HGF基因对血管内皮祖细胞增殖的影响

发布时间：2018-01-26 05:01

本文关键词： 肝细胞生长因子血管内皮祖细胞基因转染细胞培养大鼠　出处：《天津医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的离体条件下从大鼠骨髓中分离出单个核细胞,使用特殊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pIRES2-EGFP-HGF质粒,以阳离子脂质体(Lipofectamine~(TM)2000)介导pIRES2-EGFP-HGF质粒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。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,1天、2天、3天、5天、7天浓度分别为638.48±66.91、1230.33±96.35、2097.88±138.32、4017.20±169.92、1869.87±101.22(pg/ml),而空载质粒组和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5天和7天,转染组血管内皮祖细胞增殖明显加快,与空载质粒组及阴性对照组相比均有统计学意义(P0.05)。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.通过阳离子脂质体介导,可成功的将含有HGF基因的质粒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。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]:Purpose Mononuclear cells were isolated from rat bone marrow in vitro. The mononuclear cells were made to endothelial progenitor cells by using special culture medium. HGF gene was transfected into primary cultured rat vascular endothelial progenitor cells. To observe the expression of HGF in supernatant of transfected cells and the effect of HGF gene on the proliferation of primary cultured endothelial progenitor cells. Method 1. Bone marrow of femur and tibia of 4-6 weeks old Wistar rats were isolated by density gradient centrifugation. The endothelial progenitor cells were differentiated into vascular endothelial progenitor cells induced by EGM-2MV. The growth of cultured cells was observed by inverted phase contrast microscope. Cell uptake of DiL-acLDL-1 was observed by fluorescence microscope, and FITC-UEA-1 was used to identify the cells from a functional point of view. The cell surface antigen CD133 was determined dynamically by flow cytometry. Flk-1 VE-cadherin CD144) and CD31 further identified the cultured cells as vascular endothelial progenitor cells. 2. PIRES2-EGFP-HGF plasmid was extracted and amplified. PIRES2-EGFP-HGF plasmid was transfected into vascular endothelial progenitor cells with cationic liposome Lipofectaminetamine2 / TMN 2000 and the transfection efficiency was calculated. The expression of HGF protein after transfection was detected by ELISA method. The effect of HGF gene on the proliferation of EPCs was detected by MTT assay. Results 1. Vascular endothelial progenitor cells from rat bone marrow were isolated and cultured successfully, and identified by cell function test and flow cytometry. 2.The 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 supernatant of cell culture in HGF transfection group. The concentration of HGF was 638.48 卤66.91 ~ 1230.33 卤96.35 on day 1, day 2, day 3 and day 5 and day 7, respectively. 2097.88 卤138.32 卤4017.20 卤169.92 卤1869.87 卤101.22 g / ml). The expression of HGF was not detected in the no-load plasmid group and the negative control group. After transfection, HGF gene could significantly promote the proliferation of primary cultured endothelial progenitor cells, and the proliferation of vascular endothelial progenitor cells in transfection group accelerated significantly 5 and 7 days after transfection. Compared with the blank plasmid group and the negative control group, there was significant difference (P 0.05). Conclusion 1. Mononuclear cells were isolated from rat bone marrow in vitro and vascular endothelial progenitor cells were induced. 2. The plasmid containing HGF gene could be successfully transfected into rat vascular endothelial progenitor cells by cationic liposome, and HGF protein could be detected in culture supernatant. It is proved that the transfected target gene can effectively express in the cells and promote the proliferation of EPCs, which provides the experimental basis for the next step of gene stem cell transplantation in the treatment of limb ischemic diseases.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346

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