本文選題：內(nèi)皮祖細(xì)胞　切入點(diǎn)：干細(xì)胞抗原　出處：《中南大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：分離提取小鼠內(nèi)皮祖細(xì)胞,測(cè)定其表面干細(xì)胞抗原-1(Sca-1)的表達(dá)并觀察不同濃度香煙提取物(CSE)對(duì)內(nèi)皮祖細(xì)胞增殖能力和Sca-1表達(dá)的影響。 方法：從12只健康4周雄性近交系SPF級(jí)C57BL/6J小鼠體內(nèi)取四肢長(zhǎng)骨骨髓細(xì)胞培養(yǎng),于第七天取出貼壁細(xì)胞做內(nèi)皮祖細(xì)胞鑒定并測(cè)定其細(xì)胞表面Sca-1表達(dá)情況。將內(nèi)皮祖細(xì)胞植入孔板中,分四組,用原培養(yǎng)基繼續(xù)培養(yǎng)的為對(duì)照組；其余三組為低濃度組、中濃度組、高濃度組,分別用濃度為1.0%,2.5%,5.0%的香煙提取物溶液干預(yù)。分別在干預(yù)后第3小時(shí),第6小時(shí),第24小時(shí)用MTT比色法測(cè)定活細(xì)胞數(shù)量。用Western Blot法測(cè)定對(duì)照組與CSE干預(yù)組Sca-1表達(dá)差異。 結(jié)果：1、小鼠源性內(nèi)皮祖細(xì)胞在第七天貼壁后呈現(xiàn)類圓形、紡綞形、梭形或多邊形及“鋪路石”樣外觀。它們能攝取DiI-acLDL,胞漿呈紅色；與FITC-UEA-I連接,胞膜呈綠色,陽(yáng)性率為55.30%。并能表達(dá)CD34.CD133.和Flk-1陽(yáng)性,CD34+CD133+CD34+Flk-1+. CD34+CD133+Flk-1+日性率分別為12.28%、0.098%、3.85%。 2、MTT比色法測(cè)得對(duì)照組、低濃度組、中濃度組、高濃度組分別在3小時(shí)的OD值為(0.1404±0.0074)、(0.1900±0.0125)、(0.2137±0.0147)、(0.0158±0.0112)：6小時(shí)的OD值分別為(0.1444±0.0117)(0.1729±0.0132)(0.0529±0.0156)(0.0054±0.0046)；24小時(shí)的OD值分別為(0.1580±0.0013)(0.0834±0.0158)(0.0169±0.0062)(0.0008±0.0011)。CSE干預(yù)組OD值均較空白對(duì)照組顯著降低(p均0.05),差異有統(tǒng)計(jì)學(xué)意義且與對(duì)照組相比,各干預(yù)組OD值的變化與CSE之間存在濃度、時(shí)間依賴性 3、流式細(xì)胞術(shù)顯示內(nèi)皮祖細(xì)胞表達(dá)Sca-1的陽(yáng)性率為98.87%。Western-Blot法結(jié)果顯示與對(duì)照組相比,CSE干預(yù)組的Sca-1蛋白表達(dá)顯著下降(0.5657±0.0545vs0.2883±0.0658P0.05) 結(jié)論：采用密度梯度離心法和EGM-2MV培養(yǎng)體系能夠較好的從小鼠骨髓中分離獲取并培養(yǎng)出內(nèi)皮祖細(xì)胞。小鼠源性內(nèi)皮祖細(xì)胞表面可高度表達(dá)Sca-1。香煙提取物可同時(shí)降低內(nèi)皮祖細(xì)胞增殖能力和Sca-1的表達(dá)。
[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 鹵0.0074, 0.1900 鹵0.0125, 0.2137 鹵0.0147, 0.0158 鹵0.011210: 6, respectively 0.1444 鹵0.0117, 0.1729 鹵0.01320.0529 鹵0.01566.054 鹵0.0046min for 24 hours, respectively. The OD values of the intervention group were 0.1580 鹵0.001375, 0.0834 鹵0.015834 鹵0.0620.0008 鹵0.0062O 0.0008 鹵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 鹵0.0545 vs 0.2883 鹵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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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