超声空化效应促人骨髓间充质干细胞体内归巢的实验研究

发布时间：2018-08-22 10:17

【摘要】：第一部分骨髓间充质干细胞中环氧化酶2及聚蛋白多糖酶1沉默与胰岛素样生长因子1表达【目的】观察携带基因环氧化酶2、聚蛋白多糖酶1的sh RNA干扰载体和携带胰岛素样生长因子1的过表达慢病毒载体在骨髓间充质干细胞中的表达情况。【方法】应用重组慢病毒技术构建携带沉默基因环氧化酶2、聚蛋白多糖酶1、过表达基因胰岛素样生长因子1和绿色荧光蛋白基因的重组慢病毒表达载体,并用其转染体外培养的第三代人骨髓间充质干细胞(实验组),并以无目的基因的慢病毒载体转染人骨髓间充质干细胞和未做处理的人骨髓间充质干细胞分别作为阴性对照组和空白组进行对比。【结果】慢病毒转染3d后,荧光显微镜下可见人骨髓间充质干细胞发出绿色荧光,阴性对照组、实验组转染效率均达90%以上。RT-PCR、Western blot和ELISA检测结果显示,环氧化酶2和聚蛋白多糖酶1在人骨髓间充质干细胞的基因和蛋白水平有受到了明显抑制,胰岛素样生长因子1基因和蛋白水平的表达均高于阴性对照组及空白组。【结论】应用慢病毒可在人骨髓间充质干细胞成功沉默环氧化酶2和聚蛋白多糖酶1基因,同时将胰岛素样生长因子1高表达,为系统性治疗类风湿性关节炎带来新的希望。第二部分超声空化效应促人骨髓间充质干细胞体内归巢的实验研究【目的】研究观察超声空化效应对慢病毒介导h COX-2 sh RNA、h Aggrecanse-1sh RNA、h IGF-1基因转染人骨髓间充质干细胞移植的可行性及干细胞在兔关节炎模型体内归巢的影响。【方法】关节炎模型造模成功的新西兰兔分为3组:转染基因人骨髓间充质干细胞+微泡+超声组(实验组)、转染基因人骨髓间充质干细胞+超声组(对照组)、转染基因人骨髓间充质干细胞+无处理组(空白组),移植细胞后检测兔后肢膝关节滑膜组织学观察及RT-PCR基因检测。【结果】超声空化效应可以增加慢病毒介导h COX-2 sh RNA、h Aggrecanse-1sh RNA、h IGF-1基因转染人骨髓间充质干细胞在兔关节炎模型进入靶器官的比例。【结论】为临床应用骨髓间充质干细胞治疗遗传免疫性疾病提供了有效途径及实验基础。
[Abstract]:Part I silencing and expression of insulin-like growth factor 1 in bone marrow mesenchymal stem cells by cyclooxygenase 2 and polyproteoglycan enzyme 1 [objective] to observe the expression of cyclooxygenase 2, polyproteoglycan 1 and sh RNA interference vector. Overexpression of lentivirus vector carrying insulin-like growth factor 1 in bone marrow mesenchymal stem cells. [methods] Recombinant lentivirus technique was used to construct cyclooxygenase 2, polyproteoglycan 1, and excess genes carrying silencing genes. Recombinant lentivirus expression vector expressing insulin-like growth factor 1 and green fluorescent protein gene. The third generation of human bone marrow mesenchymal stem cells (experimental group) were transfected with it and transfected into human bone marrow mesenchymal stem cells and untreated human bone marrow mesenchymal stem cells respectively. The negative control group and the blank group were compared. [results] after 3 days of lentivirus transfection, Fluorescence microscope showed that human bone marrow mesenchymal stem cells emit green fluorescence, but negative control group. The transfection efficiency of the experimental group was above 90%. The results of Western blot and ELISA detection showed that the transfection efficiency of the experimental group was above 90%. The gene and protein levels of human bone marrow mesenchymal stem cells were significantly inhibited by cyclooxygenase 2 and polyproteoglycan 1. The expression of insulin-like growth factor 1 gene and protein were higher than those of negative control group and blank group. [conclusion] Lentivirus can successfully silence cyclooxygenase 2 and polyproteoglycan 1 genes in human bone marrow mesenchymal stem cells. At the same time, the high expression of insulin-like growth factor 1 brings new hope for systemic treatment of rheumatoid arthritis. Part 2: experimental study on the effect of ultrasound cavitation on homing of human bone marrow mesenchymal stem cells in vivo [objective] to study the effect of ultrasonic cavitation on lentivirus mediated transfer of human bone marrow mesenchymal stem cells into h COX-2 sh RNA-hRNA-h IGF-1 gene The feasibility of transplantation and the effect of stem cells on homing in rabbits with arthritis. [methods] New Zealand rabbits with successful arthritis model were divided into three groups: microbubble ultrasound group of transfected human bone marrow mesenchymal stem cells (experiment) The ultrasonic group was transfected with human bone marrow mesenchymal stem cells (control group), and the control group was transfected with human bone marrow mesenchymal stem cells (blank group). The synovial tissue of rabbit hind limb and knee joint and RT-PCR base were detected after transplantation. [results] Ultrasonic cavitation effect could increase the proportion of human bone marrow mesenchymal stem cells transfected with lentivirus-mediated h COX-2 sh RNA-hRNA-h IGF-1 gene into target organs in rabbit arthritis model. [conclusion] it is a clinical application of bone marrow. Mesenchymal stem cells provide an effective approach and experimental basis for the treatment of hereditary immune diseases.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.2

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