脂肪间充质干细胞对博来霉素诱导的硬皮病小鼠模型的治疗作用

doi:10.3969/j.issn.1671-167X.2016.06.009

北京大学学报(医学版) ›› 2016, Vol. 48 ›› Issue (6): 970-976. doi: 10.3969/j.issn.1671-167X.2016.06.009

脂肪间充质干细胞对博来霉素诱导的硬皮病小鼠模型的治疗作用

窦晶莉1,2 ，白力2，庞春艳2，张文兰2，张伟2，王永福1,2△

(1. 包头医学院第一附属医院风湿免疫科, 内蒙古自治区包头014010；2. 内蒙古自治区自体免疫学重点实验室，内蒙古自治区包头014010)

出版日期:2016-12-18 发布日期:2016-12-18
通讯作者: 王永福 E-mail:wyf5168@ hotmail.com
基金资助:
国家自然科学基金项目（81360464）和内蒙古自然科学基金项目(2016MS08109)资助

Therapeutic effect of adipose tissue-derived stem cells on bleomycin-induced mice of scleroderma

DOU Jing-li1,2，BAI Li2，PANG Chun-yan2，ZHANG Wen-lan2，ZHANG Wei2，WANG Yong-fu1,2△

(1. Department of Rheumatology, the First Affiliated Hospital of Baotou Medical College, Baotou 014010, Inner Mongolia, China; 2. Key Laboratory of Autoimmunity of Inner Mongolia, Baotou 014010, Inner Mongolia, China)

Online:2016-12-18 Published:2016-12-18
Contact: WANG Yong-fu E-mail:wyf5168@ hotmail.com
Supported by:
Supported by the National Natural Science Foundation of China(81360464) and the Inner Mongolia Autonomous Region Natural Science Foundation Projects(2016MS08109)

摘要/Abstract

摘要：

目的：研究脂肪间充质干细胞（adipose tissuederived stem cells，ADSCs)对博来霉素（bleomycin,BLM）诱导的硬皮病小鼠的治疗效果及其可能的作用机制。方法：选取 C57BL/6J 雌性小鼠 24 只，采用皮下注射 BLM ［2 mg/(kg·d)］建立硬皮病小鼠模型，随机分为正常对照组、BLM 组、ADSCs(皮下)组和 ADSCs（静脉）组，每组6只，正常对照组给予生理盐水2 mL/(kg·d)皮下注射，其余3组分别给予BLM皮下注射，连续3周，然后，ADSCs皮下和尾静脉注射组分别给予皮下和尾静脉注射 ADSCs（2×105个细胞）治疗，每4天1次，共3次。流式细胞术检测小鼠脾细胞中的辅助T细胞17（T-helper 17,TH17）及调节性T细胞（regulatory T cell ，Treg），实时荧光定量聚合酶联反应（ polymerase chain reaction,PCR）法检测小鼠肺组织中相关细胞因子的表达水平，酶联免疫吸附法（enzyme-linked immuno sorbent assay,ELISA）检测小鼠血清中白介素（interleukin,IL)-6、转化生长因子β（transforming growth factor-β,TGF-β）的含量，苏木精-伊红染色(hematoxylin eosin staining，HE ）观察小鼠的皮肤和肺组织病理学改变。结果：流式细胞术结果显示，BLM 组较正常对照组小鼠脾细胞中 TH17 和 Treg所占比例升高(15.30%±1.29 % vs. 4.32%±0.79%；9.90%±1.95% vs. 5.18%±1.35%，P均<0.05)，经 ADSCs 治疗后，TH17 所占比例降低(5.02%±0.83%,6.00%±0.82% vs.15.30%±1.29%， P均<0.05)，而 Treg所占比例升高(14.32%±1.59%,11.09%±4.31%vs. 9.90%±1.95%， P均<0.05)。与正常对照组相比，BLM 组小鼠肺组织中 IL-17、IL-6、肿瘤坏死因子α(tumor necrosis factor-α，TNF-α)的 mRNA 表达水平及血清中 IL-6 的含量均升高［3.54±0.30,10.65±0.66, 5.37±0.52 vs. 1.00±0.00; (21.2±1.74) ng/L vs. (16.87±1.09) ng/L， P均<0.05］，ADSCs 治疗后，其表达量下降［1.63±0.45,1.50±0.29 vs.3.54±0.30;3.11±0.85,2.98±0.76 vs.10.65±0.66;1.45±0.47,1.59±0.41 vs. 5.37±0.52; （17.87±1.45） ng/L, (17.61±1.16) ng/L vs. (21.2±1.74) ng/L， P均<0.05］，ADSCs(皮下)组和ADSCs（静脉）组比较差异无统计学意义（P>0.05)。BLM 组小鼠血清中 TGF-β 的含量较正常对照组升高［(33.95±2.49) ng/L vs. (28.8±2.29) ng/L， P<0.05］，但小鼠肺组织中 TGF-β mRNA 的表达水平与正常对照组之间差异无统计学意义( 1.17±0.11 vs.1.00±0.00， P>0.05)，经 ADSCs 治疗后 TGF-β基因和蛋白的表达无明显变化［1.25±0.11,1.26±0.12 vs.1.17±0.11; (31.84±2.04) ng/L, (31.25±2.36) ng/L vs. (33.95±2.49) ng/L，P>0.05］。HE 染色结果显示，BLM组与正常对照组相比，肺组织有大量炎性细胞浸润，肺泡间隔断裂或消失；皮肤真皮层增厚,有大量的胶原纤维积聚，经ADSCs治疗后小鼠肺组织炎症有所改善，皮肤的真皮层变薄，胶原纤维积聚减少。结论： ADSCs 可以有效缓解硬皮病小鼠肺的炎症和皮肤的纤维化症状，其抗炎和抗纤维化作用与其免疫调节功能有关。

