间充质干细胞移植治疗糖尿病大鼠早期视网膜病变实验研究
发布时间：2018-10-18 10:45
【摘要】：目的：本课题首次将fBM-MSC用于DM动物模型早期视网膜病变的治疗，通过两种间充质干细胞、两种途径移植治疗DM大鼠早期视网膜病变的疗效观察,旨在探索更为安全有效的供体细胞类型和移植方法，为MSC治疗DR提供理论基础和实验依据。 方法：全程无血清间充质培养环境下体外分离、培养及鉴定hUC-MSC和fBM-MSC,利用HIV慢病毒表达载体转染GFP，将表达GFP的P5代hUC-MSC和fBM-MSC通过玻璃体腔注射和尾静脉注射两种途径移植到STZ诱导4w的糖尿病大鼠体内，检测各组移植前及移植后1w、4w、8w大鼠血糖、体重、FFA等大体情况，石蜡切片HE染色测量内层视网膜和外核层厚度变化，，冰冻切片免疫荧光染色观察供体细胞向视网膜的迁移定植及müller细胞的增生情况，western-blotting检测视网膜GFAP和Rhodopsin的蛋白表达，F-ERG记录各组视网膜功能的变化，统计对比后得出结论。结果：1.从人脐带和胎儿骨髓中均易分离培养出MSC，细胞纯度高，形态 均一且具有典型MSC形态特征，表达MSC特异抗原标志，体外扩增能力强且能保持其低分化状态和多向分化潜能；2.hUC-BMC和fBM-BMC分别采取玻璃体腔注射和尾静脉注射两种途径移植实验观察期内均无降血糖作用；3.hUC-MSC和fBM-MSC经过同种途径移植后表现出相似的生物学特点，经玻璃体腔移植后均可移行定植于糖尿病4w的大鼠视网膜，移植后4w左右移行细胞数量较多，主要位于视网膜神经纤维层和神经节细胞层，8w内细胞最远移行至视网膜外丛状层；而尾静脉注射移植组和正常对照组在实验观察期视网膜内未发现细胞。各组观察期内均未见成瘤或是其它不良病变；4.糖尿病大鼠4w左右就出现了视网膜组织结构、功能及特异蛋白表达水平的改变;5.玻璃体腔注射和全身静脉注射均是安全简便有效的干细胞移植途径，适用于临床，其中玻璃体腔注射途径更有利于供体细胞在病变视网膜内的移行定植。本实验中两种MSC通过两种途径移植于成模4w糖尿病大鼠后在8w观察期内均对视网膜结构、功能及蛋白表达具有保护作用，4w时效果较明显，其中hUC-MSC优于fBM-MSC，玻璃体腔注射移植效果明显好于尾静脉。 结论：1.从人脐带和胎儿骨髓中均易分离培养出MSC，细胞纯度高，体外扩增能力强且能保持其低分化状态和多向分化潜能；2.hUC-MSC和fBM-MSC能够感知和适应DM早期视网膜病变微环境，经玻璃体腔移植后均可移行定植于病变视网膜；3.两种MSC通过两种途径移植对于DM早期视网膜病变均有治疗效果，4w左右效果较明显，其中hUC-MSC优于fBM-MSC，玻璃体腔注射移植效果明显好于尾静脉；4.hUC-MSC在DR等视网膜变性疾病治疗领域具有广阔的应用前景。
[Abstract]:Objective: in this study, fBM-MSC was used for the treatment of early retinopathy in DM animal model for the first time. Two kinds of mesenchymal stem cells (MSCs) and two ways of transplantation were used to treat early retinopathy in DM rats. The purpose of this study was to explore more safe and effective donor cell types and transplantation methods, and to provide theoretical and experimental basis for the treatment of DR by MSC. Methods: the whole process of serum-free mesenchymal culture was isolated in vitro. HUC-MSC and fBM-MSC, were cultured and identified. GFP, was transfected with HIV lentivirus expression vector to transfer hUC-MSC and fBM-MSC expressing GFP into STZ induced diabetic rats by vitreous injection and caudal vein injection. The blood glucose, body weight and FFA were measured before transplantation and 1 week and 8 weeks after transplantation. The thickness of inner retina and outer nuclear layer were measured by HE staining in paraffin sections. The migration and implantation of donor cells to the retina and the proliferation of m 眉 ller cells were observed by immunofluorescence staining in frozen sections. The expression of GFAP and Rhodopsin in the retina was detected by western-blotting, and the changes of retinal function in each group were recorded by F-ERG. The result is 1: 1. MSC, cells were easily isolated and cultured from human umbilical cord and fetal bone marrow with high purity, uniform morphology and typical MSC morphological characteristics, and expressed MSC specific antigen markers. 2.hUC-BMC and fBM-BMC had no hypoglycemic effect by vitreous injection and caudal vein injection respectively. 3.hUC-MSC and fBM-MSC showed similar biological characteristics after transplantation in the same way. After vitreous transplants, they could be transplanted to the retina of rats with diabetes mellitus for 4 weeks, and the number of transitional cells was large about 4 weeks after transplantation. The cells were mainly located in the retinal nerve fiber layer and ganglion cell layer, and the cells were furthest transferred to the outer plexiform layer of the retina within 8 weeks, but no cells were found in the retina in the caudal vein transplantation group and the normal control group. No tumor or other adverse lesions were found in each group during the observation period. 4. The changes of retinal tissue structure, function and specific protein expression were observed in diabetic rats at about 4 weeks. Vitreous injection and systemic intravenous injection are safe, simple and effective approaches for stem cell transplantation, which are suitable for clinical use. The vitreous injection approach is more conducive to donor cell transplantation in the diseased retina. In this experiment, two kinds of MSC were transplanted into the model of 4w diabetic rats through two ways, and both of them had protective effects on the structure, function and protein expression of retina during the observation period of 8 weeks, and the effects were obvious at 4 weeks. The effect of hUC-MSC was better than that of fBM-MSC,. Conclusion 1. MSC, cells were easily isolated and cultured from human umbilical cord and fetal bone marrow. 2.hUC-MSC and fBM-MSC could sense and adapt to the microenvironment of DM early retinopathy. After vitreous cavity transplantation, it can be transplanted to the lesion retina. Two kinds of MSC were transplanted through two ways to treat the early retinopathy of DM. The effect of hUC-MSC was better than that of the caudal vein in 4 weeks. The effect of hUC-MSC was better than that of fBM-MSC, by intravitreal injection. 4.hUC-MSC has a broad application prospect in the treatment of retinal degeneration such as DR.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R587.2;R774.1
