空气细颗粒物中Ni对血管内皮细胞作用的研究
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摘要
目的：1.分析比较镍污染区和对照区主要污染物、空气细颗粒物(PM2.5)浓度及PM2.5中Ni和V的含量是否有差异；2.观察空气细颗粒物中Ni在体内和体外对血管内皮细胞数目和功能的影响,从而探讨空气细颗粒物中Ni致心血管疾病的可能机制。
方法：1.选择镍污染区和对照区,用智能中流量采样器于2009年12月和2010年6-7月两次在两个区采集并分析空气中PM2.5浓度及PM2.5中Ni和V两种元素的含量；2.将采集的PM2.5制备成不同浓度的混悬液(0μg/ml、25μg/ml、50μg/ml. 100μg/ml),对ECV-304细胞染毒,MTT法测定细胞存活率,Transwell迁移小室测定细胞迁移率,同时测定细胞上清液中LDH的活力；3.从镍污染区和对照区配对选取20名研究对象,收集外周血后用流式细胞仪测定全血中CD34+/KDR+、CD34+/KDR+/CD133+和CD34+/KDR+/CD45三个细胞群数目,以检测外周血中血管内皮前体细胞数目；Transwell迁移小室测定血管内皮前体细胞迁移率及细胞集落形成,以检测血管内皮前体细胞功能。
结果：1.大气细颗粒物监测结果：①镍污染区和对照区主要污染物PM10、PM2SO2和NO2浓度无统计学意义(P>0.05)；②镍污染区PM2.5中Ni浓度显著高于对照区,为对照区的69倍(P<0.001)；两区V浓度无统计学差异(P>0.05)。2.空气PM2.中Ni的细胞毒理学研究结果：①用镍污染区和对照区PM2.5染毒的ECV-304细胞存活率均随着染毒浓度上升而下降(P<0.05)；在同一染毒时间和染毒剂量下,镍污染区PM2.5染毒的ECV-304细胞存活率低于对照区(P<0.05)；②镍污染区和对照区PM2.5染毒的ECV-304细胞上清中LDH活力均随染毒浓度增大而逐渐上升(P<0.05)；同一染毒时间和染毒剂量,镍污染区PM2.5染毒的ECV-304细胞上清中LDH活力均高于对照区(P均<0.01)。③随着染毒浓度的上升,镍污染区和对照区PM2.5染毒的ECV-304细胞迁移数目逐渐减少,迁移率逐渐下降(P<0.05)；同一染毒时间和染毒剂量,镍污染区PM2.5染毒的ECV-304细胞迁移数低于对照区(P<0.05)。3、人群流行病学研究结果：镍污染区研究对象外周血循环内皮前体细胞数显著低于对照区(P<0.05),细胞迁移数显著低于对照区为(P<0.01)集落形成单位数显著低于对照区(P<0.05)。
结论：1.监测期间镍污染区和对照区PM2.5浓度相近,镍污染区中Ni浓度是对照区的69倍；2.镍污染区PM2.5(Ni)可导致血管内皮细胞存活率减少、细胞功能减弱；3.镍污染区居民血管内皮前体细胞的数目减少、功能降低。提示PM2.5中镍可能是损伤血管内皮前体细胞、导致心血管疾病发生的主要成分。
Objective:1. To compare the concentration of the main pollutant、PM2.5 and Ni and V in PM2.5 in the nickel-contaminated area and control area; 2.To probe the effect of the nickel in ambient PM2.5 on the vascular endothelial cells' number and function in vitro and vivo and explore its possible mechanism.
Methods:1.In the nickel-contaminated area and control area PM2.5 were collected from December 2009 and June to July of 2010 and then the concentration of PM2.5 as well as Ni and V were tested; 2.The ambient PM2.5 of nickel-contaminated area and control area were made into suspensions for different concentrations (0μg/ml,25μg/ml,50μg/ml,100μg/ml). After being exposed, the survival rate of ECV-304 cells was determined by MTT assay. Then Transwell Migration cabinet was applied to scan its mobility and the activity, and the LDH activity in cells' supernatant was also tested; 3.Twenty research objects were selected from the nickel-contaminated area and control area. Using the Flow cytometric analysis, CD34+/KDR+, CD34+/KDR+/CD133+ and CD34+/KDR+/CD45- three cell groups in the blood samples from the 20 objects were tested, then the number of the endothelial precursor cells was accounted, and its mobility and the activity were determined by Transwell Migration cabinet, besides that the numbers of the colony forming unit(CFU) was also tested.
