心钠肽对内皮细胞缺氧复氧损伤的保护作用的实验研究
发布时间：2018-08-26 17:32
【摘要】： 目的：探讨心钠肽(Atrial Natriuretic Peptide,ANP)对培养的人脐静脉内皮细胞缺氧再复氧损伤后的保护作用。 方法：取冻存的人脐静脉内皮细胞株复苏,传代培养,培养至第三代后,将细胞按同种浓度接种到24孔培养板上,正常环境下培养一天,隔天按实验分组换液：分为三组：1.正常对照组(n=6)；2.缺氧复氧组(n=6)缺氧6h再复氧6h；3.缺氧复氧+不同浓度ANP组：0.001μg/ml(n=6)；0.005μg/ml(n=6)；0.01μg/ml (n=6)；0.05μg/ml(n=6)；0.1μg/ml(n=6)缺氧6h再复氧6h；各组细胞在倒置显微镜下观察细胞形态,取各组细胞培养上清液分别检测丙二醛(MDA)、乳酸脱氢酶(LDH)、一氧化氮(NO)、内皮素-1(ET-1)及超氧化物歧化酶(SOD)活性。用SPSS13.0软件进行统计学处理。 结果： 1.细胞形态学改变：缺氧复氧组与对照组相比,内皮细胞形状上不规则,细胞边界不清,细胞间隙变宽,可见脱落的细胞、细胞碎片及空泡。ANP干预组较缺氧复氧组在形态上发生形变的细胞更少,细胞间隙更窄,脱落的细胞更少,且随着药物浓度的增加,形态上趋于正常。 2.缺氧复氧组与正常对照组比较：细胞培养液乳酸脱氢酶(LDH)活性较对照组明显增高(69.35±5.66U/L vs 31.04±3.43 U/L,P0.01);ANP干预组各亚组较缺氧复氧组LDH活性明显降低(P0.01),Pearson相关分析示LDH活性与ANP浓度呈负相关,Pearson相关系数为-0.771(P0.05)。缺氧复氧组培养液中MDA含量较对照组明显升高(5.94±0.58nmol/ml vs 1.69±0.16nmol/ml,P0.01)；ANP干预组各亚组MDA含量较缺氧复氧组明显降低(P0.01),MDA含量与ANP浓度做Pearson相关分析显示：Pearson系数为-0.809(P0.01)。缺氧复氧组SOD活性较对照组明显降低(15.74±2.17U/Lvs 47.08±4.23U/L,P0.01)。ANP干预组各亚组细胞内SOD活性较缺氧复氧组明显升高(P0.01),当ANP浓度在(0.001～0.05μg/ml)时,pearson相关分析显示：培养液SOD活性与ANP浓度呈正相关：Pearson系数为0.736(P0.05)。缺氧复氧组细胞培养液中NO含量较对照组明显降低(36.81±3.78μmol/L vs 89.78±7.01μmol/L,P0.01),ET-1含量较对照组明显升高(1368.64±99.07pg/L vs 305.52±35.30pg/L,P0.01)；ANP干预组各亚组较缺氧复氧组ET-1含量明显减少,而NO含量明显升高(P0.01, P0.01)。Pearson相关分析显示：ANP浓度在(0.001～0.05μg/ml)时,各亚组中NO含量与ANP浓度呈正相关Pearson系数为0.912(P0.01),而ET-1含量与ANP浓度呈负相关：Pearson系数为-0.732(P0.01)。 结论： ①缺氧复氧组较正常组人脐静脉内皮细胞收缩变圆,细胞间隙增宽,细胞脱落破碎,光学显微镜下可见粗颗粒及空泡。ANP能使细胞形态结构恢复正常。 ②ANP能够降低缺氧复氧损伤后培养液中LDH浓度,对细胞膜具有保护作用；ANP能够降低内皮细胞培养液中MDA含量并且提高内皮细胞培养液中SOD活性,从而抑制缺氧复氧对内皮细胞造成的氧化性损伤。 ③ANP能够增加人脐静脉内皮细胞缺氧复氧损伤后培养液中NO含量,同时降低培养液中ET-1含量,起到保护内皮功能的作用。
[Abstract]:AIM: To investigate the protective effect of atrial natriuretic peptide (ANP) on cultured human umbilical vein endothelial cells (HUVECs) after hypoxia-reoxygenation injury.
