低剂量辐射对骨髓间充质干细胞归巢特性的影响
发布时间：2018-10-14 17:47
【摘要】： 第一部分:人胎儿骨髓间充质干细胞分离纯化及基本生物学特性研究 目的观察人胎儿骨髓间充质干细胞(FMSCs)体外分离、培养方法,并对其生物学特性进行初步分析。 方法取4个月胎龄的流产胎儿,无菌条件下分离胎儿四肢骨,PBS冲洗骨髓腔;采用Ficoll法分离胎儿骨髓腔冲洗液,离心后取单个核细胞层培养、扩增、传代,观察细胞生物学特性;采用MTT法观察细胞生长曲线及分裂指数曲线。取第3代细胞,在含新生牛血清、地塞米松、β-甘油磷酸钠、抗坏血酸的DMEM-LG培养基中诱导其成骨分化;在含新生牛血清、地塞米松、胰岛素的DMEM-LG培养基中诱导其成脂分化。 结果接种48h后,贴壁细胞呈梭形、多角形、成纤维细胞样形态。10天后开始传代,传代培养的潜伏期为24-36h,对数增殖期约为2-3天,第6天进入平台期。传代至15代后生长速度变慢;FCM检测显示,细胞高表达CD29、CD44及CD49e分子,低表达CD106、CD54、CD11b,而不表达CD34。成骨、成脂培养基诱导3周后,Von Kossa染色呈黑色,油红染色成橘红色。 结论采用Ficoll密度梯度离心、贴壁筛选法及单克隆培养法可以分离、培养、扩增出具有高度同源性的胎儿骨髓间充质干细胞;并在体外成功的诱导其向成骨细胞、脂肪细胞分化。 第二部分:低剂量辐射对骨髓间充质干细胞体内分布和归巢特性的影响 目的观察骨髓间充质干细胞治疗脊髓损伤动物模型及经X线照射后的分布和归巢特性。 方法取部分昆明小鼠做骨髓间充质干细胞(BMMSCs)分离培养;部分按照改良的Allen's打击法建立脊髓损伤模型,并随机分成照射组和对照组;CFDA SE标记后的原代BMMSCs于照射组照射后30min时,给所有脊髓损伤模型的损伤局部进行细胞移植。分别于细胞移植后24h和96h在荧光显微镜下观察BMMSCs移植后在受者体内的分布及归巢特性。 结果细胞移植24h后,所有脊髓损伤模型的损伤部位均可见被植入的BMMSCs,但照射组明显多于对照组;96h后,照射组脊髓损伤部为仍可见被植入的BMMSCs,但其他组织未见该细胞;对照组没有检测到供体BMMSCs。 结论在脊髓损伤模型中,被移植后进入循环的间充质干细胞可以靶向分布到各损伤部位;经X线照射后,BMMSCs可以通过增殖能力的增强来维持机体内环境的稳定。
[Abstract]:Part I: isolation and purification of human fetal bone marrow mesenchymal stem cells and their basic biological characteristics objective to observe the methods of isolation and culture of human fetal bone marrow mesenchymal stem cells (FMSCs) in vitro. The biological characteristics were analyzed preliminarily. Methods Fetal limb bone was isolated from aborted fetus at 4 months of gestational age, and PBS was used to wash the medullary cavity of fetus, and Ficoll method was used to separate the lavage fluid of fetal medullary cavity. The mononuclear cell layer was cultured, amplified and subcultured after centrifugation, and the cell biological characteristics were observed. Cell growth curve and mitotic index curve were observed by MTT method. The osteogenic differentiation was induced in the DMEM-LG medium containing newborn bovine serum, dexamethasone, sodium 尾 -glycerophosphate and ascorbic acid, and adipogenic differentiation was induced in DMEM-LG medium containing newborn bovine serum, dexamethasone and insulin. Results 48 h after inoculation, the adherent cells were fusiform, polygonal and fibroblast-like. 10 days later, the incubation period was 24-36 hours, the logarithmic proliferation period was 2-3 days, and the sixth day entered the plateau phase. After passage to 15 generations, the growth rate became slower. FCM detection showed that the cells expressed CD29,CD44 and CD49e molecules, but not CD34. but low expression of CD106,CD54,CD11b,. After 3 weeks of induction, Von Kossa staining was black and oil red was orange. Conclusion the fetal bone marrow mesenchymal stem cells with high homology can be isolated and cultured by Ficoll density gradient centrifugation, adherent screening and monoclonal culture, and can be successfully induced to differentiate into osteoblasts and adipocytes in vitro. Part two: effects of low dose radiation on the distribution and homing characteristics of bone marrow mesenchymal stem cells objective to observe the distribution and homing characteristics of bone marrow mesenchymal stem cells in the treatment of spinal cord injury. Methods (BMMSCs) of bone marrow mesenchymal stem cells was isolated from some Kunming mice, and the spinal cord injury model was established by modified Allen's attack method. The mice were randomly divided into irradiation group and control group, the primary BMMSCs labeled by; CFDA SE were treated with 30min after irradiation. All spinal cord injury models were transplanted with cells. The distribution and homing characteristics of BMMSCs were observed under fluorescence microscope at 24 h and 96 h after transplantation, respectively. Results 24 hours after transplantation, implanted BMMSCs, could be seen in all the injured sites of spinal cord injury model, but it was more in irradiation group than in control group, 96 hours later, the injured part of spinal cord in irradiation group was still implanted BMMSCs, but no cells were found in other tissues. No donor BMMSCs. was detected in the control group Conclusion in the model of spinal cord injury, mesenchymal stem cells entering the circulation after transplantation can be targeted to various injury sites, and BMMSCs can maintain the stability of the internal environment through the enhancement of proliferative ability after X-ray irradiation.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329