Results:1. Ambient monitoring results①There were not significant difference about the concentration of PM10, PM2.5, SO2 and NO2 between nickel-contaminated and control area (P>0.05):①The concentration of Ni in PM2.5 was 69 times' higher in nickel-contaminated area than in the control area(P<0.001), the concentration of V in nickel-contaminated area was almost the same as in the control area (P>0.05).2.The results of cytotoxicological study of nickel (Ni) in ambient PM2.5①With the increasing dose of exposure, ECV-304 cells' survival rate decreased gradually both in nickel-contaminated area and the control area(P<0.05), under the same exposure time and exposure dose, there were significant difference between the ECV-304 cells, survival rate coming from these two areas.②As the exposure dose increased, ECV-304 cells' LDH activity in cell supernatant fluid increased gradually(P<0.05). At the same exposure time and exposure doses, LDH activity of ECV-304 also showed significant difference in cell supernatant fluid (P<0.01).③With the increasing of exposure doses, ECV-304 cells migration number and its rate decreased gradually both in nickel-contaminated area and the control area (P<0.05); at the same exposure time and exposure dose, ECV-304 cells' migration number and its rate in nickel-contaminated area was lower than in the control area (P<0.01).3.The results of epidemiological studies①The concentration of EPCs of subjects from nickel-contaminated area were lower than from the control area(P<0.05), the number of the migration of endothelial precursor cells from nickel-contaminated area was lower than in the control area (P<0.01), the cell formed unit of the endothelial precursor cells in nickel-contaminated area was also lower than in the control area(P<0.05).
Conclusion:1.During the monitoring period nickel-contaminated area shows similar concentration of PM2.5 with control area. The concentration of Ni of PM2.5 in nickel-contaminated area was 69 times as in the control area.2. After being exposed to PM2.5(Ni), the endothelial cells' survival rate reduced, and their function is changed in nickel-contaminated area.3. The number and function of endothelial progenitor cell from people who live in the nickel-contaminated area were decreased, which suggested that nickel in PM2.5 may injure endothelial progenitor cells and played an important role in cardiovascular disease.
方法：1.选择镍污染区和对照区,用智能中流量采样器于2009年12月和2010年6-7月两次在两个区采集并分析空气中PM2.5浓度及PM2.5中Ni和V两种元素的含量；2.将采集的PM2.5制备成不同浓度的混悬液(0μg/ml、25μg/ml、50μg/ml. 100μg/ml),对ECV-304细胞染毒,MTT法测定细胞存活率,Transwell迁移小室测定细胞迁移率,同时测定细胞上清液中LDH的活力；3.从镍污染区和对照区配对选取20名研究对象,收集外周血后用流式细胞仪测定全血中CD34+/KDR+、CD34+/KDR+/CD133+和CD34+/KDR+/CD45三个细胞群数目,以检测外周血中血管内皮前体细胞数目；Transwell迁移小室测定血管内皮前体细胞迁移率及细胞集落形成,以检测血管内皮前体细胞功能。
结果：1.大气细颗粒物监测结果：①镍污染区和对照区主要污染物PM10、PM2SO2和NO2浓度无统计学意义(P>0.05)；②镍污染区PM2.5中Ni浓度显著高于对照区,为对照区的69倍(P<0.001)；两区V浓度无统计学差异(P>0.05)。2.空气PM2.中Ni的细胞毒理学研究结果：①用镍污染区和对照区PM2.5染毒的ECV-304细胞存活率均随着染毒浓度上升而下降(P<0.05)；在同一染毒时间和染毒剂量下,镍污染区PM2.5染毒的ECV-304细胞存活率低于对照区(P<0.05)；②镍污染区和对照区PM2.5染毒的ECV-304细胞上清中LDH活力均随染毒浓度增大而逐渐上升(P<0.05)；同一染毒时间和染毒剂量,镍污染区PM2.5染毒的ECV-304细胞上清中LDH活力均高于对照区(P均<0.01)。③随着染毒浓度的上升,镍污染区和对照区PM2.5染毒的ECV-304细胞迁移数目逐渐减少,迁移率逐渐下降(P<0.05)；同一染毒时间和染毒剂量,镍污染区PM2.5染毒的ECV-304细胞迁移数低于对照区(P<0.05)。3、人群流行病学研究结果：镍污染区研究对象外周血循环内皮前体细胞数显著低于对照区(P<0.05),细胞迁移数显著低于对照区为(P<0.01)集落形成单位数显著低于对照区(P<0.05)。
结论：1.监测期间镍污染区和对照区PM2.5浓度相近,镍污染区中Ni浓度是对照区的69倍；2.镍污染区PM2.5(Ni)可导致血管内皮细胞存活率减少、细胞功能减弱；3.镍污染区居民血管内皮前体细胞的数目减少、功能降低。提示PM2.5中镍可能是损伤血管内皮前体细胞、导致心血管疾病发生的主要成分。
Objective:1. To compare the concentration of the main pollutant、PM2.5 and Ni and V in PM2.5 in the nickel-contaminated area and control area; 2.To probe the effect of the nickel in ambient PM2.5 on the vascular endothelial cells' number and function in vitro and vivo and explore its possible mechanism.
