脐带间充质干细胞治疗狼疮性肾炎的作用及机制
发布时间：2018-09-04 17:18
【摘要】：[目的]系统性红斑狼疮(Systemic Lupus Erythematosus,SLE)是一种病因及发病机制上不明确,累及多器官复杂的自身免疫性疾病。SLE最常见的临床表现是狼疮性肾炎(lupus nephritis,LN),在SLE死亡病例中占到60%～70%,狼疮性肾炎的病因和发病机制尚不十分清楚,目前认为细胞因子网络失衡或细胞因子异常表达是其发病的重要因素之一。间充质干细胞(MSC)治疗LN有一定效果。多项实验提示血或尿中骨桥蛋白(OPN)的表达和LN损伤程度及病情变化有着密切关系,对LN有重要的诊断和疗效判断价值。本课题的目的将探讨脐带组织来源间充质干细胞(UC-MSC)治疗LN的临床疗效和相关作用机制;探究骨桥蛋白(OPN)在UC-MSC治疗LN过程中的作用及相关机制。[方法]1.将13例确诊为LN且WHO分型为Ⅲ/Ⅳ型的患者给予HUC-MSC或安慰剂替代治疗(方案通过了伦理委员会),应用ELISA测定两组LN患者治疗后2、6月血清中OPN、AP-1、Sp-1、NF-KB、MMP-2和MMP-9在蛋白水平的变化。2. (1)将24只22周龄雌性MRL/lpr小鼠随机分为实验组(输注标记多聚赖氨酸修饰的超顺磁性氧化铁 SPIO-PLL (super paramagnetic iron oxide polylysine)的HUC-MSCs)和对照组(未输注HUC-MSCs), MRI扫描观察治疗后UC-MSCs分布情况;(2)观察两组MRL/lpr小鼠在处理前后不同时间点体重、尿蛋白及相关免疫学指标变化;(3)用携带目的基因绿色荧光蛋白GFP (green fluorescent protein)的慢病毒感染靶细胞,进行人脐带干细胞标记,注射进入MRL/lpr狼疮小鼠体内,观察UC-MSCs移植对MRL/lpr小鼠肾脏OPN表达影响。3.将24只22周龄雌性MRL/lpr小鼠随机分为两组:对照组和UC-MSCs治疗组(治疗组第18周进行1X106 P3代UC-MSCs尾静脉注射,对照组注射PBS)。12只22周龄的正常C57BL/6小鼠作为正常对照组。应用RT-qPCR,western-blot,免疫组化的方法检测HUC-MSCs移植治疗MRL/lpr狼疮小鼠前后与对照组肾脏组织中OPN、AP-1和Sp-1的表达水平,探索OPN的表达与AP-1和Sp-1相关性,同时检测NF-KB,MMP-2和MMP-9的mRNA水平和蛋白水平,并探索与OPN表达的相关性。[结果]1. HUC-MSC移植治疗LN患者2个月后,血清中OPN,MMP-2, MMP-9的表达水平与对照组相比均表现出降低。HUC-MSC移植治疗LN患者6个月后,治疗组OPN,MMP-2和MMP-9的表达水平与对照组相比,显著降低,差异均具有显著性(P0. 05)。2.血清中转录因子AP-1, SP-1和NF-κB/P65的表达水平:治疗后6个月,与治疗后2个月相比,治疗组的AP-1, SP-1和NF-κB的表达水平均有下降趋势。3. MRL/lpr狼疮小鼠HUC-MSC移植治疗后对比发现,治疗组小鼠24 h尿蛋白量,血清中抗ds-DNA抗体滴度,抗核抗体(ANA)滴度均显著降低,体重有明显增加。4. HUC-MSC移植能显著降低OPN、MMP-2和MMP-9在MRL/lpr小鼠血清中的表达(P0. 05)。血清中转录因子AP-1、SP-1和NF-κ B/p65的表达水平显著降低(P0. 05)。5. HUC-MSC移植可显著降低MRL/lpr狼疮小鼠肾脏组织中OPN,MMP-2, MMP-9及转录因子AP-1,SP-1和NF- κ B在蛋白及RNA的表达水平。6. HUC-MSC移植治疗显著降低MRL/lpr狼疮小鼠在肾小球系膜细胞、肾小管中OPN,MMP-2, MMP-9及转录因子AP-1,SP-1和NF-κB的表达水平。[结论]HUC-MSC 移植可有效降低 LN 患者血清中 OPN, AP-1, SP-1,NF-κB/P65, MMP-2和MMP-9的表达水平,对LN患者治疗有一定作用;HUC-MSC移植治疗MRL/lpr狼疮性小鼠肾炎有一定疗效,治疗组血清中OPN的表达量显著降低,血清中转录因子AP-1、SP-1、MMP-2、MMP-9和NF-κB/p65的表达在蛋白和RNA水平降低,在狼疮小鼠在肾小球系膜细胞、肾小管中表达也显著降低。提示HUC-MSC移植治疗狼疮肾炎可能是通过降低转录因子AP-1、SP-1和NF-κB的表达,抑制OPN的表达,OPN表达的减少抑制了 NF-κB通路的活化,进而降低通路下游MMP-2和MMP-9的表达,减少肾脏的损伤。该研究为HUC-MSC移植治疗LN提供了一定的依据。
[Abstract]:[Objective] Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease involving multiple organs with unclear etiology and pathogenesis. The most common clinical manifestation of SLE is lupus nephritis (LN), which accounts for 60% to 70% of SLE deaths. The etiology and pathogenesis of lupus nephritis are still unknown. It is not clear that the imbalance of cytokine network or abnormal expression of cytokines is one of the important factors in the pathogenesis of LN. Mesenchymal stem cells (MSC) have a certain effect on the treatment of LN. A number of experiments have suggested that the expression of osteopontin (OPN) in blood or urine is closely related to the degree of LN injury and the changes of LN condition. It is important for the diagnosis and treatment of LN. Objective: To investigate the clinical efficacy and mechanism of umbilical cord tissue-derived mesenchymal stem cells (UC-MSC) in the treatment of LN, and to explore the role and mechanism of osteopontin (OPN) in the treatment of LN by UC-MSC. [Methods] 1. HUC-MSC or placebo replacement were given to 13 patients with LN and WHO type III/IV. The levels of OPN, AP-1, Sp-1, NF-KB, MMP-2 and MMP-9 in serum of two groups of LN patients were measured by ELISA. 