HGF基因促进内皮祖细胞增殖的体外研究
发布时间：2018-11-22 11:49
【摘要】：目的 体外条件下从大鼠骨髓中分离出单个核细胞,使用专用的培养基使其向内皮祖细胞(endothelial progenitor cells,EPCs)分化生长,并将HGF基因转染原代培养的大鼠血管内皮祖细胞,观察转染后细胞上清液中HGF的表达及HGF基因对原代培养血管内皮祖细胞增殖、迁移及血管形成能力的影响。 方法 1.取4-6周龄Wistar大鼠(120-150g)双下肢股骨及胫骨骨髓,利用密度梯度离心法分离单个核细胞,并使用内皮系专用培养液EGM-2MV诱导培养,使其分化为血管内皮祖细胞。通过倒置相差显微镜观察培养细胞的生长情况,通过荧光显微镜观察细胞摄取DiL-acLDL,结合FITC-UEA-1,从功能角度鉴定细胞,并通过流式细胞术动态测定细胞表面抗原CD133、flk-1、VE-cadherin(CD144)及CD31进一步鉴定所培养细胞为血管内皮祖细胞。 2.提取和扩增pCMV-HGF质粒,以缺陷性腺病毒(Ad-GFP)为载体介导HGF基因转染血管内皮祖细胞并计算转染效率；采用ELISA法检测转染后HGF蛋白的表达情况；用MTT法检测HGF基因对EPCs增殖的促进作用；Transwell检测细胞的迁移能力；3D培养法观察细胞体外血管形成能力。 结果 1.成功分离和培养出大鼠骨髓血管内皮祖细胞,并通过细胞功能检测和流式细胞术检测表面抗原来鉴定。 2.成功将HGF基因转染大鼠骨髓血管内皮祖细胞,荧光显微镜下可见绿色荧光蛋白的表达；在HGF转染组细胞培养上清中检测到HGF的表达,第1天、2天、4天、7天、11天浓度分别为22.15±3.77ng/ml、39.42±7.32ng/ml、99.09±9.89ng/ml、311.87±26.56 ng/ml、224.72±20.91ng/ml,而空载腺病毒组、阴性对照组没有检测出HGF的表达；转染后HGF基因对原代培养的血管内皮祖细胞生长有显著的促增殖作用,转染4天和7天,转染组血管内皮祖细胞增殖明显加快,与空载腺病毒组和阴性对照组相比均有统计学意义(P0.05)；转染后细胞迁移能力增强；3D培养可见血管样结构形成。 结论 1.体外条件下能够从大鼠骨髓中成功分离出单个核细胞并诱导培养出血管内皮祖细胞。 2.通过腺病毒介导,可成功的将HGF基因转染入大鼠血管内皮祖细胞,并且在培养上清液中可检测到HGF蛋白,证实转染后的目的基因能够在细胞中有效的表达并促进EPCs增殖、迁移及血管形成。为下一步利用该基因进行基因-干细胞移植治疗肢体缺血性疾病提供实验基础。
[Abstract]:Objective to isolate mononuclear cells from rat bone marrow in vitro and to differentiate and grow into endothelial progenitor cells (endothelial progenitor cells,EPCs) by using a special medium. The HGF gene was transfected into primary cultured rat vascular endothelial progenitor cells. The expression of HGF in supernatant of transfected cells and the effect of HGF gene on the proliferation, migration and angiogenesis of primary cultured endothelial progenitor cells were observed. Method 1. The bone marrow of femur and tibia of 4-6 week old Wistar rats (120-150g) was isolated by density gradient centrifugation. The mononuclear cells were induced by EGM-2MV and differentiated into endothelial progenitor cells (EPCs). The growth of cultured cells was observed by inverted phase contrast microscope, the uptake of DiL-acLDL, and FITC-UEA-1, was observed by fluorescence microscope, and the cell surface antigen CD133, was dynamically determined by flow cytometry. Flk-1,VE-cadherin (CD144) and CD31 further identified the cultured cells as vascular endothelial progenitor cells. 2. PCMV-HGF plasmid was extracted and amplified and HGF gene was transfected into vascular endothelial progenitor cells mediated by defective adenovirus (Ad-GFP) and the transfection efficiency was calculated. The expression of HGF protein after transfection was detected by ELISA method. MTT assay was used to detect the effect of HGF gene on the proliferation of EPCs; Transwell was used to detect the migration ability of cells; and 3D culture method was used to observe the ability of cells to form blood vessels in vitro. Result 1. Vascular endothelial progenitor cells from rat bone marrow were isolated and cultured successfully and identified by cell function test and flow cytometry. 2. HGF gene was successfully transfected into rat bone marrow vascular endothelial progenitor cells and the expression of green fluorescent protein was observed under fluorescence microscope. The expression of HGF was detected in the culture supernatant of HGF transfection group. The concentration of HGF was 22.15 卤3.77ng / ml / ml 39.42 卤7.32ng / ml / ml 99.09 卤9.89ng / ml / ml + 311.87 卤26.56 ng/ml, on day 1, day 2, day 4, day 7 and day 11, respectively. The expression of HGF was not detected in the control group (224.72 卤20.91 ng / ml), while in the non-loaded adenovirus group, the expression of HGF was not detected in the negative control group. After transfection, HGF gene significantly promoted the proliferation of vascular endothelial progenitor cells in primary culture. After 4 and 7 days of transfection, the proliferation of vascular endothelial progenitor cells was significantly accelerated in the transfected group. Compared with the control group and the non-loaded adenovirus group, there was statistical significance (P0.05). After transfection, the migration ability of the cells was enhanced, and vascular like structure was found in 3D culture. Conclusion 1. Mononuclear cells were isolated from rat bone marrow in vitro and vascular endothelial progenitor cells were induced. 2. HGF gene was successfully transfected into rat vascular endothelial progenitor cells mediated by adenovirus, and HGF protein was detected in culture supernatant. It was proved that the transfected target gene could effectively express in the cells and promote the proliferation of EPCs. Migration and angiogenesis. To provide experimental basis for the treatment of limb ischemic disease by gene-stem cell transplantation.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