Methods:1.In the nickel-contaminated area and control area PM2.5 were collected from December 2009 and June to July of 2010 and then the concentration of PM2.5 as well as Ni and V were tested; 2.The ambient PM2.5 of nickel-contaminated area and control area were made into suspensions for different concentrations (0μg/ml,25μg/ml,50μg/ml,100μg/ml). After being exposed, the survival rate of ECV-304 cells was determined by MTT assay. Then Transwell Migration cabinet was applied to scan its mobility and the activity, and the LDH activity in cells' supernatant was also tested; 3.Twenty research objects were selected from the nickel-contaminated area and control area. Using the Flow cytometric analysis, CD34+/KDR+, CD34+/KDR+/CD133+ and CD34+/KDR+/CD45- three cell groups in the blood samples from the 20 objects were tested, then the number of the endothelial precursor cells was accounted, and its mobility and the activity were determined by Transwell Migration cabinet, besides that the numbers of the colony forming unit(CFU) was also tested.
Results:1. Ambient monitoring results①There were not significant difference about the concentration of PM10, PM2.5, SO2 and NO2 between nickel-contaminated and control area (P>0.05):①The concentration of Ni in PM2.5 was 69 times' higher in nickel-contaminated area than in the control area(P<0.001), the concentration of V in nickel-contaminated area was almost the same as in the control area (P>0.05).2.The results of cytotoxicological study of nickel (Ni) in ambient PM2.5①With the increasing dose of exposure, ECV-304 cells' survival rate decreased gradually both in nickel-contaminated area and the control area(P<0.05), under the same exposure time and exposure dose, there were significant difference between the ECV-304 cells, survival rate coming from these two areas.②As the exposure dose increased, ECV-304 cells' LDH activity in cell supernatant fluid increased gradually(P<0.05). At the same exposure time and exposure doses, LDH activity of ECV-304 also showed significant difference in cell supernatant fluid (P<0.01).③With the increasing of exposure doses, ECV-304 cells migration number and its rate decreased gradually both in nickel-contaminated area and the control area (P<0.05); at the same exposure time and exposure dose, ECV-304 cells' migration number and its rate in nickel-contaminated area was lower than in the control area (P<0.01).3.The results of epidemiological studies①The concentration of EPCs of subjects from nickel-contaminated area were lower than from the control area(P<0.05), the number of the migration of endothelial precursor cells from nickel-contaminated area was lower than in the control area (P<0.01), the cell formed unit of the endothelial precursor cells in nickel-contaminated area was also lower than in the control area(P<0.05).
Conclusion:1.During the monitoring period nickel-contaminated area shows similar concentration of PM2.5 with control area. The concentration of Ni of PM2.5 in nickel-contaminated area was 69 times as in the control area.2. After being exposed to PM2.5(Ni), the endothelial cells' survival rate reduced, and their function is changed in nickel-contaminated area.3. The number and function of endothelial progenitor cell from people who live in the nickel-contaminated area were decreased, which suggested that nickel in PM2.5 may injure endothelial progenitor cells and played an important role in cardiovascular disease.
引文
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