2. UC-MSCs of IC iron oxide polylysine group and control group (not infused with HUC-MSCs), the distribution of UC-MSCs was observed by MRI scan after treatment; (2) The changes of body weight, urinary protein and related immunological indexes were observed before and after treatment in two groups of MRL/lpr mice; (3) The slowness of GFP carrying the target gene green fluorescent protein (GFP) was observed. Human umbilical cord stem cells were labeled and injected into MRL/lpr lupus mice to observe the effect of UC-MSCs transplantation on OPN expression in kidney of MRL/lpr mice. Twelve 22-week-old normal C57BL/6 mice were injected with PBS as normal control group.The expression of OPN,AP-1 and Sp-1 in renal tissues of HUC-MSCs transplantation treated MRL/lpr mice and control group were detected by RT-qPCR,Western-blot and immunohistochemistry. The expression levels of OPN, MMP-2 and MMP-9 in serum of patients with LN after HUC-MSC transplantation were lower than those of the control group 2 months later. After 6 months of HUC-MSC transplantation, the expression levels of OPN, MMP-2 and MMP-9 in the treatment group were significantly lower than those in the control group. Serum levels of transcription factors AP-1, SP-1 and NF-kappa B/P65: 6 months after treatment, compared with 2 months after treatment, the expression levels of AP-1, SP-1 and NF-kappa B in the treatment group showed a downward trend. 3. Compared with HUC-MSC transplantation in lupus mice treated with MRL/lpr, 24 hours urinary protein and serum protein levels in the treatment group were found. HUC-MSC transplantation significantly reduced the expression of OPN, MMP-2 and MMP-9 in the serum of MRL/lpr mice (P 0.05). The expression levels of transcription factors AP-1, SP-1 and NF-kappa B/p65 in the serum were significantly decreased (P 0.05). 5. HUC-MSC transplantation significantly reduced the expression of MRL/lpr wolf. Expression of OPN, MMP-2, MMP-9 and transcription factors AP-1, SP-1 and NF-kappa B in renal tissues of mice with lupus erythematosus. 6. HUC-MSC transplantation significantly decreased the expression of OPN, MMP-2, MMP-9 and transcription factors AP-1, SP-1 and NF-kappa B in glomerular mesangial cells, renal tubules of mice with lupus erythematosus. The expression levels of OPN, AP-1, SP-1, NF-kappa B/P65, MMP-2 and MMP-9 in LN patients have a certain effect on the treatment of LN patients; HUC-MSC transplantation has a certain effect on the treatment of lupus nephritis in MRL/lpr mice; the expression level of OPN in serum of treatment group is significantly reduced, and the expression of transcription factors AP-1, SP-1, MMP-2, MMP-9 and NF-kappa B/p65 in protein and RNA. These results suggest that HUC-MSC transplantation may reduce the expression of transcription factors AP-1, SP-1 and NF-kappa B, inhibit the expression of OPN. The decrease of OPN expression inhibits the activation of NF-kappa B pathway, thereby reducing the expression of MMP-2 and MMP-9 downstream of the pathway. This study provides a basis for HUC-MSC transplantation in the treatment of LN.