METHODS: The cryopreserved human umbilical vein endothelial cells were resuscitated, subcultured and cultured until the third generation. The cells were inoculated on 24-well culture plate at the same concentration for one day under normal conditions. The cells were divided into three groups according to the experiment: 1. normal control group (n = 6); 2. hypoxia-reoxygenation group (n = 6) hypoxia-reoxygenation 6 h; 3. hypoxia-reoxygenation + reoxygenation 6 h. Different concentrations of ANP group: 0.001 ug/ml (n = 6); 0.005 ug/ml (n = 6); 0.01 ug/ml (n = 6); 0.05 ug/ml (n = 6); 0.1 ug/ml (n = 6) hypoxia for 6 h and reoxygenation for 6 h; cells in each group were observed under an inverted microscope and supernatants of cell culture were taken to detect malondialdehyde (MDA), lactate dehydrogenase (LDH), nitric oxide (NO), endothelin-1 (ET-1) and endothelin-1 (ET-1) respectively. Superoxide dismutase (SOD) activity was analyzed by SPSS13.0 software.
Result:
1. Cell morphological changes: Compared with the control group, the endothelial cells in the hypoxia-reoxygenation group had irregular shape, unclear cell boundaries, wider cell gap, and visible exfoliated cells, cell fragments and vacuoles. Compared with the hypoxia-reoxygenation group, the ANP intervention group had fewer morphologically deformed cells, narrower cell gap and fewer exfoliated cells, and with the drug treatment. The concentration increased to normal morphology.
2. Compared with the normal control group, the activity of lactate dehydrogenase (LDH) in the cell culture medium was significantly higher (69.35 +5.66U/L vs 31.04 +3.43 U/L, P 0.01); the activity of LDH in each subgroup of the ANP intervention group was significantly lower than that in the hypoxia-reoxygenation group (P 0.01); the Pearson correlation analysis showed that LDH activity was negatively correlated with the concentration of ANP, and the Pearson correlation coefficient was - 0. 771 (P 0.05). The content of MDA in the culture medium of the hypoxia-reoxygenation group was significantly higher than that of the control group (5.94.58 nmol/ml vs 1.69.16 nmol/ml, P 0.01); the content of MDA in each subgroup of the ANP intervention group was significantly lower than that of the hypoxia-reoxygenation group (P 0.01). Pearson coefficient was - 0.809 (P 0.01). The SOD activity in each subgroup of the ANP intervention group was significantly higher than that of the hypoxia-reoxygenation group (P 0.01). When the concentration of ANP was (0.001-0.05 ug/ml), the Pearson correlation analysis showed that the SOD activity in the culture medium was positively correlated with the concentration of ANP: the Pearson coefficient was 0.736 (P 0.05). The content of NO in cell culture medium was significantly lower than that in control group (36.81 The results showed that the Pearson coefficient was 0.912 (P 0.01) when the concentration of ANP was (0.001-0.05 ug/ml), but the content of ET-1 was negatively correlated with the concentration of ANP: the Pearson coefficient was - 0.732 (P 0.01).
Conclusion:
(1) In hypoxia-reoxygenation group, the human umbilical vein endothelial cells (HUVECs) contracted and became round, the intercellular space widened, the cells shed and fragmented, and coarse particles and vacuoles were observed under optical microscope.
(2) ANP can reduce the concentration of LDH in the culture medium after hypoxia-reoxygenation injury and protect the cell membrane; ANP can reduce the content of MDA in the culture medium of endothelial cells and increase the activity of SOD in the culture medium of endothelial cells, thereby inhibiting the oxidative damage of endothelial cells caused by hypoxia-reoxygenation.