关键词: 干细胞, 间充质, 硬皮病, 免疫调节, 细胞因子类

Abstract:

Objective： To investigate the effects and mechanisms of adipose-derived stem cells (ADSCs) on bleomycin-induced mice of scleroderma. Methods: In the study, 24 C57BL/6J female mice were randomly divided into control group, bleomycin（BLM）group, ADSCs (hypodermic injection) group and ADSCs (intravenous injection) group . BLM ［2 mg/(kg·d)］ was injected into the mice to establish the model of scleroderma. There were 6 mice in each group .The control group mice were injected with normal saline 2 mL/(kg·d) by subcutaneously. The rest of the three groups were injected with BLM. ADSCs groups were injected with ADSCs (2×105) subcutaneously and intravenously, respectively. T-helper 17 (Th17 ) and regulatory T cell (Treg cell) of spleen cells were detected by flow cytometry. The levels of cytokines in the lung tissue and in the serum were detected by real-time fluorescence quantification. Real-time polymerase chain reaction（PCR） and enzyme-linked immuno sorbent assay（ELISA）. The pathology change of skin and lung tissue was observed by hematoxylin eosin （HE）staining. Results: The proportion of Th17 and Treg increased in BLM group than in control group(15.30%±1.29% vs.4.32%±0.79%; 9.90%±1.95% vs.5.18%±1.35%, P<0.05), the expression of Th17 significantly decreased (5.02%±0.83%, 6.00%±0.82% vs.15.30%±1.29%, P<0.05) and the expression of Treg increased after the ADSCs therapy (14.32%±1.59%, 11.09%±4.31% vs. 9.90%±1.95%, P<0.05). The expression levels of IL-17，IL-6，tumor necrosis factor -α （TNF-α）mRNA in the lung tissue and IL-6 in the serum increased in BLM group than in control group ［3.54±0.30, 10.65±0.66, 5.37±0.52 vs. 1.00±0.00; (21.2±1.74) ng/L vs. (16.87±1.09) ng/L, P<0.05］. The expression of these cytokines significant decreased after the ADSCs therapy ［1.63±0.45,1.50±0.29 vs.3.54±0.30; 3.11±0.85, 2.98±0.76 vs.10.65±0.66;1.45±0.47, 1.59±0.41 vs. 5.37±0.52; (17.87±1.45) ng/L, (17.61±1.16) ng/L vs. (21.2±1.74) ng/L, P<0.05］. But there was no obvious difference between ADSCs (hypodermic injection) group and ADSCs (intravenous injection) group（P>0.05). The expression of TGF-β in the serum increased in BLM group than in control group［(33.95±2.49) ng/L vs. (28.8± 2.29) ng/L, P<0.05］, however, the expression of TGF-β mRNA had no significant differences than that of control group (1.17±0.11 vs.1.00±0.00, P>0.05). The expression of TGF-β mRNA and protein had no significant differences than that of BLM group ［1.25±0.11,1.26±0.12 vs.1.17±0.11; (31.84±2.04) ng/L, (31.25±2.36) ng/L vs. (33.95±2.49) ng/L, P>0.05］. HE staining showed that the inflammation of lung tissue was relieved and the dermal thickness and collagen deposition were decreased after the ADSCs therapy. Conclusion: ADSCs could effectively alleviate inflammation of the lungs and fibrosis of skin; the effects of antiinflammatory and antifibrosis were associated with immune regulating function.

Key words: Stem cells, mesenchymal, Scleroderma, Immune regulation, Cytokines

中图分类号:

R593.25

窦晶莉，白力，庞春艳，张文兰，张伟，王永福. 脂肪间充质干细胞对博来霉素诱导的硬皮病小鼠模型的治疗作用[J]. 北京大学学报(医学版), 2016, 48(6): 970-976.

DOU Jing-li，BAI Li，PANG Chun-yan，ZHANG Wen-lan，ZHANG Wei，WANG Yong-fu. Therapeutic effect of adipose tissue-derived stem cells on bleomycin-induced mice of scleroderma[J]. Journal of Peking University(Health Sciences), 2016, 48(6): 970-976.

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脂肪间充质干细胞对博来霉素诱导的硬皮病小鼠模型的治疗作用

Therapeutic effect of adipose tissue-derived stem cells on bleomycin-induced mice of scleroderma

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