(66.00±9.06)个/200倍镜,BaP③组(49.80±10.72)个/200倍镜,均P<0.01],BaP染毒组间黏附能力差异亦有统计学意义(两两比较,均P<0.05)};与正常对照组相比,EPC的迁移能力亦呈BaP浓度依赖性降低{正常对照组[(46.10±4.51)个/400倍镜]显著高于BaP各组[BaP①组(35.50±4.95)个/400倍镜,BaP②组(26.80±4.08)个/400倍镜,BaP③组(19.50±2.84)个/400倍镜,均P<0.01],BaP染毒组间迁移能力差异亦有统计学意义(两两比较,均P<0.01)};与正常对照组比较,EPC的成血管能力亦呈BaP浓度依赖性降低{正常对照组[(33.20±3.70)个/100倍镜]显著高于BaP各组[BaP①组(22.00±3.39)个/100倍镜,BaP②组(16.20±2.59)个/100倍镜,BaP③组(10.80±2.39)个/100倍镜,均P<0.01],BaP染毒组间成血管能力差异亦有统计学意义(两两比较,均P<0.05)}。同时细胞培养上清液中SOD的活力也呈BaP浓度依赖性地降低{正常对照组[(22.6±2.19)U/ml]高于BaP各组[BaP①组(15.94±1.68)U/ml,BaP②组(12.5±1.58)U/ml,BaP③组(6.9±1.55)U/ml,均P<0.01],BaP染毒组间SOD活力差异亦有统计学意义(两两比较,均P<0.01)}。结论 BaP体外诱导显著影响EPC的多种生物学功能,其机制可能与氧化损伤有关。

Objective To evaluate the effects of benzo（a）pyrene（BaP） on biological functions of endothelial progenitor cell（EPC） from human umbilical cord blood. Methods Mononuclear cell were isolated from cord blood by density gradient centrifugation, and EPCs were obtained by adherent culture. BydDouble-staining with Dil-ac-LDL and lentil lectin EPCs were identified. EPCs of third passage were collected and treated with BaP. Cell Counting Kit-8（CCK-8） assay was used to analyze the viability of EPC. The cell adhesion ability was examined. Migration ability was evaluated through a transwell chamber, and vasculogenesis capacity of EPC was measured in Matrigel. The activity of SOD in culture supernatants of EPC was detected, using univariate variate and LSD-t test for statistical analysis. Results EPC cultured under specified condition formed colony with cells positive for ac-LDL and lentil lectin. Compared with the normal control group, cell proliferation capacity of EPC was obviously inhibited by BaP in a dose-dependent manner. The OD was 0.66 ± 0.04, 0.55 ± 0.04, and 0.35 ± 0.05 respectively for BaP at different doses, all significantly lower than the control group（1.02 ± 0.04, P ＆lt; 0.01）. The proliferation capacity was also inhibited by BaP（P ＆lt; 0.01）. Compared with the normal control group, cell adhesion ability of EPC was obviously inhibited by BaP in a dose-dependent manner. The numbers of adhesion cells in the specified filed under the microscope were 117.50 ± 17.16, 80.00 ± 14.46, 66.00 ± 9.06, 49.80 ± 10.72 respectively for the control and three BaP groups（P 〈 0.01）. The adhesion ability of EPCs was inhibited by BaP. Compared with the normal control group, cell migration ability of EPC was obviously inhibited by BaP in a dose-dependent manner. The numbers of migrated cells in the specific filed were 46.10 ± 4.51, 35.50 ± 4.95, 26.80 ± 4.08 and 19.50 ± 2.84 respectively for the four groups（P ＆lt; 0.01）. Compared with the normal control group, cell vasculogenesis capacity of EPC was obviously inhibited by BaP in a dose-dependent manner. Finally, the activity of SOD was also inhibited by BaP in a dose-dependent manner, with a SOD activity of 22.6 ± 2.19 U/ml, 15.94 ± 1.68U/ml, 12.5 ± 1.58U/ml, and 6.9 ± 1.55U/ml, respectively for the four groups（P 〈 0.01）. Conclusions The biological functions of EPC could be inhibited notably by BaP, and the mechanism might be related to oxidant-mediated stress responses.