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牙髓间充质干细胞治疗自身免疫性肝炎小鼠模型的效果及其免疫调控机制
DOI: 10.12449/JCH250719
伦理学声明：本研究方案于2023年6月14日经由首都医科大学附属北京积水潭医院动物研究委员会批准，批号：202306006，符合实验室动物管理与使用准则。
利益冲突声明：本文不存在任何利益冲突。
作者贡献声明：李茵负责设计论文框架，起草论文；宋光元负责实验操作，研究过程的实施； 李晓东负责数据收集，统计学分析、绘制图表；史婉婉负责论文修改；王贵强负责拟定写作思路，指导撰写文章并最后定稿。
Therapeutic effects of dental pulp stem cells in a mouse model of autoimmune hepatitis and related immunoregulatory mechanisms
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摘要:
目的 探讨牙髓间充质干细胞（DPSC）在体内外实验中对自身免疫性肝炎的治疗作用及相关机制。 方法 首先通过体外共培养体系评估DPSC的免疫调节作用；然后进行动物实验，将32只小鼠随机分成健康对照组、模型组、阳性药物组、DPSC治疗组，每组8只。检测各组血清ALT、AST及炎症因子水平，HE染色评估肝脏病理损伤。计量资料符合正态分布多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。 结果 体外实验结果显示DPSC的CD105、CD73和CD90阳性率分别为99.97%、100%和99.53%；而CD34、HLA-DR和CD45阳性率分别为0.56%、0.17%和0，符合间充质干细胞特征。DPSC可显著抑制辅助性T细胞（Th）1和Th17的增殖，抑制率分别为31.32%、45.76%；DPSC可促进调节性T细胞（Treg）（CD4+CD25+FoxP3+）的增殖，促进率为52.29%。DPSC对淋巴细胞的增殖抑制率为93.70%。在自身免疫性肝炎小鼠模型中，DPSC治疗组小鼠血清ALT、AST水平较模型组显著下降66.8%和60.0%（t值分别为3.321、2.907，P值分别为0.007 5、0.017 5），炎症相关因子TNF-α和IL-1β显著下降57.5%和71.3%（t值分别为2.484、2.796，P值分别为0.039 8、0.020 6），组织病理学结果显示门静脉周桥接坏死改善不明显（t=1.969，P=0.098）。 结论 DPSC通过免疫调节有效缓解免疫性肝损伤，为临床转化提供了实验依据。 -
关键词:
- 间质干细胞 /
- 牙髓 /
- 肝炎， 自身免疫性 /
- 小鼠， 近交BALB/C /
- 免疫调节
Abstract:Objective To investigate the therapeutic effect of dental pulp stem cells （DPSCs） on autoimmune hepatitis in invivo and in vitro experiments and the related mechanism. Methods An in vitro co-culture system was used to evaluate the immunoregulatory effect of DPSCs， and 32 mice were randomly divided into healthy control group， model group， positive drug group， and DPSCs treatment group， with 8 mice in each group. The serum levels of alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， and inflammatory factors were measured， and HE staining was used to assess liver pathological injury. An analysis of variance was used for comparison of normally distributed continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. Results The in vitro experiment showed that the positive rates of CD105， CD73， and CD90 in DPSCs were 99.97%， 100%， and 99.53%， respectively， while the positive rates of CD34， HLA-DR， and CD45 were 0.56%， 0.17%， and 0， respectively. DPSCs significantly inhibited the proliferation of Th1 and Th17 subsets， with inhibition rates of 31.32% and 45.76%， respectively； DPSCs promoted the proliferation of Treg （CD4+CD25+FoxP3+）， with a promoting rate of 52.29%. DPSCs had an inhibition rate of 93.70% on the proliferation of lymphocytes. In the mouse model of autoimmune hepatitis， compared with the model group， the DPSCs treatment group had significant reductions in the serum levels of ALT and AST， with reduction rates of 66.8% and 60.0%， respectively （t=3.321 and 2.907， P=0.007 5 and 0.017 5） and significant reductions in the inflammatory factors tumor necrosis factor-α and interleukin-1β， with reduction rates of 57.5% and 71.3%， respectively （t=2.484 and 2.796， P=0.039 8 and 0.020 6）， and histopathological examination showed no significant improvement in periportal bridging necrosis （t=1.969， P=0.098）. Conclusion DPSCs effectively alleviate immune-mediated liver injury through immunoregulation， which provides an experimental basis for clinical translation. -
Key words:
- Mesenchymal Stem Cells /
- Dental Pulp /
- Hepatitis， Autoimmune /
- Mice， Inbred BALB C /
- Immunoregulation
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注： a，牙髓分离；b，DPSC细胞培养光镜照片（×10）。
图 1 DPSC的培养与鉴定
Figure 1. Cultivation and identification of DPSC
注： a，成脂分化（油红O染色，×20）；b，成骨分化（茜素红染色，×4）；c，成软骨分化（阿利新蓝染色，×10）。
图 3 DPSC的三系分化能力
Figure 3. Osteogenic， adipogenic and chondrogenic differentiation of DPSC
注： a，Th1流式细胞仪结果；b，Th17流式细胞仪结果；c，Treg流式细胞仪结果；d，经CFSE染色的PBMC扩增分析。
图 4 DPSC在体外的免疫调节作用
Figure 4. Immunoregulation of DPSC in vitro
图 5 干细胞治疗ConA诱导的自身免疫性肝损伤的动物实验流程图
Figure 5. Flowchart of stem cell treatment for ConA-induced autoimmune liver injury in mouse
注： a，体质量变化；b，肝重量变化。
图 6 干细胞治疗前后小鼠体质量和肝质量变化
Figure 6. Changes in body weight and liver weight of mice before and after stem cell treatment
图 7 DPSC改善ConA诱导免疫性肝损伤小鼠的转氨酶和炎症因子
Figure 7. DPSC improve transaminase and inflammatory factors in ConA-induced immune liver injury mice
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