内皮素-1对大鼠骨髓内皮前体细胞增殖、细胞周期、NO分泌及凋亡的影响

发布时间：2018-04-12 22:32

本文选题：内皮素 + 内皮前体细胞　；参考：《第四军医大学》2008年硕士论文

【摘要】： 研究背景及目的内皮素家族包括内皮素-1(ET-1)、内皮素-2(ET-2)和内皮素-3(ET-3),均是含有21个氨基酸残基的短肽。ET-1主要由血管内皮细胞分泌,在脉管系统作用最强,是已知最有力的血管收缩剂。ET-1是一种多功能调节肽,具有广泛的生物学效应,具有介导炎症、刺激其它激素分泌、促有丝分裂、刺激细胞增殖、移行和黏附等多种功能,通过自分泌和旁分泌方式经受体发挥作用。许多临床和实验研究证实ET-1作为一种血管活性肽,在血管生成过程中也发挥作用。ET-1通过直接作用于内皮细胞上的ETB受体,在新生血管的不同阶段发挥调节作用,包括细胞增殖、迁移和蛋白酶的合成等,在体内实验中ET-1也能刺激新血管生成。内皮前体细胞(EPC)又称内皮祖细胞,是一类能循环、增殖并分化为血管内皮细胞,但尚未表达成熟血管内皮细胞表型特征的前体细胞。研究发现,EPC不仅参与人胚胎血管生成,同时也参与出生后血管新生和内皮损伤后的修复过程。当发生血管损伤时,内皮前体细胞从骨髓释放入外周血,迁移至受损部位,掺入血管壁中,分化为成熟内皮细胞,修复受损血管,参与血管新生,改善甚至恢复缺血组织的血供。本研究拟探讨体外培养条件下,不同浓度ET-1对骨髓来源EPC的增殖、细胞周期、NO分泌和凋亡的影响,以期从新的角度理解ET-1对心血管系统的作用。实验方法第一部分EPC的培养和鉴定: 1、取大鼠股骨骨髓,以密度梯度离心法分离出单核细胞,在添加了VEGF和bFGF的M199培养基中培养,并定期观察; 2、DiⅠ-ac-LDL和FITC-UEA-Ⅰ免疫荧光双染,鉴定细胞表型。第二部分ET-1对EPC增殖、细胞周期、NO分泌和凋亡的影响: 1、取培养5天的细胞,分别给予不同浓度ET-1干预不同时间; 2、MTT检测细胞增殖能力; 3、流式细胞术检测细胞周期; 4、硝酸还原酶法检测NO分泌水平; 5、流式细胞术检测细胞凋亡率。统计学分析:数值均以x±s表示。数据采用SPSS11.0统计软件进行统计分析,应用单因素方差分析进行统计学分析,有统计学意义的采用LSD-t检验进一步比较。实验结果 1、细胞鉴定示细胞呈DiⅠ-ac-LDL和FITC-UEA-Ⅰ双阳性,证实所得到的细胞为EPC。 2、EPC增殖能力在ET-1干预后48h开始明显增强(P0.05),干预72h和96h后明显高于对照组(P0.01);在浓度为10-6mol/L时最为显著(P0.01)。 3、ET-1能够促进EPC向S期和G2/M期的转化,在浓度为10-6mol/L时达到最大效应(P0.01)。 4、EPC分泌NO能力在ET-1刺激后48h开始明显增强(P0.05),干预72h和96h后明显高于对照组(P0.01);在浓度为10-6mol/L时最为显著(P0.01)。 5、ET-1能够诱导EPC凋亡,干预48h时最显著(P0.01),72h、96h作用减弱。结论 1、ET-1促进EPC的增殖,作用呈浓度和时间依赖性。 2、ET-1促进EPC由G0/G1期向S期和G2/M期的转化,作用呈浓度依赖性。 3、ET-1提高细胞NO分泌功能,作用呈浓度和时间依赖性。 4、ET-1能诱导EPC凋亡,在干预48h时作用最强,之后其促凋亡作用减弱。 5、ET-1可显著促进EPC增殖及凋亡,但以促进增殖效应为主。
[Abstract]:Background and purpose of research
The endothelin family including endothelin -1 (ET-1), endothelin -2 (ET-2) and endothelin -3 (ET-3), is a short peptide.ET-1 containing 21 amino acid residues mainly secreted by vascular endothelial cells in the vasculature, the strongest effect is the most powerful known vasoconstrictor.ET-1 is a multifunctional regulatory peptide, which has extensive biological effects. It mediates inflammation, stimulate the secretion of other hormones, mitogenic stimulation, cell proliferation, adhesion and other shift function, through autocrine and paracrine manner through the receptors. Many clinical and experimental studies confirmed that ET-1 as a vasoactive peptide, also play a role.ET-1 through the direct effect on the endothelial cells of the ETB receptor in angiogenesis play a regulatory role in the different stages of angiogenesis, including cell proliferation, migration and protein synthesis in vivo, ET-1 can also stimulate new blood Guan Shengcheng.
Endothelial progenitor cells (EPC) also known as endothelial progenitor cells, which can circulate, proliferate and differentiate into vascular endothelial cells, progenitor cells but not expression of mature endothelial cells phenotype. The study found that EPC is not only involved in embryonic angiogenesis and in postnatal angiogenesis and endothelial repair after injury process. When the occurrence of vascular injury, endothelial progenitor cells derived from bone marrow into peripheral blood and migrate to the site of injury, the incorporation of the vessel wall, differentiate into mature endothelial cells, repair damaged blood vessels involved in angiogenesis, restore or improve the blood supply of ischemic tissue.
