兰州大学机构库 >学院待认领
HGF促动脉血管形成的机理研究
Alternative TitleThe study on the mechanism of HGF- mediated arteriogenesis
高玉芳
Thesis Advisor哈小琴
2009-05-12
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword肝细胞生长因子 NOTCH信号通路 动脉生成 基因转录 皮祖细胞
Abstract摘 要 目的: (1)确定Ad-HGF感染人股动脉内皮细胞(HFAECs)的最佳感染强度,初步探讨肝细胞生长因子(HGF)对HFAECs的Notch1和Dll4基因转录的调节作用,为进一步探讨HGF与Notch信号通路的关系奠定基础; (2)探讨HGF对HFAECs的Dll4-Notch-Hey2信号通路的激活作用,及此通路激活后对HFAECs增殖和迁移能力的影响,从血管新生的角度来阐明Notch信号调控HGF促动脉形成的作用及机制。 (3)体外分离培养及鉴定人内皮祖细胞(EPCs),为下一步从血管发生的角度探讨HGF是否通过Notch信号通路调控祖细胞的分化来促动脉形成的机理提供体外细胞模型。 方法: 第一部分 体外培养HFAECs,并用不同感染强度(MOI)(50,100,150,200,250,300pfu/cell)的携带绿色荧光蛋白基因的重组腺病毒(Ad-GFP)转染HFAECs,通过流式细胞仪(检测转染效率)和MTT法(检测细胞损伤程度)筛选和确定最佳MOI(高转染效率且对细胞低损伤),作为下一步携带HGF基因的重组腺病毒(Ad-HGF)转染细胞的最优条件。Ad-HGF以最佳的MOI转染HFAECs48 h后,收集细胞上清,用ELISA法检测HGF蛋白的表达水平,选取HGF表达水平最佳的细胞提取总RNA,通过反转录PCR(RT-PCR)来检测Notch1和Dll4基因的转录水平。以转染Ad-GFP的细胞作为对照。 第二部分 基于前期研究,Ad-HGF以最佳MOI(200 pfu/cell)转染HFAECs后0h、24h、48h、72h,分别收集细胞上清,用ELISA法检测不同时间点HGF蛋白的表达水平;同时收集细胞提取总RNA,通过RT-PCR检测不同时间点HFAECs中Notch1、Dll4和Hey2基因的转录水平;并采用MTT法和Transwell迁移实验分别观察HFAECs增殖和迁移能力的变化及与Dll4-Notch-Hey2信号通路激活的关系;均以转染Ad-GFP的细胞作对照。 第三部分 无菌、肝素抗凝,取脐血( 兰州军区总医院妇产科提供,产妇均知情同意)作为EPCs的标本来源。用PBS等倍稀释后取7 mL脐血缓慢加入装有3mL淋巴细胞分离液的离心管中,2000r/min离心25min,取云雾状灰白色层单个核细胞。将细胞接种在人纤维粘连蛋白包被的6孔板中,培养于M-199培养基中。观察贴壁细胞的形态变化;采用免疫细胞化学法鉴定EPCs。 实验数据采用SPSS13.0软件进行统计分析。 结果: (1)以高转染效率且对细胞低损伤为标准,确定最佳MOI为200 pfu/cell;ELISA法检测HGF表达水平的结果显示,Ad-GFP组:(3.672±0.810) ng/ml;Ad-HGF组:(86.318±3.864) ng/ml。两组比较转染后48 h的HGF表达水平有统计学差异(P<0.05); (2)Ad-HGF转染HFAECs48 h后,RT-PCR结果显示,通过紫外凝胶分析仪,可观察到Ad-HGF组的Notch1和Dll4基因转录水平明显上调;应用凝胶成像分析系统分析凝胶成像图中目的基因的相对强度,结果显示:Ad-GFP组和Ad-HGF组的数据分别为:Dll4,0.788±0.021和0.936±0.018;Notch1,0和0.458±0.023,两组间进行单侧t检验分析,P<0.05,有统计学意义。Ad-HGF组目的基因的相对强度明显高于Ad-GFP组; (3)Ad-HGF转染HFAECs不同时间段后,ELISA检测HGF表达水平的结果显示:Ad-HGF有效的导入了细胞,且HGF表达水平48 h内有时间依赖性,72h开始下降; (4)Ad-HGF...
Other AbstractAbstract Objective: (1) To investigate the regulation of Dll4 and Notch1 mediated by Hepatocyte growth factor(HGF)in human femoral artery endothelial cells(HFAECs), and to establish the basis for further investigating the mechanism of HGF promoting arteriogenesis; (2) To investigate the relationship between the proliferation and migration ability of artery endothelial cells and Dll4-Notch-Hey2 signaling pathway in the process of arteriogenesis, and elucidate the mechanism of HGF promoting arteriogenesis. (3) Endothelial progenitor cells(EPCs) were isolated,cultured and identified in vitro, and which were used as a study model from the point of angiogenesis, to further investigate that HGF promoted the formation of artery whether via Notch signal pathway regulating the differentiation of progenitor cells or not. Methods: Part 1 HFAECs were cultured in vitro and infected with the different multiplicity of infection(MOI) of Ad-GFP (50,100,150,200,250,300 pfu/cell). The optimal MOI was screened and determined by MTT (detecting the degree of cell damage ) and flow cytometry (detecting the rate of transfection) ,and used as the optimal MOI of Ad-HGF. At 48 h after HFAECs were infected with the optimal MOI of Ad-HGF, HGF expression level in the supernatant was detected by ELISA;meanwhile, the transcription of Notch1 and Dll4 genes was detected by reverse transcription-PCR(RT-PCR). Ad-GFP–infected HFAECs were used as negative controls. Part 2 On account of prophase study,cells and supernatant were harvested at the indicated times(0,24,48,72 h) after HFAECs were infected with the 200 pfu/cell of Ad-HGF. HGF expression in the supernatant was detected by ELISA and the transcription of Notch1,Dll4 and Hey2 genes was analyzed by RT-PCR. The changes in the proliferation,survivorship and migration ability of HFAECs, and the relationship between Dll4-Notch-Hey2 signaling pathway and them was detected respectively by MTT and Transwell migration experiment.Ad-GFP–infected HFAECs were used as negative controls. Part 3 Cord blood, asepstic, anticoagulated by Heparin, was used as the example resource of EPCs. After diluting with PBS(1:1), 7 mL of cord blood was added slowly into centrifuge tube which containing 3mL of lymphocyte isolation. To centrifuge for 30 min at 2000 r/min and collect the mononuclearcells in cloudiness buffy coat. Cells were inoculated in 6-well plate coated with human plasma fibronectin purified protein and cultured in M-199 medium. To o...
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Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/224106
Collection学院待认领
Affiliation临床医学院
Recommended Citation
GB/T 7714
高玉芳. HGF促动脉血管形成的机理研究[D]. 兰州. 兰州大学,2009.
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