Ghrelin对ox-LDL诱导内皮细胞损伤的保护及机制研究

发布时间：2018-06-04 02:10

本文选题：动脉粥样硬化 + 人脐静脉内皮细胞　；参考：《南华大学》2011年硕士论文

【摘要】：目的: 通过建立ox-LDL体外诱导的人脐静脉内皮细胞损伤模型,观察ox-LDL对血管内皮细胞功能和形态的影响,探讨Ghrelin对ox-LDL诱导人脐静脉内皮细胞凋亡和功能损伤的保护作用及其机制,为Ghrelin应用于动脉粥样硬化疾病的治疗提供理论及实践依据。方法: 1.采用细胞消化方法从足月新生儿脐静脉分离内皮细胞后,于含20%胎牛血清的M199完全培养基中进行培养传代。传至第4-5代的人脐静脉内皮细胞分别进行瑞氏-吉姆萨染色和人VIII因子相关抗原免疫组化检测。 2.实验包括剂量因素分析与时间因素分析两个方面,各组人脐静脉内皮细胞孵育24h后,分别收集内皮细胞及细胞培养液,以Hoechest 33258染色、Western-blot法和硝酸还原酶法检测血管内皮细胞凋亡情况,细胞中caspase-3蛋白水平和一氧化氮含量。采用ELISA法分别检测内皮细胞中IL-6、IL-8和TNF-α的表达水平。提取细胞的总RNA,采用半定量RT-PCR方法检测细胞间黏附分子-1(ICAM-1)和血管黏附分子-1(VCAM-1)基因在人脐静脉内皮细胞中的含量。结果: 1. Ghrelin拮抗ox-LDL诱导的人脐静脉内皮细胞凋亡 ①Hoechst33258染色荧光检测发现,与正常对照组相比,ox-LDL组HUVEC中出现大量凋亡细胞,凋亡率明显增加(p0.01),细胞核染色质固缩,聚集于核膜下或碎裂成致密的颗粒状,呈亮蓝色强荧光;与ox-LDL组比较,1、10、100ng/ml Ghrelin预处理组HUVEC细胞凋亡率分别降低30%、44%、56%,1h、6h、12h Ghrelin预处理组凋亡率分别降低40%、60%、72%,各组间差异均具有显著统计学意义(P 0.05)。 ②Western-blot检测表明, ox-LDL可上调细胞中caspase-3表达(P0.01);而经过Ghrelin预处理后,1、10、100ng/ml组HUVEC细胞中ox-LDL所诱导的caspase-3表达分别降低17%、23%、45%,1h、6h、12h组则分别降低32%、47%、52%,组间比较均有统计学意义(P0.05)。 ③硝酸还原酶法显示,ox-LDL可显著减少内皮细胞培养液中NO含量,差异有统计学意义(P0.01);经过Ghrelin预处理后,相较于ox-LDL组,1、10、100ng/ml组细胞培养液中NO含量分别增加25%、54%、85%,1h、6h、12h组则分别增加30%、46%、68%,各组间差异均有统计学意义(P0.05)。 2. Ghrelin抑制ox-LDL诱导的人脐静脉内皮细胞炎症因子表达 ①ELISA结果显示,ox-LDL组IL-6表达水平较对照组显著增加(P0.01);而与ox-LDL组比较,1、10、100ng/ml Ghrelin预处理组IL-6表达分别降低19%、35%、60%,1h、6h、12h Ghrelin预处理组则分别降低36%、45%、56%,各组间比较差异均有显著性(P0.01或0.05)。 ②ox-LDL组IL-8含量较对照组显著增加(P0.01);经过Ghrelin预处理后,1、10、100ng/ml组IL-8水平分别降低31%、52%、84%,1h、6h、12h组则分别降低56%、68%、79%,组间比较差异均有统计学意义(P0.01或0.05)。 ③Ghrelin预处理抑制ox-LDL诱导的内皮细胞中TNF-α表达(P0.05),相对于ox-LDL组,1、10、100ng/ml Ghrelin组TNF-α表达水平分别降低7%、19%、30%,组间比较有显著差异(P0.05);1h、6h、12h Ghrelin组TNF-α表达含量分别降低18%、28%、33%,其中6h和12h组间差异无统计学意义(P0.05)。 3.Ghrelin抑制ox-LDL诱导的人脐静脉内皮细胞粘附分子表达 ①半定量RT-PCR结果表明,ox-LDL组内皮细胞VCAM-1表达水平较空白对照组明显升高(P0.05),而1、10、100ng/ml Ghrelin预处理可使内皮细胞VCAM-1 mRNA含量,相较于ox-LDL组分别减少33%、52%、63%,1h、6h、12h Ghrelin预处理组则分别减少48%、65%、72%,其中10ng/ml与100ng/ml组,6h与12h组之间比较无显著差异(P0.05)。 ②RT-PCR结果显示,ox-LDL可显著增加内皮细胞中ICAM-1表达,与空白对照组之间差异有统计学意义(P0.01),Ghrelin预处理可抑制ox-LDL诱导的内皮细胞ICAM-1 mRNA表达(P0.05),其中1、10、100ng/ml Ghrelin预处理组减少百分比分别为18%、46%、57%,1h、6h、12h Ghrelin预处理组则分别降低48%、63%、69%,其是10ng/ml与100ng/ml组,6h与12h组之间ICAM-1 mRNA水平比较差异无显著性(P0.05)。结论: Ghrelin对ox-LDL诱导的人脐静脉内皮细胞凋亡有拮抗作用,可能与抑制ox-LDL引起的细胞内caspase-3蛋白水平升高及NO合成下降有关。对ox-LDL诱导的人脐静脉内皮细胞炎症因子IL-6、IL-8、TNF-α和粘附分子VCAM-1、ICAM-1的表达均有抑制作用。
[Abstract]:Objective:
