Transplantation of human umbilical cord mesenchymal stem cells in the treatment of systemic lupus erythematosus

doi:10.3969/j.issn.2095-4344.2015.14.008

Abstract

Abstract:

BACKGROUND: Systemic lupus erythematosus is an autoimmune disease characterized as an emergence of a variety of autoantibodies in serum and multi-system and multi-organ lesions. Currently, there is a lack of effective treatment options, and umbilical cord mesenchymal stem cells are a promising therapy for systemic lupus erythematosus based on cell biological roles.

OBJECTIVE: To observe the therapeutic efficacy of human umbilical cord mesenchymal stem cell transplantation in the treatment of systemic lupus erythematosus in mice.

METHODS: Human umbilical cord mesenchymal stem cells were isolated and cultured followed by labeling with DiR fluorescence. Experimental mice were divided into normal control group (C57BL mice), model control group (C57BL/lpr mice), low-, medium- and high-dose umbilical cord mesenchymal stem cell therapy groups (C57BL/lpr mice), with 10 mice in each group. Mice in the low-, medium- and high-dose groups were respectively injected 0.5×10⁶, 1×10⁶, 2×10⁶ human umbilical cord mesenchymal stem cells, once a week, for 3 consecutive weeks. At the end of treatment, blood samples were collected to measure antinuclear antibody, anti-histone antibody, anti-double stranded DNA antibody changes; OPG and Foxp3 gene expression changes were detected by quantitative PCR method.

RESULTS AND CONCLUSION: After treatment, the levels of anti-nuclear antibodies, anti-histone antibodies and anti-double stranded DNA antibodies in the peripheral blood of mice were all declined in the low-, medium- and high-dose groups, while the number of peripheral blood CD4⁺CD25⁺T cells was significantly elevated. OPG and Foxp3 gene expression was also increased dramatically in the low-, medium- and high-dose groups, which was similar to that in the normal control group and significantly different from that in the model control group (P < 0.01). Experimental findings demonstrate that after transplantation of human umbilical cord mesenchymal stem cells, all relevant indicators in C57BL/lpr mice recovered to the normal levels, and the high-dose treatment group had the most obvious effect.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: Umbilical Cord, Mesenchymal Stem Cell Transplantation, Lupus Erythematosus, Systemic

CLC Number:

R394.2

Ruan Guang-ping, Yao Xiang, Liu Ju-fen, Wang Jin-xiang, Hu Yuan-yuan, Li Zi-an, Yang Jian-yong, Pang Rong-qing, Pan Xing-hua. Transplantation of human umbilical cord mesenchymal stem cells in the treatment of systemic lupus erythematosus [J]. Chinese Journal of Tissue Engineering Research, 2015, 19(14): 2172-2178.

Figures/Tables 4

2.1 人脐带间充质干细胞的形态培养第7天，可见细胞贴壁生长，呈长梭形(图1A)。传到第3代时，可见细胞贴壁生长较多，呈长梭形，均匀分布(图1B)。 2.2 人脐带间充质干细胞的流式鉴定结果流式细胞仪检测结果表明人脐带间充质干细胞表达CD90、CD29、CD13，不表达CD31、CD34，符合间充质干细胞的特征(图2)。 2.3 实验动物数量分析参加实验小鼠数量50只，均进入结果分析，中途无脱落。 2.4 治疗后正常对照组、模型对照组和3个治疗组的抗核抗体水平见图3。模型对照组抗核抗体明显高于正常对照组和治疗组。5组之间两两比较，正常对照组与模型对照组相比，P < 0.01，模型对照组与其他4组相比，P < 0.01，但正常对照组与高剂量治疗组相比，P=0.083，说明高剂量组治疗后小鼠的抗核抗体接近正常对照组。 2.5 治疗后正常对照组、模型对照组和3个治疗组的抗组蛋白抗体水平见图4。模型对照组抗核抗体明显高于正常对照组和治疗组。5组之间两两比较，正常对照组与模型对照组相比，P < 0.01，治疗组与模型对照组相比，P < 0.01，但正常对照组与高剂量治疗组相比，P=0.043，说明高剂量组治疗后小鼠的抗组蛋白抗体降低，但与正常对照组相比差异仍有显著性意义。 2.6 治疗后正常对照组、模型对照组和3个治疗组的抗双链DNA抗体水平见图5。模型对照组抗双链DNA抗体明显高于正常对照组和治疗组。5组之间两两比较，正常对照组与模型对照组相比，P < 0.01，模型对照组与其他4组相比，P < 0.01，但正常对照组与高剂量治疗组相比，P=0.163，说明高剂量组治疗后小鼠的抗核抗体接近正常对照组。 2.7 治疗后正常对照组、模型对照组和3个治疗组的调节性T细胞和Foxp3阳性细胞的流式分析检测结果 2.7.1 CD4+CD25+T细胞的变化模型对照组的调节性T细胞明显降低，经过治疗后调节性T细胞有所升高，而高剂量治疗组的升高最明显(图6)，模型对照组调节性T细胞阳性率为7.9%，比正常对照组27.2%明显降低。治疗后低、中、高剂量组调节性T细胞分别升高到8.3%，10.2%，12.4%。 2.7.2 Foxp3阳性细胞的变化模型对照组的Foxp3阳性细胞明显减少，经过治疗后Foxp3阳性细胞增多，而高剂量治疗组升高到接近正常对照组(图7)。模型对照组Foxp3细胞阳性率为12%，比正常对照组59.5%明显降低。治疗后低、中、高剂量组Foxp3阳性细胞分别升高到47.6%，50.4%，50.4%。 2.8 治疗后正常对照组、模型对照组和3个治疗组的CD3和CD38双阳性细胞的流式检测结果模型对照组的CD3和CD38双阳性细胞明显增多，经过治疗后CD3和CD38双阳性细胞减少，而高剂量组降低到接近正常对照组(图8)。模型对照组CD3和CD38双阳性细胞为3.7%，比正常对照组1.5%明显升高。治疗后低、中、高剂量组CD3和CD38双阳性细胞分别降低到3.6%，2.9%，1.7%。 2.9 定量PCR检测外周血OPG和Foxp3两个基因表达变化从图9可看出，模型对照组OPG、Foxp3基因表达降低，治疗后恢复到正常水平。 2.10 治疗后治疗组小鼠的脏器体外成像 2.10.1 细胞的标记细胞标记后荧光显微镜观察，细胞染上了深红色荧光(图10A)。细胞标记后活体成像：取 0.1 mL细胞(1×105个)加到平皿上，用活体成像仪观察，可见细胞标记上了深红色荧光(图10B)。 2.10.2 小鼠内脏的体外成像标记DiR的间充质干细胞回输给系统性红斑狼疮模型小鼠，2周后取脏器进行体外成像，在肾脏观察到细胞聚集，说明细胞参与到肾脏病变的修复中，在肝脏也看到一些细胞，说明部分细胞滞留于肝脏，在肝脏被吞噬，其他器官未见标记细胞，说明细胞静脉回输后主要在肝和肾脏聚集(图11)。"

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