【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R593.242
本文编号：2222829
[Abstract]:[Objective] Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease involving multiple organs with unclear etiology and pathogenesis. The most common clinical manifestation of SLE is lupus nephritis (LN), which accounts for 60% to 70% of SLE deaths. The etiology and pathogenesis of lupus nephritis are still unknown. It is not clear that the imbalance of cytokine network or abnormal expression of cytokines is one of the important factors in the pathogenesis of LN. Mesenchymal stem cells (MSC) have a certain effect on the treatment of LN. A number of experiments have suggested that the expression of osteopontin (OPN) in blood or urine is closely related to the degree of LN injury and the changes of LN condition. It is important for the diagnosis and treatment of LN. Objective: To investigate the clinical efficacy and mechanism of umbilical cord tissue-derived mesenchymal stem cells (UC-MSC) in the treatment of LN, and to explore the role and mechanism of osteopontin (OPN) in the treatment of LN by UC-MSC. [Methods] 1. HUC-MSC or placebo replacement were given to 13 patients with LN and WHO type III/IV. The levels of OPN, AP-1, Sp-1, NF-KB, MMP-2 and MMP-9 in serum of two groups of LN patients were measured by ELISA. 2. UC-MSCs of IC iron oxide polylysine group and control group (not infused with HUC-MSCs), the distribution of UC-MSCs was observed by MRI scan after treatment; (2) The changes of body weight, urinary protein and related immunological indexes were observed before and after treatment in two groups of MRL/lpr mice; (3) The slowness of GFP carrying the target gene green fluorescent protein (GFP) was observed. Human umbilical cord stem cells were labeled and injected into MRL/lpr lupus mice to observe the effect of UC-MSCs transplantation on OPN expression in kidney of MRL/lpr mice. Twelve 22-week-old normal C57BL/6 mice were injected with PBS as normal control group.The expression of OPN,AP-1 and Sp-1 in renal tissues of HUC-MSCs transplantation treated MRL/lpr mice and control group were detected by RT-qPCR,Western-blot and immunohistochemistry. The expression levels of OPN, MMP-2 and MMP-9 in serum of patients with LN after HUC-MSC transplantation were lower than those of the control group 2 months later. After 6 months of HUC-MSC transplantation, the expression levels of OPN, MMP-2 and MMP-9 in the treatment group were significantly lower than those in the control group. Serum levels of transcription factors AP-1, SP-1 and NF-kappa B/P65: 6 months after treatment, compared with 2 months after treatment, the expression levels of AP-1, SP-1 and NF-kappa B in the treatment group showed a downward trend. 3. Compared with HUC-MSC transplantation in lupus mice treated with MRL/lpr, 24 hours urinary protein and serum protein levels in the treatment group were found. HUC-MSC transplantation significantly reduced the expression of OPN, MMP-2 and MMP-9 in the serum of MRL/lpr mice (P 0.05). The expression levels of transcription factors AP-1, SP-1 and NF-kappa B/p65 in the serum were significantly decreased (P 0.05). 5. HUC-MSC transplantation significantly reduced the expression of MRL/lpr wolf. Expression of OPN, MMP-2, MMP-9 and transcription factors AP-1, SP-1 and NF-kappa B in renal tissues of mice with lupus erythematosus. 6. HUC-MSC transplantation significantly decreased the expression of OPN, MMP-2, MMP-9 and transcription factors AP-1, SP-1 and NF-kappa B in glomerular mesangial cells, renal tubules of mice with lupus erythematosus. The expression levels of OPN, AP-1, SP-1, NF-kappa B/P65, MMP-2 and MMP-9 in LN patients have a certain effect on the treatment of LN patients; HUC-MSC transplantation has a certain effect on the treatment of lupus nephritis in MRL/lpr mice; the expression level of OPN in serum of treatment group is significantly reduced, and the expression of transcription factors AP-1, SP-1, MMP-2, MMP-9 and NF-kappa B/p65 in protein and RNA. These results suggest that HUC-MSC transplantation may reduce the expression of transcription factors AP-1, SP-1 and NF-kappa B, inhibit the expression of OPN. The decrease of OPN expression inhibits the activation of NF-kappa B pathway, thereby reducing the expression of MMP-2 and MMP-9 downstream of the pathway. This study provides a basis for HUC-MSC transplantation in the treatment of LN.
【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R593.242
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