干扰素-γ预处理增强hUC-MSC免疫调节活性及其移植治疗小鼠系统性红斑狼疮的实验研究

发布时间：2018-06-16 11:33

本文选题：人脐带间充质干细胞 + 干扰素-γ　；参考：《青岛大学》2017年硕士论文

【摘要】：目的:探讨干扰素-γ预处理对人脐带间充质干细胞免疫调节活性的影响,及其对系统性红斑狼疮小鼠的治疗作用。方法:hUC-MSC经过IFN-γ(20ng/ml)预处理或者单独培养24小时,实时荧光定量核酸扩增检测系统和蛋白质印迹法检测细胞吲哚2,3双加氧酶的表达水平。24只20周左右大的雌性Fas-/-敲除的SLE模型小鼠被随机分为3组:PBS组、IFN-γ未处理h UC-MSC组、IFN-γ预处理h UC-MSC组。分别通过尾静脉注射等剂量(0.2ml)的PBS,IFN-γ未处理h UC-MSC(1x106)悬液以及IFN-γ(20ng/ml)预处理24h的h UC-MSC(1x106)的细胞悬液。收集Fas-/-小鼠24h尿液,送检验科检测尿蛋白水平。Fas-/-小鼠眼眶静脉取血,利用Elisa试剂盒测定Fas-/-小鼠血清抗双链DNA抗体(ds-DNA)水平以及血清肌酐酸(IMP)水平;免疫组织化学检测肾脏是否有免疫复合物Ig G及C3沉积。采用流式细胞仪检测小鼠血液中CD3+CD4+T细胞、CD3+CD8+T细胞、CD4+CD25+T细胞水平。结果:IFN-γ未处理h UC-MSC在RNA水平和蛋白水平均不表达IDO,而IFN-γ预处理h UC-MSC表达IDO。细胞移植后连续观察8周,发现IFN-γ预处理h UC-MSC组和IFN-γ未处理h UC-MSC组Fas-/-小鼠尿蛋白、血清IMP和血清ds-DNA水平均明显低于PBS组(P0.05或P0.01)。与移植治疗之前比,PBS组Fas-/-小鼠尿蛋白、血清IMP和血清ds-DNA水平显著提高(P0.05或P0.01);IFN-γ未处理h UC-MSC组Fas-/-小鼠尿蛋白、血清IMP和血清ds-DNA水平并没有发生显著性改变(P0.05);而IFN-γ预处理h UC-MSC组Fas-/-小鼠尿蛋白、血清IMP和血清ds-DNA水平则显著下降的趋势(P0.05或P0.01)。PBS组Fas-/-小鼠肾小球系膜区域呈现大量Ig G、C3免疫复合物,IFN-γ未处理h UC-MSC组Fas-/-小鼠肾小球系膜区域呈现少量Ig G、C3免疫复合物,IFN-γ预处理h UC-MSC组Fas-/-小鼠肾小球系膜区域没有Ig G、C3免疫复合物。IFN-γ未处理h UC-MSC组Fas-/-小鼠CD3+CD4+T细胞、CD3+CD8+T细胞水平与PBS组Fas-/-小鼠相比没有显著性变化(P0.05),IFN-γ预处理h UC-MSC显著地降低了Fas-/-小鼠CD3+CD4+T细胞、CD3+CD8+T细胞(P0.01)。与PBS组Fas-/-小鼠相比,IFN-γ未处理h UC-MSC升高了Fas-/-小鼠CD4+CD25+T水平(P0.01),同时IFN-γ预处理h UC-MSC组Fas-/-小鼠CD4+CD25+T水平明显高于IFN-γ未处理hUC-MSC组(P0.01)。结论:IFN-γ预处理hUC-MSC可显著提高hUC-MSC的抗炎活性,及其对SLE小鼠的治疗疗效,对系统性红斑狼疮疾病的治疗具有更好的前景。
[Abstract]:Aim: to investigate the effect of interferon-纬 preconditioning on the immunomodulatory activity of human umbilical cord mesenchymal stem cells and its therapeutic effect on systemic lupus erythematosus mice. Methods Twenty one HUC-MSC was pretreated with IFN- 纬 (20 ng / ml) or cultured alone for 24 hours. Real-time fluorescence quantitative nucleic acid amplification system and Western blotting were used to detect the expression of indocyanine 3-dioxygenase. Twenty-four female Fas-r-knockout SLE mice aged about 20 weeks were randomly divided into 3 groups: 1: 10% PBS group without treatment of IFN- 纬 for h UC-MSC group was pretreated with IFN- 纬 for h UC-MSC group. The cell suspensions of hUC-MSC1x106) were pretreated with the same dose of PBS- IFN- 纬 (0.2 ml) and IFN- 纬 (20ng / ml) for 24 h, respectively, and the cell suspensions of hUC-MSC1x106) were pretreated with IFN- 纬 20 ng / ml for 24 h. The urine samples of Fas-r / -mice were collected for 24 hours, and the urine protein level. Fas-r-mouse orbital vein blood was collected. The serum levels of anti-ds-DNA and creatinine IMP were measured by Elisa kit. Immunoglobulin G and C 3 deposition were detected by immunohistochemistry. The levels of CD 3 CD 4 T cells, CD 3 CD 8 T cells and CD 4 CD 25 T cells in the blood of mice were detected by flow cytometry. Results the hUC-MSC did not express IDO at RNA level and protein level, but IFN- 纬 pretreated hUC-MSC expressed IDO. After 8 weeks of cell transplantation, it was found that the levels of urinary protein, serum IMP and serum ds-DNA were significantly lower in IFN- 纬 pretreated hUC-MSC group and untreated hUC-MSC group than in PBS group (P0.05 or P0.01). Compared with those before transplantation, the levels of urinary protein, serum IMP and serum ds-DNA of Fas-r-mice in PBS group were significantly higher than those in control group (P 0.05 or P 0.01) IFN- 纬 untreated Fas-r-mouse urine protein in HUC-MSC group. The levels of serum IMP and serum ds-DNA did not change significantly (P 0.05), but IFN- 纬 pretreated the urinary protein of Fas-r-mices in UC-MSC group. The level of serum IMP and serum ds-DNA decreased significantly (P0.05) or Fas-r-mil of P0.01U / PBS group showed a large number of immunoreactive immune complexes (IFN- 纬) in glomerular Mesangial region of mice. The glomerular Mesangial region of HUC-MSC group showed a small amount of Ig GfC 3 immune complex in untreated h UC-MSC group. No immunoglobulin G C 3 immune complex. IFN- 纬 untreated Fas-r mouse CD3 CD4 T cell CD3 CD8 T cell level in hUC-MSC group was not significantly changed compared with PBS group Fas-r-mouse group (P0.05 IFN- 纬 preconditioning h UC-MSC). CD3 CD 4 T cells and CD 3 CD 8 T cells in Fas-R-mice were significantly decreased (P 0. 01). Compared with PBS group, untreated hUC-MSC with IFN- 纬 increased CD4 CD25T level of Fas-r-mouse and P0.01T of IFN- 纬 pretreated h UC-MSC group, and the CD4 CD25T level of IFN- 纬 pretreated hour UC-MSC group was significantly higher than that of hUC-MSC group without IFN- 纬 treatment (P0.01). Conclusion the anti-inflammatory activity of hUC-MSC and its therapeutic effect on SLE mice can be significantly improved by preconditioning hUC-MSC with 10% IFN- 纬, and has a better prospect for the treatment of systemic lupus erythematosus.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.241

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