本文编号：2271174
[Abstract]:Part I: isolation and purification of human fetal bone marrow mesenchymal stem cells and their basic biological characteristics objective to observe the methods of isolation and culture of human fetal bone marrow mesenchymal stem cells (FMSCs) in vitro. The biological characteristics were analyzed preliminarily. Methods Fetal limb bone was isolated from aborted fetus at 4 months of gestational age, and PBS was used to wash the medullary cavity of fetus, and Ficoll method was used to separate the lavage fluid of fetal medullary cavity. The mononuclear cell layer was cultured, amplified and subcultured after centrifugation, and the cell biological characteristics were observed. Cell growth curve and mitotic index curve were observed by MTT method. The osteogenic differentiation was induced in the DMEM-LG medium containing newborn bovine serum, dexamethasone, sodium 尾 -glycerophosphate and ascorbic acid, and adipogenic differentiation was induced in DMEM-LG medium containing newborn bovine serum, dexamethasone and insulin. Results 48 h after inoculation, the adherent cells were fusiform, polygonal and fibroblast-like. 10 days later, the incubation period was 24-36 hours, the logarithmic proliferation period was 2-3 days, and the sixth day entered the plateau phase. After passage to 15 generations, the growth rate became slower. FCM detection showed that the cells expressed CD29,CD44 and CD49e molecules, but not CD34. but low expression of CD106,CD54,CD11b,. After 3 weeks of induction, Von Kossa staining was black and oil red was orange. Conclusion the fetal bone marrow mesenchymal stem cells with high homology can be isolated and cultured by Ficoll density gradient centrifugation, adherent screening and monoclonal culture, and can be successfully induced to differentiate into osteoblasts and adipocytes in vitro. Part two: effects of low dose radiation on the distribution and homing characteristics of bone marrow mesenchymal stem cells objective to observe the distribution and homing characteristics of bone marrow mesenchymal stem cells in the treatment of spinal cord injury. Methods (BMMSCs) of bone marrow mesenchymal stem cells was isolated from some Kunming mice, and the spinal cord injury model was established by modified Allen's attack method. The mice were randomly divided into irradiation group and control group, the primary BMMSCs labeled by; CFDA SE were treated with 30min after irradiation. All spinal cord injury models were transplanted with cells. The distribution and homing characteristics of BMMSCs were observed under fluorescence microscope at 24 h and 96 h after transplantation, respectively. Results 24 hours after transplantation, implanted BMMSCs, could be seen in all the injured sites of spinal cord injury model, but it was more in irradiation group than in control group, 96 hours later, the injured part of spinal cord in irradiation group was still implanted BMMSCs, but no cells were found in other tissues. No donor BMMSCs. was detected in the control group Conclusion in the model of spinal cord injury, mesenchymal stem cells entering the circulation after transplantation can be targeted to various injury sites, and BMMSCs can maintain the stability of the internal environment through the enhancement of proliferative ability after X-ray irradiation.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329
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