By establishing a model of human umbilical vein endothelial cell injury induced by ox-LDL in vitro, the effects of ox-LDL on the function and morphology of vascular endothelial cells were observed, and the protective effect of Ghrelin on the apoptosis and functional damage of human umbilical vein endothelial cells induced by ox-LDL and its mechanism were discussed, which provided the theory and reality for the treatment of Ghrelin in the treatment of atherosclerotic diseases. Practice the basis.
Method:
1. the cell digestion method was used to isolate the endothelial cells from the umbilical vein of the full-term newborns, and was cultured in the M199 complete medium containing 20% fetal bovine serum. To the 4-5 generation, the human umbilical vein endothelial cells were stained by Rayleigh Giemsa and the human VIII factor related antigen were detected by immunohistochemistry.
2. the experiment included two aspects of dose factor analysis and time factor analysis. After incubation of umbilical vein endothelial cells in each group for 24h, endothelial cells and cell culture fluid were collected, and Hoechest 33258 was stained, Western-blot and nitrate reductase were used to detect the apoptosis of vascular endothelial cells, and the level of caspase-3 protein and nitric oxide contained in the cells. The expression level of IL-6, IL-8 and TNF- alpha in endothelial cells was detected by ELISA method. The total RNA of the cells was extracted and the content of -1 (ICAM-1) and -1 (VCAM-1) gene of vascular adhesion molecules in human umbilical vein endothelial cells were detected by semi quantitative RT-PCR method.
Result:
1. Ghrelin antagonize ox-LDL induced apoptosis of human umbilical vein endothelial cells
(1) Hoechst33258 staining fluorescence detection showed that a large number of apoptotic cells appeared in the HUVEC group of ox-LDL group compared with the normal control group, the apoptosis rate increased significantly (P0.01), the chromatin of the nucleus was condensed, and the nuclei gathered under the nuclear membrane or fragmented into dense granules, showing a bright blue strong fluorescence. Compared with the ox-LDL group, the 1,10100ng/ml Ghrelin preconditioning group withered the HUVEC cells. The mortality rate decreased by 30%, 44%, 56%, respectively. The apoptosis rates of 1H, 6h, 12h Ghrelin pretreatment groups were reduced by 40%, 60% and 72%, respectively, and the differences between the groups were statistically significant (P 0.05).