The aim of this study is to investigate the effects of different concentrations of ET-1 on proliferation, cell cycle, NO secretion and apoptosis of bone marrow derived EPC in vitro, in order to understand the role of ET-1 in cardiovascular system from a new perspective.
Experimental method
The first part of the culture and identification of EPC:
1, the bone marrow of the rat femur was taken and the mononuclear cells were isolated by density gradient centrifugation, and were cultured in the M199 medium adding VEGF and bFGF and observed regularly.
2, Di I -ac-LDL and FITC-UEA- I were immunofluorescent double staining to identify the cell phenotype.
Second the effects of ET-1 on EPC proliferation, cell cycle, NO secretion and apoptosis:
1, the cells were cultured for 5 days, and the different concentrations of ET-1 were given for different time, respectively.
2, MTT was used to detect cell proliferation.
3, flow cytometry was used to detect cell cycle.
4, the nitric acid reductase method was used to detect the level of NO secretion.
5, the rate of cell apoptosis was detected by flow cytometry.
Statistical analysis: the values were all expressed as x + s. The data were statistically analyzed by SPSS11.0 statistical software. Statistical analysis was performed by one-way ANOVA, and LSD-t test was used for statistical comparison.
experimental result
1, cell identification showed that the cells were Di I -ac-LDL and FITC-UEA- I positive, which proved that the cells obtained were EPC..
2, the proliferation ability of EPC began to increase significantly after ET-1 intervention (P0.05). After intervention of 72h and 96h, the proliferation of 48h was significantly higher than that of the control group (P0.01), while 10-6mol/L was the most significant (P0.01) when the concentration was 10-6mol/L.
3, ET-1 can promote the transformation of EPC into S phase and G2/M phase, and reach the maximum effect (P0.01) when the concentration is 10-6mol/L.
4, the ability of EPC secreting NO increased significantly after ET-1 stimulation (P0.05). After intervention of 72h and 96h, the level of 48h was significantly higher than that of control group (P0.01). When 10-6mol/L concentration was the most significant (P0.01).
5, ET-1 can induce EPC apoptosis and the most significant (P0.01) in 48h intervention, and the effect of 72h and 96h is weakened.
conclusion
1, ET-1 promotes the proliferation of EPC in a concentration and time dependent manner.
2, ET-1 promotes the transformation of EPC from phase G0/G1 to S and G2/M phase, and the effect is concentration dependent.
3, ET-1 increased the function of NO secretion, and the effect was concentration and time dependent.
4, ET-1 could induce apoptosis of EPC, and the effect was strongest when 48h was intervened, and then the effect of apoptosis was weakened.
5, ET-1 can significantly promote the proliferation and apoptosis of EPC, but mainly promote the proliferation effect.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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