[Abstract]:Objective: in this study, fBM-MSC was used for the treatment of early retinopathy in DM animal model for the first time. Two kinds of mesenchymal stem cells (MSCs) and two ways of transplantation were used to treat early retinopathy in DM rats. The purpose of this study was to explore more safe and effective donor cell types and transplantation methods, and to provide theoretical and experimental basis for the treatment of DR by MSC. Methods: the whole process of serum-free mesenchymal culture was isolated in vitro. HUC-MSC and fBM-MSC, were cultured and identified. GFP, was transfected with HIV lentivirus expression vector to transfer hUC-MSC and fBM-MSC expressing GFP into STZ induced diabetic rats by vitreous injection and caudal vein injection. The blood glucose, body weight and FFA were measured before transplantation and 1 week and 8 weeks after transplantation. The thickness of inner retina and outer nuclear layer were measured by HE staining in paraffin sections. The migration and implantation of donor cells to the retina and the proliferation of m 眉 ller cells were observed by immunofluorescence staining in frozen sections. The expression of GFAP and Rhodopsin in the retina was detected by western-blotting, and the changes of retinal function in each group were recorded by F-ERG. The result is 1: 1. MSC, cells were easily isolated and cultured from human umbilical cord and fetal bone marrow with high purity, uniform morphology and typical MSC morphological characteristics, and expressed MSC specific antigen markers. 2.hUC-BMC and fBM-BMC had no hypoglycemic effect by vitreous injection and caudal vein injection respectively. 3.hUC-MSC and fBM-MSC showed similar biological characteristics after transplantation in the same way. After vitreous transplants, they could be transplanted to the retina of rats with diabetes mellitus for 4 weeks, and the number of transitional cells was large about 4 weeks after transplantation. The cells were mainly located in the retinal nerve fiber layer and ganglion cell layer, and the cells were furthest transferred to the outer plexiform layer of the retina within 8 weeks, but no cells were found in the retina in the caudal vein transplantation group and the normal control group. No tumor or other adverse lesions were found in each group during the observation period. 4. The changes of retinal tissue structure, function and specific protein expression were observed in diabetic rats at about 4 weeks. Vitreous injection and systemic intravenous injection are safe, simple and effective approaches for stem cell transplantation, which are suitable for clinical use. The vitreous injection approach is more conducive to donor cell transplantation in the diseased retina. In this experiment, two kinds of MSC were transplanted into the model of 4w diabetic rats through two ways, and both of them had protective effects on the structure, function and protein expression of retina during the observation period of 8 weeks, and the effects were obvious at 4 weeks. The effect of hUC-MSC was better than that of fBM-MSC,. Conclusion 1. MSC, cells were easily isolated and cultured from human umbilical cord and fetal bone marrow. 2.hUC-MSC and fBM-MSC could sense and adapt to the microenvironment of DM early retinopathy. After vitreous cavity transplantation, it can be transplanted to the lesion retina. Two kinds of MSC were transplanted through two ways to treat the early retinopathy of DM. The effect of hUC-MSC was better than that of the caudal vein in 4 weeks. The effect of hUC-MSC was better than that of fBM-MSC, by intravitreal injection. 4.hUC-MSC has a broad application prospect in the treatment of retinal degeneration such as DR.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R587.2;R774.1
【共引文献】
相关期刊论文 前6条
1 尤青青;戴春;;骨髓间充质干细胞在肾脏疾病中的应用进展[J];中国中西医结合肾病杂志;2013年08期
2 谭笑;汪年松;;干细胞治疗糖尿病肾病的研究进展[J];中国中西医结合肾病杂志;2013年08期
3 王晶;梁维邦;;干细胞移植治疗帕金森病的研究进展[J];东南大学学报(医学版);2013年06期
4 丁慰祖;姚慧萍;刘嫣;钱钧;许宇东;叶芳;;大鼠糖尿病模型的建立及其视网膜功能早期改变的研究[J];临床眼科杂志;2014年01期
5 汪年松;姜珍珍;;干细胞在肾脏疾病中的研究进展[J];中国中西医结合肾病杂志;2014年03期
6 卢辰;李华;刘源R
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