【摘要】： 目的：探討心鈉肽(Atrial Natriuretic Peptide,ANP)對(duì)培養(yǎng)的人臍靜脈內(nèi)皮細(xì)胞缺氧再復(fù)氧損傷后的保護(hù)作用。 方法：取凍存的人臍靜脈內(nèi)皮細(xì)胞株復(fù)蘇,傳代培養(yǎng),培養(yǎng)至第三代后,將細(xì)胞按同種濃度接種到24孔培養(yǎng)板上,正常環(huán)境下培養(yǎng)一天,隔天按實(shí)驗(yàn)分組換液：分為三組：1.正常對(duì)照組(n=6)；2.缺氧復(fù)氧組(n=6)缺氧6h再復(fù)氧6h；3.缺氧復(fù)氧+不同濃度ANP組：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再復(fù)氧6h；各組細(xì)胞在倒置顯微鏡下觀察細(xì)胞形態(tài),取各組細(xì)胞培養(yǎng)上清液分別檢測丙二醛(MDA)、乳酸脫氫酶(LDH)、一氧化氮(NO)、內(nèi)皮素-1(ET-1)及超氧化物歧化酶(SOD)活性。用SPSS13.0軟件進(jìn)行統(tǒng)計(jì)學(xué)處理。 結(jié)果： 1.細(xì)胞形態(tài)學(xué)改變：缺氧復(fù)氧組與對(duì)照組相比,內(nèi)皮細(xì)胞形狀上不規(guī)則,細(xì)胞邊界不清,細(xì)胞間隙變寬,可見脫落的細(xì)胞、細(xì)胞碎片及空泡。ANP干預(yù)組較缺氧復(fù)氧組在形態(tài)上發(fā)生形變的細(xì)胞更少,細(xì)胞間隙更窄,脫落的細(xì)胞更少,且隨著藥物濃度的增加,形態(tài)上趨于正常。 2.缺氧復(fù)氧組與正常對(duì)照組比較：細(xì)胞培養(yǎng)液乳酸脫氫酶(LDH)活性較對(duì)照組明顯增高(69.35±5.66U/L vs 31.04±3.43 U/L,P0.01);ANP干預(yù)組各亞組較缺氧復(fù)氧組LDH活性明顯降低(P0.01),Pearson相關(guān)分析示LDH活性與ANP濃度呈負(fù)相關(guān),Pearson相關(guān)系數(shù)為-0.771(P0.05)。缺氧復(fù)氧組培養(yǎng)液中MDA含量較對(duì)照組明顯升高(5.94±0.58nmol/ml vs 1.69±0.16nmol/ml,P0.01)；ANP干預(yù)組各亞組MDA含量較缺氧復(fù)氧組明顯降低(P0.01),MDA含量與ANP濃度做Pearson相關(guān)分析顯示：Pearson系數(shù)為-0.809(P0.01)。缺氧復(fù)氧組SOD活性較對(duì)照組明顯降低(15.74±2.17U/Lvs 47.08±4.23U/L,P0.01)。ANP干預(yù)組各亞組細(xì)胞內(nèi)SOD活性較缺氧復(fù)氧組明顯升高(P0.01),當(dāng)ANP濃度在(0.001～0.05μg/ml)時(shí),pearson相關(guān)分析顯示：培養(yǎng)液SOD活性與ANP濃度呈正相關(guān)：Pearson系數(shù)為0.736(P0.05)。缺氧復(fù)氧組細(xì)胞培養(yǎng)液中NO含量較對(duì)照組明顯降低(36.81±3.78μmol/L vs 89.78±7.01μmol/L,P0.01),ET-1含量較對(duì)照組明顯升高(1368.64±99.07pg/L vs 305.52±35.30pg/L,P0.01)；ANP干預(yù)組各亞組較缺氧復(fù)氧組ET-1含量明顯減少,而NO含量明顯升高(P0.01, P0.01)。Pearson相關(guān)分析顯示：ANP濃度在(0.001～0.05μg/ml)時(shí),各亞組中NO含量與ANP濃度呈正相關(guān)Pearson系數(shù)為0.912(P0.01),而ET-1含量與ANP濃度呈負(fù)相關(guān)：Pearson系數(shù)為-0.732(P0.01)。 結(jié)論： ①缺氧復(fù)氧組較正常組人臍靜脈內(nèi)皮細(xì)胞收縮變圓,細(xì)胞間隙增寬,細(xì)胞脫落破碎,光學(xué)顯微鏡下可見粗顆粒及空泡。ANP能使細(xì)胞形態(tài)結(jié)構(gòu)恢復(fù)正常。 ②ANP能夠降低缺氧復(fù)氧損傷后培養(yǎng)液中LDH濃度,對(duì)細(xì)胞膜具有保護(hù)作用；ANP能夠降低內(nèi)皮細(xì)胞培養(yǎng)液中MDA含量并且提高內(nèi)皮細(xì)胞培養(yǎng)液中SOD活性,從而抑制缺氧復(fù)氧對(duì)內(nèi)皮細(xì)胞造成的氧化性損傷。 ③ANP能夠增加人臍靜脈內(nèi)皮細(xì)胞缺氧復(fù)氧損傷后培養(yǎng)液中NO含量,同時(shí)降低培養(yǎng)液中ET-1含量,起到保護(hù)內(nèi)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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R363

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本文編號(hào)：2205645