【作者】 顾志峰；

【导师】 孙凌云；

【作者基本信息】 南京中医药大学 ， 中西医结合临床， 2009， 博士

【摘要】 目的:1.探讨系统性红斑狼疮(SLE)患者骨髓间质干细胞(MSCs)的超微结构及细胞骨架的形态学特征及探讨脐带间质干细胞(UC-MSCs)对MRL/lpr狼疮鼠的治疗作用和可能机制。方法:体外培养SLE患者的MSCs后,透射电子显微镜(TEM)、激光共聚焦显微镜(LCM)观察超微结构及肌动蛋白、纽蛋白的表达;24只18周龄雌性MRL/lpr小鼠,分为3组:对照组(G1)、UC-MSCs 1次治疗组(G2)、UC-MSCs 3次治疗组(G3)。观察体重,考马斯亮蓝法检测24h尿蛋白含量,酶联免疫吸附法(ELISA)检测抗双链DNA(ds-DNA)抗体水平、血清和尿液单核细胞趋化蛋白-1(MCP-1)水平,观察肾脏、肺、颌下腺病理改变蛋白印迹法检测肾脏MCP-1、高迁移率族蛋白B1(HMGB-1)、基质金属蛋白酶-2(MMP-2)和脾脏Foxp3蛋白水平的表达,实时定量PCR检测肾脏MCP-1、HMGB-1、MMP-2 mRNA和脾脏Poxp3基因表达,免疫组化检测MCP-1以及HMGB-1、MMP-2在肾脏的表达。流式细胞仪检测脾脏、淋巴结中CD4~+CD25~+Foxp3~+T细胞百分率,TUNEL法检测肾脏细胞的凋亡。结果:SLE MSCs透射电子显微镜下多数细胞内呈衰老征象,可见大量自噬体;细胞边缘应力纤维增多,排列紊乱,组成粗大的强荧光团,核周围应力纤维减少;纽蛋白在细胞边缘排列紊乱,细胞核周围增多;UC-MSCs移植后显著降低MRL/lpr狼疮鼠24h尿蛋白定量、血肌酐,改善肾脏、肺、颌下腺病理。G1组抗ds-DNA抗体滴度较G3组显著升高。HMGB-1mRNA表达量与血清抗ds-DNA抗体水平(r=0.469,P=0.021)及24h尿蛋白定量(r=0.766,P=0.000)相关。UC-MSCs移植能显著降低MCP-1、HMGB-1、MMP-2在MRL/lpr狼疮鼠肾脏的表达,上调脾脏CD4~+Foxp3~+T淋巴细胞的表达。UC-MSCs移植后系膜区细胞、肾小管间质细胞、肾血管周围浸润的炎性细胞凋亡增加。结论:SLE MSCs超微结构及细胞骨架的改变是细胞扩增不良的原因之一;UC-MSCs对MRL/lpr狼疮鼠有显著疗效,安全且无排斥反应;肾小球HMGB-1高表达与MRL/lpr鼠LN相关,抑制MCP-1、HMGB-1、MMP-2的表达及上调调节性T细胞表达,促进肾脏系膜区细胞凋亡,可能与UC-MSCs移植改善LN有关。更多还原

【Abstract】 ObjectiveTo explore the morphological character of bone marrow-derived mesenchymal stem cells (MSCs) in patients with systemic lupus erythematosus(SLE).To investigate the potential therapeutic effect and the possible mechanisms of human umbilical cord mesenchymal stem cells (UC-MSCs) transplantation in the MRL/lpr mice.MethodsBone marrow MSCs were isolated from two SLE patients and two healthy controls. Ultrastructural morphology was investigated using transmission electron microscope (TEM). Expression of actin and vinculin were assessed using laser Confocal microscopy (LCM). Twenty four 18-week-old female MRL/lpr mice were divided into 3 groups as following : the group 1(G1) had been treated with 0.5ml Phosphate buffer saline(PBS) as control,the group 2(G2)had been transplanted with 1×10~6 human UC- MSCs through caudal vein, and the group 3 (G3)had been transplanted with 1×10~6 UC- MSCs for three times via the caudal vein. Twenty-four hours proteinuria and bodyweights were assessed every two weeks. The levels of serum anti-ds-DNA antibody, urine and serum monocyte chemoattractant protein-1 (MCP-1) were measured using enzyme linked immunosorbent assay (ELISA) . The histopathology of the kidney, lung and sialaden was observed via immunohistochemical staining. Renal MCP-1, high mobility group box protein-1(HMGB-1), and matrix metalloproteinases-2 (MMP-2) and spleen (Foxp3) gene and protein expression were detecteded by quantitation real time polymerase chain reaction and western blot, respectively. Kidney MCP-1 and HMGB-1, MMP-2 expressions were assessed using immunohistochemistry. The percentage of CD4+ Foxp3+ T cell in spleen and lymph nodes was analyzed by flow cytometry. The kidney cell apoptosis was detected using TUNEL method.ResultsMSCs in patients with SLE have signs of aging: lots of autophagosomes, dense F-actin arranged disorderly at the edge of cytoplasm, dense vinculin arranged disorderly in the cytoplasm. In comparison to mice which received saline alone, mice received three weekly UC-MSCs showed marked reduction of 24h proteinuria, decrease in serum creatinine, and lower anti-ds-DNA antibody level with reduction of renal injury such as crescent formation. Less but significant decreases in these parameters were also observed in mice received single infusion. In comparison to control ground mice, UC-MSCs significantly reduced the extent of interstitial pneumonitis and sialadenitis in the MRL/lpr mice. Expression of HMGB-1 mRNA was positively correlated with the level of serum anti ds-DNA antibodies (r=0.469, P=0.021) and the 24 hours proteinuria (r=0.766, P=0.000). Marked inhibition of expression of MCP-1, HMGB-1 and MMP-2 were observed in the mice treated with UC-MSCs. The G2and G3 MRL/lpr mice had higher percentages of CD4+ Foxp3+ T cells in spleen and lower in lymphoid nodes as compared with controls (P<0.01).The expressions of Foxp3 protein and mRNA of spleen in G2 and G3 were both significantly higher than those in G1 (P<0.05).There was also a significantly difference between G2 and G3(P<0.05). The percentage of apoptotic cells in both the peritubular and perivascular infiltrating cells was also higher in G2 and G3 than in the control group. Besides, in both G2 and G3 mice, the number of apoptotic cells in glomerular mesangium increased significantly, as compared with that in G1 group(P<0.05).ConclusionsThe change of ultrastructure and cytoskeleton may explain why the MSCs from SLE patient showed an abnormal ability of cell expansion in vitro. These findings indicate that UC- MSCs has pleiotropic therapeutic effects on lupus nephritis, pneumonitis, and sialadenitis, suggesting a potential application of UC-MSCs in the treatment of human lupus. High expression of HMGB-1 may contribute to the pathogenesis of lupus nephritis in MRL/lpr mice. By upregulating Treg population and inducing inflammatory cell apoptosis, inhibition of MCP-1, HMGB-1, or MMP-2 may account for some of the therapeutic mechanisms of UC-MSCs in treating MRL/lpr mice.更多还原