三种血小板生成关键因子共转染人骨髓基质细胞的实验研究

发布时间：2018-06-07 14:37

本文选题：TPO + IL-6　；参考：《第三军医大学》2012年硕士论文

【摘要】：目的：通过构建TPO-pEGFP-N1、IL-6-pcDNA3.1(+)、IL-11-pcDNA3.1(-)三个真核表达载体，并用脂质体转染法共转染人骨髓基质细胞，观察三个真核表达载体在人骨髓基质细胞中的表达情况。方法： 1、分别对TPO、IL-6、IL-11三个基因片段克隆，再用PCR技术将克隆出来的TPO、IL-6、IL-11基因片段和pEGFP-N1、pcDNA3.1(+)、pcDNA3.1(-)这三个质粒分别连接，构建成TPO-pEGFP-N1、IL-6-pcDNA3.1(+)、IL-11-pcDNA3.1(-)三个真核表达载体，并进行酶切鉴定及测序分析。 2、用贴壁筛选法对人骨髓基质细胞进行分离，选用分裂能力强的第三代细胞做为转基因的靶细胞，，用脂质体转染法将TPO-pEGFP-N1、 IL-6-pcDNA3.1(+)、IL-11-pcDNA3.1(-)三个真核表达载体共转染人骨髓基质细胞，并采用细胞免疫组化及Western blot法等方法检测多基因转染人骨髓基质细胞后在mRNA和蛋白水平的表达。结果： 1、酶切及测序结果证实，本实验成功构建TPO-pEGFP-N1、IL-6-pcDNA3.1(+)、IL-11-pcDNA3.1(-)三个真核表达载体。 2、人骨髓基质细胞的体外培养形态呈异质性，原代细胞呈集落生长，传代后则均匀生长。 3、TPO-pEGFP-N1、IL-6-pcDNA3.1(+)、IL-11-pcDNA3.1(-)三个真核表达载体转染人骨髓基质细胞后，荧光显微镜下可观察到TPO-pEGFP-N1的表达，25%细胞发出绿色荧光；免疫细胞化学染色检测出IL-6及IL-11在细胞内表达；经Western blot检测：TPO蛋白表达(TPO/GAPDH IOD比值)在正常对照组为(0.17±0.01)，空载体转染组为(0.18±0.01)，多基因转染组为(0.62±0.01)；IL-6蛋白表达(IL-6/GAPDH IOD比值)在正常对照组为(0.14±0.01)，空载体转染组为(0.18±0.04)，多基因转染组为(0.34±0.06)；IL-11蛋白表达(IL-11/GAPDH IOD比值)在正常对照组为的(0.8±0.23)，空载体转染组为(0.19±0.14)，多基因转染组为(1.62±0.23)。多基因转染组细胞的蛋白水平明显高于对照组(P均0.01)，表明转染成功。结论： 1、通过分子生物学方法可成功构建TPO-pEGFP-N1、 IL-6-pcDNA3.1(+)、IL-11-pcDNA3.1(-)三个真核表达载体。 2、TPO、IL-6、IL-11三个基因可共转染人骨髓基质细胞，并在细胞内有效表达，将转染后的人骨髓基质细胞作为血小板体外培养的滋养细胞可能对血小板生成起促进作用。
[Abstract]:Objective: Three eukaryotic expression vectors of TPO-pEGFP-N1mIL-6-pcDNA3.1were constructed and co-transfected into human bone marrow stromal cells by liposome transfection. The expression of three eukaryotic expression vectors in human bone marrow stromal cells was observed. Methods: 1. TPO-pEGFP-N1IL-6-pcDNA3.1 (pEGFP-N1) plasmid was cloned into three eukaryotic expression vectors of TPO-pEGFP-N1IL-6-pcDNA3.1by PCR technique, and identified by restriction endonuclease digestion, and sequenced and analyzed by restriction endonuclease digestion and sequencing. The three eukaryotic expression vectors of TPO-pEGFP-N1IL-6-pcDNA3.1 (TPO-pEGFP-N1IL-6-pcDNA3.1m-1) were constructed. 2. Human bone marrow stromal cells were isolated by adherent screening, and the third generation cells with strong mitotic ability were selected as target cells. Three eukaryotic expression vectors, TPO-pEGFP-N1, IL-6-pcDNA3.1 (TPO-pEGFP-N1, IL-6-pcDNA3.1) were co-transfected into human bone marrow stromal cells by liposome transfection, and the three eukaryotic expression vectors (TPO-pEGFP-N1, IL-6-pcDNA3.1) were co-transfected into human bone marrow stromal cells. The expression of mRNA and protein in human bone marrow stromal cells was detected by immunohistochemistry and Western blot. Results: 1. The results of restriction endonuclease digestion and sequencing confirmed that three eukaryotic expression vectors, TPO-pEGFP-N1, IL-6-pcDNA3.1 (TPO-pEGFP-N1), were successfully constructed. 2. The morphology of human bone marrow stromal cells was heterogeneity in vitro, and the primary cells grew in colony, and then grew homogeneously after passage. (3) after transfection of three eukaryotic expression vectors of TPO-pEGFP-N1 and IL-6-pcDNA3.1into human bone marrow stromal cells, 25% of the cells expressed TPO-pEGFP-N1 were observed to emit green fluorescence under fluorescence microscope, and IL-6 and IL-11 were detected by immunocytochemical staining. The ratio of TPO / GAPDH IOD in normal control group, empty vector transfection group and polygene transfection group was 0.17 卤0.01, 0.62 卤0.01 and 0.62 卤0.01 respectively. The ratio of TPO / GAPDH IOD was 0.14 卤0.01 in normal control group, 0.18 卤0.04 in empty vector transfection group and 0.18 卤0.04 in polygene transfection group. The ratio of IL-11 / GAPDH IOD was 0.34 卤0.06 in the control group, 0.19 卤0.14 in the empty vector transfection group and 1.62 卤0.23 in the multigene transfection group. The protein level of the cells in the polygene transfection group was significantly higher than that in the control group (P < 0.01), indicating that the transfection was successful. Conclusion: 1.Three eukaryotic expression vectors of TPO-pEGFP-N1, IL-6-pcDNA3.1 were successfully constructed by molecular biological methods. 2TPO-IL-6 / IL-11 gene can be co-transfected into human bone marrow stromal cells and expressed effectively in the cells. The transfection of human bone marrow stromal cells as trophoblastic cells in vitro may promote platelet-forming.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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