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[2mg/(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 m L/(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-βmRNA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。
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 femalemice 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.
作者
窦晶莉
白力
庞春艳
张文兰
张伟
王永福
DOU Jing-li BAI Li PANG Chun-yan ZHANG Wen-lan ZHANG Wei WANG Yong-fu(Department of Rheumatology, the First Affiliated Hospital of Baotou Medical College, Baotou 014010, Inner Mongolia, China Key Laboratory of Autoimmunity of Inner Mongolia, Baotou 014010, Inner Mongolia, China)
出处
《北京大学学报（医学版）》
CAS
CSCD
北大核心
2016年第6期970-976,共7页
Journal of Peking University:Health Sciences
基金
国家自然科学基金项目(81360464)
内蒙古自然科学基金项目(2016MS08109)资助~~