(2) Western-blot test showed that ox-LDL could up regulate the expression of Caspase-3 in cells (P0.01), and after Ghrelin pretreatment, the caspase-3 expression induced by ox-LDL in 1,10100ng/ml group HUVEC cells decreased by 17%, 23%, 45%, 1H, 6h, and 32%, 47%, 52% respectively.
(3) the nitrate reductase method showed that ox-LDL could significantly reduce the content of NO in endothelial cell culture, and the difference was statistically significant (P0.01). After Ghrelin pretreatment, the NO content increased by 25%, 54%, 85%, 1H, 6h, and 12h group, respectively, in group 1,10100ng/ml group, and 30%, 46%, 68% respectively. The differences in each group were statistically significant. Meaning (P0.05).
2. Ghrelin inhibits ox-LDL induced expression of inflammatory factors in human umbilical vein endothelial cells
(1) ELISA results showed that the expression level of IL-6 in group ox-LDL was significantly higher than that in the control group (P0.01). Compared with the ox-LDL group, the IL-6 expression in the 1,10100ng/ml Ghrelin preconditioning group decreased by 19%, 35%, 60%, 1H, 6h, and 12h Ghrelin preconditioning group was 36%, 45%, 56% respectively.
The content of IL-8 in group ox-LDL was significantly higher than that in the control group (P0.01). After Ghrelin pretreatment, the IL-8 level in 1,10100ng/ml group decreased by 31%, 52%, 84%, 1H, 6h, and 12h group decreased by 56%, 68%, 79% respectively. The differences were statistically significant (P0.01 or 0.05).
(3) Ghrelin pretreatment inhibited the expression of TNF- alpha (P0.05) in the endothelial cells induced by ox-LDL. Compared with the ox-LDL group, the expression of TNF- a decreased by 7%, 19%, 30%, respectively (P0.05), and 1H, 6h, and 18%, 28%, 33%, respectively. Meaning (P0.05).
3.Ghrelin inhibits ox-LDL induced expression of adhesion molecules in human umbilical vein endothelial cells
The results of semi quantitative RT-PCR showed that the expression level of VCAM-1 in endothelial cells in ox-LDL group was significantly higher than that in the blank control group (P0.05), while 1,10100ng/ml Ghrelin pretreatment could reduce the content of VCAM-1 mRNA in endothelial cells by 33%, 52%, 63%, 1H, 6h, respectively, and decreased by 48%, 65%, 72% respectively. There was no significant difference between group 6h and group 12h (P0.05).
The results of RT-PCR showed that ox-LDL could significantly increase the expression of ICAM-1 in endothelial cells, and there was a significant difference between the control group and the blank control group (P0.01). Ghrelin preconditioning could inhibit the expression of ICAM-1 mRNA in endothelial cells induced by ox-LDL (P0.05), and the percentage of 1,10100ng/ml Ghrelin pretreatment group was 18%, 46%, 57%, respectively. The treatment group decreased by 48%, 63%, 69%, respectively, in the 10ng/ml and 100ng/ml groups. There was no significant difference in the level of ICAM-1 mRNA between the 6h and 12h groups (P0.05).
Conclusion:
Ghrelin can antagonize the apoptosis of human umbilical vein endothelial cells induced by ox-LDL, which may be related to the increase of caspase-3 protein level and the decrease of NO synthesis induced by ox-LDL. The expression of inflammatory factors IL-6, IL-8, TNF- alpha, and adhesion molecule VCAM-1, ICAM-1 expression of human umbilical vein endothelial cells induced by ox-LDL can be inhibited.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

【参考文献】