【摘要】：目的 1、探討氧化低密度脂蛋白(ox-LDL)對(duì)小鼠血管平滑肌細(xì)胞表達(dá)基質(zhì)細(xì)胞衍生因子1α(SDF-1α)的影響; 2、比較VSMC的條件培養(yǎng)液及SDF-1α對(duì)血管平滑肌細(xì)胞(VSMC)和平滑肌祖細(xì)胞(SPC)趨化遷移的影響。 方法 1、組織塊貼壁法培養(yǎng)小鼠主動(dòng)脈平滑肌細(xì)胞,用免疫組織化學(xué)方法對(duì)培養(yǎng)的平滑肌細(xì)胞進(jìn)行鑒定; 2、ox-LDL與VSMC共培養(yǎng),用免疫組織化學(xué)方法、酶聯(lián)免疫吸附試驗(yàn)(ELISA)測(cè)定VSMC表達(dá)SDF-1α蛋白的變化,用逆轉(zhuǎn)錄-聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)測(cè)定VSMC表達(dá)SDF-1αmRNA的變化,觀察劑量-時(shí)間效應(yīng); 3、趨化遷移實(shí)驗(yàn):將含有趨化因子SDF-1α的培養(yǎng)液置于Transwell小室下室,將VSMC或SPC懸液置于其上室,共孵育6h后,乙醇固定,高倍鏡下計(jì)數(shù)濾膜背面(下室面)的細(xì)胞數(shù),檢測(cè)VSMC的條件培養(yǎng)液及SDF-1α對(duì)VSMC和SPC的趨化遷移的影響。 結(jié)果 1、培養(yǎng)的小鼠血管平滑肌細(xì)胞的純度可以達(dá)到約90%。 2、小鼠血管平滑肌細(xì)胞有基礎(chǔ)水平的SDF-1α的表達(dá)。ox-LDL的濃度在0～60ng/ml范圍內(nèi),作用24h,SDF-1α蛋白的表達(dá)隨ox-LDL濃度的增加而變化,SDF-1α蛋白表達(dá)的峰值在ox-LDL濃度為45ng/ml時(shí),是ox-LDL濃度為0ng/ml時(shí)的1.4倍。在ox-LDL濃度為45ng/ml時(shí),0～48h范圍內(nèi),SDF-1α蛋白的表達(dá)變化隨時(shí)間的延長(zhǎng)而變化, SDF-1α蛋白表達(dá)的峰值在24h,是0h的1.5倍。在ox-LDL濃度為45ng/ml時(shí),0～30h范圍內(nèi),SDF-1αmRNA的表達(dá)變化隨時(shí)間的延長(zhǎng)而變化,SDF-1α基因mRNA的峰值在10h,是0h的3.2倍。 3、VSMC的條件培養(yǎng)液及SDF-1α對(duì)VSMC和SPC都具有明顯趨化作用。而且VSMC的條件培養(yǎng)液及SDF-1α對(duì)SPC的趨化作用強(qiáng)于對(duì)VSMC的趨化作用。 結(jié)論 1.、ox-LDL可增強(qiáng)小鼠血管平滑肌細(xì)胞SDF-1α的表達(dá)。 2、VSMC的條件培養(yǎng)液及SDF-1α對(duì)SPC的趨化作用明顯強(qiáng)于對(duì)VSMC的趨化作用。
[Abstract]:Objective 1 to investigate the effect of oxidized low density lipoprotein (ox-LDL) on the expression of stromal cell-derived factor-1 偽 (SDF-1 偽) in mouse vascular smooth muscle cells (VSMCs), and to compare the effects of conditioned medium of VSMC and SDF-1 偽 on the expression of (VSMC) and SDF-1 偽 in VSMCs. Effects of (SPC) chemotactic migration on smooth muscle progenitor cells. Methods 1. Smooth muscle cells of mouse aorta were cultured by tissue mass adherence method, and the cultured smooth muscle cells were identified by immunohistochemical method, 2the cultured smooth muscle cells were co-cultured with VSMC. The changes of SDF-1 偽 protein expression in VSMC and SDF-1 偽 mRNA in VSMC were detected by enzyme linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) respectively. (3) chemotactic migration experiment: the culture medium containing chemokine SDF-1 偽 was placed under the Transwell chamber, and the VSMC or SPC suspension was placed in the upper chamber. After incubation for 6 h, ethanol was fixed. The number of cells on the back of filter membrane (lower chamber surface) was counted under high power microscope, and the effects of conditioned medium of VSMC and SDF-1 偽 on chemotaxis migration of VSMC and SPC were detected. Results 1. The purity of cultured mouse vascular smooth muscle cells was about 90.2. The expression of SDF-1 偽 in vascular smooth muscle cells of mice was at a basic level. The concentration of ox-LDL was in the range of 0~60ng/ml. The expression of SDF-1 偽 protein changed with the increase of ox-LDL concentration at 24 h. The peak expression of SDF-1 偽 protein was 1.4 times higher than that of 0ng/ml when ox-LDL concentration was 45ng/ml. The expression of SDF-1 偽 protein changed with the prolongation of time when the concentration of ox-LDL was 45ng/ml. The peak expression of SDF-1 偽 protein at 24 h was 1.5 times higher than that at 0 h. The expression of SDF-1 偽 mRNA changed with the prolongation of time when the concentration of ox-LDL was 45ng/ml. The peak value of mRNA of SDF-1 偽 gene was at 10 h, which was 3.2 times of that of 0 h. The conditioned medium of VSMC and SDF-1 偽 had obvious chemotaxis on VSMC and SPC. Moreover, the conditioned medium of VSMC and SDF-1 偽 had stronger chemotaxis on SPC than on VSMC. Conclusion 1. Ox-LDL can enhance the expression of SDF-1 偽 in mouse vascular smooth muscle cells. (2) the conditioned medium of VSMC and SDF-1 偽 have stronger chemotaxis on SPC than on VSMC. 2.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R363

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本文編號(hào)：2205523