(3) ANP can increase the content of NO in the culture medium of human umbilical vein endothelial cells after hypoxia-reoxygenation injury, and decrease the content of ET-1 in the culture medium, thus protecting the endothelial function.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363
本文编号：2205645
[Abstract]:AIM: To investigate the protective effect of atrial natriuretic peptide (ANP) on cultured human umbilical vein endothelial cells (HUVECs) after hypoxia-reoxygenation injury.
METHODS: The cryopreserved human umbilical vein endothelial cells were resuscitated, subcultured and cultured until the third generation. The cells were inoculated on 24-well culture plate at the same concentration for one day under normal conditions. The cells were divided into three groups according to the experiment: 1. normal control group (n = 6); 2. hypoxia-reoxygenation group (n = 6) hypoxia-reoxygenation 6 h; 3. hypoxia-reoxygenation + reoxygenation 6 h. Different concentrations of ANP group: 0.001 ug/ml (n = 6); 0.005 ug/ml (n = 6); 0.01 ug/ml (n = 6); 0.05 ug/ml (n = 6); 0.1 ug/ml (n = 6) hypoxia for 6 h and reoxygenation for 6 h; cells in each group were observed under an inverted microscope and supernatants of cell culture were taken to detect malondialdehyde (MDA), lactate dehydrogenase (LDH), nitric oxide (NO), endothelin-1 (ET-1) and endothelin-1 (ET-1) respectively. Superoxide dismutase (SOD) activity was analyzed by SPSS13.0 software.
Result:
1. Cell morphological changes: Compared with the control group, the endothelial cells in the hypoxia-reoxygenation group had irregular shape, unclear cell boundaries, wider cell gap, and visible exfoliated cells, cell fragments and vacuoles. Compared with the hypoxia-reoxygenation group, the ANP intervention group had fewer morphologically deformed cells, narrower cell gap and fewer exfoliated cells, and with the drug treatment. The concentration increased to normal morphology.
2. Compared with the normal control group, the activity of lactate dehydrogenase (LDH) in the cell culture medium was significantly higher (69.35 +5.66U/L vs 31.04 +3.43 U/L, P 0.01); the activity of LDH in each subgroup of the ANP intervention group was significantly lower than that in the hypoxia-reoxygenation group (P 0.01); the Pearson correlation analysis showed that LDH activity was negatively correlated with the concentration of ANP, and the Pearson correlation coefficient was - 0. 771 (P 0.05). The content of MDA in the culture medium of the hypoxia-reoxygenation group was significantly higher than that of the control group (5.94.58 nmol/ml vs 1.69.16 nmol/ml, P 0.01); the content of MDA in each subgroup of the ANP intervention group was significantly lower than that of the hypoxia-reoxygenation group (P 0.01). Pearson coefficient was - 0.809 (P 0.01). The SOD activity in each subgroup of the ANP intervention group was significantly higher than that of the hypoxia-reoxygenation group (P 0.01). When the concentration of ANP was (0.001-0.05 ug/ml), the Pearson correlation analysis showed that the SOD activity in the culture medium was positively correlated with the concentration of ANP: the Pearson coefficient was 0.736 (P 0.05). The content of NO in cell culture medium was significantly lower than that in control group (36.81 The results showed that the Pearson coefficient was 0.912 (P 0.01) when the concentration of ANP was (0.001-0.05 ug/ml), but the content of ET-1 was negatively correlated with the concentration of ANP: the Pearson coefficient was - 0.732 (P 0.01).
Conclusion:
(1) In hypoxia-reoxygenation group, the human umbilical vein endothelial cells (HUVECs) contracted and became round, the intercellular space widened, the cells shed and fragmented, and coarse particles and vacuoles were observed under optical microscope.
(2) ANP can reduce the concentration of LDH in the culture medium after hypoxia-reoxygenation injury and protect the cell membrane; ANP can reduce the content of MDA in the culture medium of endothelial cells and increase the activity of SOD in the culture medium of endothelial cells, thereby inhibiting the oxidative damage of endothelial cells caused by hypoxia-reoxygenation.
(3) ANP can increase the content of NO in the culture medium of human umbilical vein endothelial cells after hypoxia-reoxygenation injury, and decrease the content of ET-1 in the culture medium, thus protecting the endothelial function.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363
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