兰州大学机构库 >学院待认领
促红细胞生成素预防培养人脐静脉内皮细胞凋亡的研究
Alternative TitleErythropoietin Inhibits Apoptosis Induced by Oxidized LDL in Human Umbilical Vein Endothelial Cells
杨小芳
Thesis Advisor王志禄
2009-05-26
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword促红细胞生成素 内皮细胞 凋亡 氧化低密度脂蛋白
Abstract目的和背景 氧化低密度脂蛋白(Oxidized LDL,Ox-LDL)可诱导冠状动脉内皮细胞凋亡,是否可诱导人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs)凋亡尚不清楚。促红细胞生成素(Erythropoietin,EPO)可以抑制心肌梗死和缺血再灌注损伤中的心肌细胞凋亡,能否抑制由Ox-LDL介导的脐静脉内皮细胞凋亡,也不清楚。本研究拟建立Ox-LDL诱导培养HUVECs凋亡模型,证实Ox-LDL诱导HUVECs凋亡,以及类凝血素内皮型Ox-LDL受体(Lectin-like endothelial ox-LDL receptor,LOX-1)在Ox-LDL诱导HUVECs凋亡过程中的作用,并研究EPO对Ox-LDL诱导HUVECs凋亡的影响。 方法 体外培养HUVECs,一组细胞分组孵育24h后加入反式或顺式LOX-1 mRNA预处理24h,再加入 100 μg/ml的Ox-LDL再孵育12h,采用Western blot检测凋亡蛋白Bcl-2/Bax的表达。另一组细胞培养24h后加不同浓度(6.25、50、100 IU /ml)重组人促红细胞生成素(Recombinant human erythropoietin,rhEPO)预处理24 h,再加入100 μg/ml Ox-LDL孵育,12 h后采用Western blot检测凋亡蛋白Bcl-2/Bax的表达,48 h后流式细胞术检测细胞凋亡率,甲基噻唑基四唑(MTT)法检测细胞活力。 结果 0.5 μmol/L反式LOX-1 mRNA预处理细胞组凋亡蛋白Bcl-2/Bax比值较Ox-LDL组增高(1.72±0.04 VS 0.55±0.02,P<0.05);0.5 μmol/L顺式LOX-1 mRNA预处理细胞组凋亡蛋白Bcl-2/Bax比值较Ox-LDL组对照无明显差异(0.59±0.02 VS 0.55±0.02,P>0.05)。rhEPO预处理+Ox-LDL组随rhEPO浓度的递增细胞存活率较Ox-LDL对照组增加(61.23%VS 57.24%,63.09% VS 57.24%,74.08% VS 57.24%,P<0.05,依次),且与rhEPO呈浓度依赖性(61.23% VS 63.09% VS 74.08%,P<0.05);rhEPO预处理+Ox-LDL组细胞凋亡率均较Ox-LDL组降低(22.10±2.10% VS 35.40±1.30%,13.30±2.30% VS 35.40±1.30%,5.70±1.10%VS35.40±1.30%,P<0.05,依次),且与rhEPO呈浓度依赖性(22.10±2.10%VS13.30±2.30%VS 5.70±1.10%,P<0.05);rhEPO预处理+Ox-LDL组凋亡蛋白Bcl-2/Bax比值较Ox-LDL组增高(1.53±0.05VS1.00±0.11,2.86±0.18VS1.00±0.11,161.45±10.56VS 1.00±0.11,P <0.05,依次),且与rhEPO呈浓度依赖性(1.53±0.05VS2.86±0.18VS 161.45±10.56,P<0.05)。 结论 Ox-LDL可以诱导HUVECs凋亡,并且通过LOX-1 mRNA来调节,rhEPO可降低Ox-LDL诱导的HUVECs的凋亡率、增高Bcl-2/Bax的比值,结果提示rhEPO可抑制Ox-LDL诱导的HUVECs凋亡。
Other AbstractObjective and Backgroud Oxidized LDL (Ox-LDL) induces apoptosis in cultured human coronary artery endothelial cells (HCAECs), whether Ox-LDL could induce human umbilical vein endothelial cells (HUVECs) apoptosis was unclear. Erythropoietin (EPO) can inhibit apoptosis of cadiocytes induced by myocardial infarction and ischemia-reperfusion injury, whether EPO can inhibit the Ox-LDL-induced apoptosis of HCVECs was unclear. This study was to set up Ox-LDL-induced apoptosis in cultured HUVECs, to examine the role of Ox-LDL and its receptor LOX-1 in the Ox-LDL-induced apoptosis in HUVECs and to determine the impact of recombinant human erythropoietin (rhEPO) on Ox-LDL-induced apoptosis in HUVECs. Methods Potential role of the Ox-LDL receptor (LOX-1) was tested by pr-treating cells with the LOX-1-specific sense or anti-sense RNA (0.5 μmol/L) for 24 hours before exposure to Ox-LDL(100 μg/ml). The impact of rhEPO on Ox-LDL-induced apoptosis was determined by assessing cell survival and the expression of intracellular pro- and anti-apoptotic proteins in HUVECs. HUVECs were cultured in vitro and treated with rhEPO (6.25, 50, or 100 IU/ml) for 24 hours and then with Ox-LDL (100 μg/ml) for 48 hours. Apoptosis was assessed by the apoptosis ratio, cell viability, Bcl-2/Bax ratio. Results The Bcl-2/Bax ratio (1.72±0.04 VS 0.55±0.02,P<0.05) was significantly increaser in pretreatment of HUVECs with antisense-LOX-1 group compared with Ox-LDL group, and that ratio (0.59±0.02 VS 0.55±0.02,P>0.05) was no significant difference between pretreatment of HUVECs with sense-LOX-1 group and Ox-LDL group.As compared to Ox-LDL group, pretreatment with rhEPO led to increased cell survival of HUVECs (61.23% VS 57.24%, 63.09% VS57.24%, 74.08% VS 57.24%, P<0.05, respectively) in a concentration-dependent manner (61.23% VS 63.09% VS 74.08%, P<0.05) and decreased the apoptosis ratio(22.10±2.10% VS 35.40±1.30%, 13.30±2.30% VS 35.40±1.30%, 5.70±1.10% VS 35.40±1.30%, P<0.05), respectivelyin a concentration-dependent manner(22.10±2.10% VS 13.30±2.30% VS 5.70 ± 1.10%,P<0.05). Consistently, the Bcl-2/Bax ratios were also increased (1.53±0.05 VS 1.00±0.11, 2.86±0.18 VS 1.00±0.11, 161.45±10.56 VS 1.00±0.11, P<0.05, respec- tively) in a similar fashion, also in a concentration-dependent manner (1.53±0.05 VS 2.86±0.18 VS 161.45±10.56, P<0.05). Conclusions Our findings suggest that apoptosis induced by Ox-LDL in Human Umbilical Vein Endothelial Cells is mediated by ...
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/223711
Collection学院待认领
Affiliation临床医学院
Recommended Citation
GB/T 7714
杨小芳. 促红细胞生成素预防培养人脐静脉内皮细胞凋亡的研究[D]. 兰州. 兰州大学,2009.
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