香烟提取物对小鼠内皮祖细胞增殖能力及干细胞抗原-1表达的影响

发布时间：2018-03-03 14:08

本文选题：内皮祖细胞　切入点：干细胞抗原　出处：《中南大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：分离提取小鼠内皮祖细胞,测定其表面干细胞抗原-1(Sca-1)的表达并观察不同浓度香烟提取物(CSE)对内皮祖细胞增殖能力和Sca-1表达的影响。方法：从12只健康4周雄性近交系SPF级C57BL/6J小鼠体内取四肢长骨骨髓细胞培养,于第七天取出贴壁细胞做内皮祖细胞鉴定并测定其细胞表面Sca-1表达情况。将内皮祖细胞植入孔板中,分四组,用原培养基继续培养的为对照组；其余三组为低浓度组、中浓度组、高浓度组,分别用浓度为1.0%,2.5%,5.0%的香烟提取物溶液干预。分别在干预后第3小时,第6小时,第24小时用MTT比色法测定活细胞数量。用Western Blot法测定对照组与CSE干预组Sca-1表达差异。结果：1、小鼠源性内皮祖细胞在第七天贴壁后呈现类圆形、纺缍形、梭形或多边形及“铺路石”样外观。它们能摄取DiI-acLDL,胞浆呈红色；与FITC-UEA-I连接,胞膜呈绿色,阳性率为55.30%。并能表达CD34.CD133.和Flk-1阳性,CD34+CD133+CD34+Flk-1+. CD34+CD133+Flk-1+日性率分别为12.28%、0.098%、3.85%。 2、MTT比色法测得对照组、低浓度组、中浓度组、高浓度组分别在3小时的OD值为(0.1404±0.0074)、(0.1900±0.0125)、(0.2137±0.0147)、(0.0158±0.0112)：6小时的OD值分别为(0.1444±0.0117)(0.1729±0.0132)(0.0529±0.0156)(0.0054±0.0046)；24小时的OD值分别为(0.1580±0.0013)(0.0834±0.0158)(0.0169±0.0062)(0.0008±0.0011)。CSE干预组OD值均较空白对照组显著降低(p均0.05),差异有统计学意义且与对照组相比,各干预组OD值的变化与CSE之间存在浓度、时间依赖性 3、流式细胞术显示内皮祖细胞表达Sca-1的阳性率为98.87%。Western-Blot法结果显示与对照组相比,CSE干预组的Sca-1蛋白表达显著下降(0.5657±0.0545vs0.2883±0.0658P0.05) 结论：采用密度梯度离心法和EGM-2MV培养体系能够较好的从小鼠骨髓中分离获取并培养出内皮祖细胞。小鼠源性内皮祖细胞表面可高度表达Sca-1。香烟提取物可同时降低内皮祖细胞增殖能力和Sca-1的表达。
[Abstract]:Aim: to isolate and extract mouse endothelial progenitor cells (EPCs), detect the expression of Sca-1), and observe the effects of cigarette extract of different concentrations on the proliferation and Sca-1 expression of EPCs. Methods: bone marrow cells were harvested from 12 healthy 4-week male inbred SPF C57BL / 6J mice. After 7th days, the adherent cells were taken out to identify the endothelial progenitor cells and the expression of Sca-1 on the surface of the endothelial progenitor cells was determined. The endothelial progenitor cells were implanted into the orifice plate and divided into four groups, which were cultured on the original medium as the control group, and the other three groups were the low concentration group, the other three groups were the low concentration group. The middle concentration group and the high concentration group were treated with the cigarette extract solution of 1.0% and 2.5%, respectively, at the 3rd and 6th hour after the intervention. The number of living cells was determined by MTT colorimetry at 24 hours, and the expression of Sca-1 in control group and CSE intervention group was detected by Western Blot method. Results the mouse endothelial progenitor cells were round, fusiform, fusiform, fusiform or polygonal and "paving stone" appearance after 7th days of adhesion. They could ingest DiI-acLDLs with red cytoplasm, and the cell membrane was green in connection with FITC-UEA-I. The positive rate of CD34.CD133.The daily rate of CD34 CD133 CD34 Flk-1. CD34 CD133 Flk-1 was 12.280.98 and 3.85, respectively. 2MTT colorimetric assay was used to determine control group, low concentration group and middle concentration group. The OD values of the high concentration group at 3 hours were 0.1404 卤0.0074, 0.1900 卤0.0125, 0.2137 卤0.0147, 0.0158 卤0.011210: 6, respectively 0.1444 卤0.0117, 0.1729 卤0.01320.0529 卤0.01566.054 卤0.0046min for 24 hours, respectively. The OD values of the intervention group were 0.1580 卤0.001375, 0.0834 卤0.015834 卤0.0620.0008 卤0.0062O 0.0008 卤0.00111.CSE were significantly lower than those of the control group (P < 0.05), and the difference was statistically significant compared with that of the control group (P < 0.01), and the OD value of the CSE intervention group was significantly lower than that of the control group (P < 0.01), and the OD value of the CSE intervention group was significantly lower than that of the control group (P < 0.01), and the OD value of the CSE intervention group was significantly lower than that of the control group (P < 0.05), which was significantly lower than that of the control group. There was a concentration and time dependent relationship between OD value and CSE in each intervention group. 3. The positive rate of Sca-1 expression in endothelial progenitor cells by flow cytometry was 98.87.Western-Blot. The results showed that the expression of Sca-1 protein decreased significantly (0.5657 卤0.0545 vs 0.2883 卤0.0658 P 0.05) in the control group compared with the control group. Conclusion: endothelial progenitor cells can be isolated and cultured from mouse bone marrow by density gradient centrifugation and EGM-2MV culture system, and Sca-1 can be highly expressed on the surface of mouse derived endothelial progenitor cells. Cigarette extract can decrease simultaneously. Low endothelial progenitor cell proliferation and Sca-1 expression.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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