本文選題：動(dòng)脈粥樣硬化 + 人臍靜脈內(nèi)皮細(xì)胞�。� 參考：《南華大學(xué)》2011年碩士論文

【摘要】：目的: 通過建立ox-LDL體外誘導(dǎo)的人臍靜脈內(nèi)皮細(xì)胞損傷模型,觀察ox-LDL對(duì)血管內(nèi)皮細(xì)胞功能和形態(tài)的影響,探討Ghrelin對(duì)ox-LDL誘導(dǎo)人臍靜脈內(nèi)皮細(xì)胞凋亡和功能損傷的保護(hù)作用及其機(jī)制,為Ghrelin應(yīng)用于動(dòng)脈粥樣硬化疾病的治療提供理論及實(shí)踐依據(jù)。 方法: 1.采用細(xì)胞消化方法從足月新生兒臍靜脈分離內(nèi)皮細(xì)胞后,于含20%胎牛血清的M199完全培養(yǎng)基中進(jìn)行培養(yǎng)傳代。傳至第4-5代的人臍靜脈內(nèi)皮細(xì)胞分別進(jìn)行瑞氏-吉姆薩染色和人VIII因子相關(guān)抗原免疫組化檢測(cè)。 2.實(shí)驗(yàn)包括劑量因素分析與時(shí)間因素分析兩個(gè)方面,各組人臍靜脈內(nèi)皮細(xì)胞孵育24h后,分別收集內(nèi)皮細(xì)胞及細(xì)胞培養(yǎng)液,以Hoechest 33258染色、Western-blot法和硝酸還原酶法檢測(cè)血管內(nèi)皮細(xì)胞凋亡情況,細(xì)胞中caspase-3蛋白水平和一氧化氮含量。采用ELISA法分別檢測(cè)內(nèi)皮細(xì)胞中IL-6、IL-8和TNF-α的表達(dá)水平。提取細(xì)胞的總RNA,采用半定量RT-PCR方法檢測(cè)細(xì)胞間黏附分子-1(ICAM-1)和血管黏附分子-1(VCAM-1)基因在人臍靜脈內(nèi)皮細(xì)胞中的含量。 結(jié)果: 1. Ghrelin拮抗ox-LDL誘導(dǎo)的人臍靜脈內(nèi)皮細(xì)胞凋亡 ①Hoechst33258染色熒光檢測(cè)發(fā)現(xiàn),與正常對(duì)照組相比,ox-LDL組HUVEC中出現(xiàn)大量凋亡細(xì)胞,凋亡率明顯增加(p0.01),細(xì)胞核染色質(zhì)固縮,聚集于核膜下或碎裂成致密的顆粒狀,呈亮藍(lán)色強(qiáng)熒光;與ox-LDL組比較,1、10、100ng/ml Ghrelin預(yù)處理組HUVEC細(xì)胞凋亡率分別降低30%、44%、56%,1h、6h、12h Ghrelin預(yù)處理組凋亡率分別降低40%、60%、72%,各組間差異均具有顯著統(tǒng)計(jì)學(xué)意義(P 0.05)。 ②Western-blot檢測(cè)表明, ox-LDL可上調(diào)細(xì)胞中caspase-3表達(dá)(P0.01);而經(jīng)過Ghrelin預(yù)處理后,1、10、100ng/ml組HUVEC細(xì)胞中ox-LDL所誘導(dǎo)的caspase-3表達(dá)分別降低17%、23%、45%,1h、6h、12h組則分別降低32%、47%、52%,組間比較均有統(tǒng)計(jì)學(xué)意義(P0.05)。 ③硝酸還原酶法顯示,ox-LDL可顯著減少內(nèi)皮細(xì)胞培養(yǎng)液中NO含量,差異有統(tǒng)計(jì)學(xué)意義(P0.01);經(jīng)過Ghrelin預(yù)處理后,相較于ox-LDL組,1、10、100ng/ml組細(xì)胞培養(yǎng)液中NO含量分別增加25%、54%、85%,1h、6h、12h組則分別增加30%、46%、68%,各組間差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。 2. Ghrelin抑制ox-LDL誘導(dǎo)的人臍靜脈內(nèi)皮細(xì)胞炎癥因子表達(dá) ①ELISA結(jié)果顯示,ox-LDL組IL-6表達(dá)水平較對(duì)照組顯著增加(P0.01);而與ox-LDL組比較,1、10、100ng/ml Ghrelin預(yù)處理組IL-6表達(dá)分別降低19%、35%、60%,1h、6h、12h Ghrelin預(yù)處理組則分別降低36%、45%、56%,各組間比較差異均有顯著性(P0.01或0.05)。 ②ox-LDL組IL-8含量較對(duì)照組顯著增加(P0.01);經(jīng)過Ghrelin預(yù)處理后,1、10、100ng/ml組IL-8水平分別降低31%、52%、84%,1h、6h、12h組則分別降低56%、68%、79%,組間比較差異均有統(tǒng)計(jì)學(xué)意義(P0.01或0.05)。 ③Ghrelin預(yù)處理抑制ox-LDL誘導(dǎo)的內(nèi)皮細(xì)胞中TNF-α表達(dá)(P0.05),相對(duì)于ox-LDL組,1、10、100ng/ml Ghrelin組TNF-α表達(dá)水平分別降低7%、19%、30%,組間比較有顯著差異(P0.05);1h、6h、12h Ghrelin組TNF-α表達(dá)含量分別降低18%、28%、33%,其中6h和12h組間差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 3.Ghrelin抑制ox-LDL誘導(dǎo)的人臍靜脈內(nèi)皮細(xì)胞粘附分子表達(dá) ①半定量RT-PCR結(jié)果表明,ox-LDL組內(nèi)皮細(xì)胞VCAM-1表達(dá)水平較空白對(duì)照組明顯升高(P0.05),而1、10、100ng/ml Ghrelin預(yù)處理可使內(nèi)皮細(xì)胞VCAM-1 mRNA含量,相較于ox-LDL組分別減少33%、52%、63%,1h、6h、12h Ghrelin預(yù)處理組則分別減少48%、65%、72%,其中10ng/ml與100ng/ml組,6h與12h組之間比較無顯著差異(P0.05)。 ②RT-PCR結(jié)果顯示,ox-LDL可顯著增加內(nèi)皮細(xì)胞中ICAM-1表達(dá),與空白對(duì)照組之間差異有統(tǒng)計(jì)學(xué)意義(P0.01),Ghrelin預(yù)處理可抑制ox-LDL誘導(dǎo)的內(nèi)皮細(xì)胞ICAM-1 mRNA表達(dá)(P0.05),其中1、10、100ng/ml Ghrelin預(yù)處理組減少百分比分別為18%、46%、57%,1h、6h、12h Ghrelin預(yù)處理組則分別降低48%、63%、69%,其是10ng/ml與100ng/ml組,6h與12h組之間ICAM-1 mRNA水平比較差異無顯著性(P0.05)。 結(jié)論: Ghrelin對(duì)ox-LDL誘導(dǎo)的人臍靜脈內(nèi)皮細(xì)胞凋亡有拮抗作用,可能與抑制ox-LDL引起的細(xì)胞內(nèi)caspase-3蛋白水平升高及NO合成下降有關(guān)。對(duì)ox-LDL誘導(dǎo)的人臍靜脈內(nèi)皮細(xì)胞炎癥因子IL-6、IL-8、TNF-α和粘附分子VCAM-1、ICAM-1的表達(dá)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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363

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本文編號(hào)：1975352