本文编号：2349289
[Abstract]:Objective to isolate mononuclear cells from rat bone marrow in vitro and to differentiate and grow into endothelial progenitor cells (endothelial progenitor cells,EPCs) by using a special medium. The HGF gene was transfected into primary cultured rat vascular endothelial progenitor cells. The expression of HGF in supernatant of transfected cells and the effect of HGF gene on the proliferation, migration and angiogenesis of primary cultured endothelial progenitor cells were observed. Method 1. The bone marrow of femur and tibia of 4-6 week old Wistar rats (120-150g) was isolated by density gradient centrifugation. The mononuclear cells were induced by EGM-2MV and differentiated into endothelial progenitor cells (EPCs). The growth of cultured cells was observed by inverted phase contrast microscope, the uptake of DiL-acLDL, and FITC-UEA-1, was observed by fluorescence microscope, and the cell surface antigen CD133, was dynamically determined by flow cytometry. Flk-1,VE-cadherin (CD144) and CD31 further identified the cultured cells as vascular endothelial progenitor cells. 2. PCMV-HGF plasmid was extracted and amplified and HGF gene was transfected into vascular endothelial progenitor cells mediated by defective adenovirus (Ad-GFP) and the transfection efficiency was calculated. The expression of HGF protein after transfection was detected by ELISA method. MTT assay was used to detect the effect of HGF gene on the proliferation of EPCs; Transwell was used to detect the migration ability of cells; and 3D culture method was used to observe the ability of cells to form blood vessels in vitro. Result 1. Vascular endothelial progenitor cells from rat bone marrow were isolated and cultured successfully and identified by cell function test and flow cytometry. 2. HGF gene was successfully transfected into rat bone marrow vascular endothelial progenitor cells and the expression of green fluorescent protein was observed under fluorescence microscope. The expression of HGF was detected in the culture supernatant of HGF transfection group. The concentration of HGF was 22.15 卤3.77ng / ml / ml 39.42 卤7.32ng / ml / ml 99.09 卤9.89ng / ml / ml + 311.87 卤26.56 ng/ml, on day 1, day 2, day 4, day 7 and day 11, respectively. The expression of HGF was not detected in the control group (224.72 卤20.91 ng / ml), while in the non-loaded adenovirus group, the expression of HGF was not detected in the negative control group. After transfection, HGF gene significantly promoted the proliferation of vascular endothelial progenitor cells in primary culture. After 4 and 7 days of transfection, the proliferation of vascular endothelial progenitor cells was significantly accelerated in the transfected group. Compared with the control group and the non-loaded adenovirus group, there was statistical significance (P0.05). After transfection, the migration ability of the cells was enhanced, and vascular like structure was found in 3D culture. Conclusion 1. Mononuclear cells were isolated from rat bone marrow in vitro and vascular endothelial progenitor cells were induced. 2. HGF gene was successfully transfected into rat vascular endothelial progenitor cells mediated by adenovirus, and HGF protein was detected in culture supernatant. It was proved that the transfected target gene could effectively express in the cells and promote the proliferation of EPCs. Migration and angiogenesis. To provide experimental basis for the treatment of limb ischemic disease by gene-stem cell transplantation.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
【引证文献】
相关硕士学位论文 前2条
1 鲁明;海拔、民族、年龄、性别对外周血EPCs数、VEGF、HIF-1α浓度的影响研究[D];兰州大学;2012年
2 孙宁;HGF基因转染血管内皮祖细胞促进血管生成的动物实验研究[D];天津医科大学;2012年
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