Chinese Journal of Tissue Engineering Research ›› 2023, Vol. 27 ›› Issue (10): 1492-1500.doi: 10.12307/2023.270
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Therapeutic effect of umbilical cord mesenchymal stem cells modified by miR-1-5p in MRL/lpr lupus mice
Ding Lili1, Hu Mingzhi1, Wu Zhihui1, Sun Xiaolin2, Wang Yongfu1
- 1Department of Rheumatology and Immunology, First Affiliated Hospital of Baotou Medical College, Baotou 014010, Inner Mongolia Autonomous Region, China; 2Central Laboratory (Key Laboratory of Autoimmunity in Inner Mongolia Autonomous Region), First Affiliated Hospital of Baotou Medical College, Baotou 014010, Inner Mongolia Autonomous Region, China
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Received:2022-02-10Accepted:2022-04-29Online:2023-04-08Published:2022-09-08 -
Contact:Wang Yongfu, MD, Professor, Department of Rheumatology and Immunology, First Affiliated Hospital of Baotou Medical College, Baotou 014010, Inner Mongolia Autonomous Region, China Sun Xiaolin, MD, Inspector-in-charge, Central Laboratory (Key Laboratory of Autoimmunity in Inner Mongolia Autonomous Region), First Affiliated Hospital of Baotou Medical College, Baotou 014010, Inner Mongolia Autonomous Region, China -
About author:Ding Lili, Master candidate, Department of Rheumatology and Immunology, First Affiliated Hospital of Baotou Medical College, Baotou 014010, Inner Mongolia Autonomous Region, China -
Supported by:the National Natural Science Foundation of China, No. 81860294 (to SXL); Natural Science Foundation of Inner Mongolia Autonomous Region, No. 2019MS08055 (to SXL); Natural Science Foundation of Inner Mongolia Autonomous Region, No. 2021MS08045 (to SXL); Science and Technology Planning Project of Inner Mongolia Autonomous Region, No. 201802089 (to SXL); Science and Technology Planning Project of Inner Mongolia Autonomous Region, No. 2019GG052 (to WYF)
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Ding Lili, Hu Mingzhi, Wu Zhihui, Sun Xiaolin, Wang Yongfu. Therapeutic effect of umbilical cord mesenchymal stem cells modified by miR-1-5p in MRL/lpr lupus mice[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(10): 1492-1500.
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2.1 UC-MSCs的形态 利用组织块培养法培养8 d，在显微镜下观察原代UC-MSCs，在组织块周围可见呈多角形或短梭形散落分布的单个细胞；12 d后细胞明显增多，细胞呈放射状或漩涡状密集生长并形成群落；16 d后细胞大多呈长梭形并可融合至70%-90%。传代后增殖速度较快，三四天可融合达到90%左右。该UC-MSCs的形态和生长规律均符合间充质干细胞的特性，见图1。"
2.2 UC-MSCs表面标记检测和诱导分化能力鉴定结果 UC-MSCs高表达 CD44、CD90、CD105(阳性率大于95%)，低表达 CD11c、CD34、CD45(阳性率小于1%) [21]。UC-MSCs成脂诱导21 d后，镜下可见空泡形成，经油红O染色后UC-MSCs内可见大量鲜红色脂滴。UC-MSCs成骨诱导21 d后，镜下可见钙化结节，经茜素红染色后UC-MSCs内可见橘红色钙化结节，见图2。"
2.3 细胞转染效果 实验按照最佳转染效率：BLOCK-iTTMAlexa FluorTM Red Fluorescent Control浓度为10 nmol/L、转染时间为 48 h。转染后，显微镜下观察miRNA Mimic转染情况，见图3。"
2.4 miR-1-5p 修饰的 UC-MSCs 对MRL/lpr 狼疮小鼠肝组织和肠组织病理变化的影响 2.4.1 苏木精-伊红染色观察各组MRL/lpr 小鼠肝脏病理变化 对照组、PBS组MRL/lpr小鼠肝组织内可见肝细胞沿中央静脉呈肝索排列，偶见肝细胞核溶解、坏死；汇管区可见中度炎性细胞浸润。与对照组相比，UC-MSCs+miR-1-5p组、UC-MSCs+miR-NC组和UC-MSCs组肝组织汇管区可见炎性细胞浸润减少，表明miR-1-5p修饰的UC-MSCs、miR-NC修饰的UC-MSCs、UC-MSCs均可缓解系统性红斑狼疮小鼠肝组织炎症反应，但miR-1-5p并未加强UC-MSCs对肝组织细胞的修复能力。UC-MSCs+miR-NC组还可见肝细胞核固缩及肝细胞水肿变性，提示miR-NC修饰后可能影响了UC-MSCs对肝组织细胞的修复能力，见图4。"
2.4.2 苏木精-伊红染色观察各组MRL/lpr小鼠肠组织病理变化 对照组、PBS组肠组织内可见肠绒毛黏膜上皮细胞轻微变性，杯状细胞数量重度减少；黏膜下层未见水肿、炎性细胞浸润；肌层细胞排列整齐，未见变性坏死；浆膜层未见增厚。与对照组相比，UC-MSCs+miR-1-5p组可见肠组织中空泡杯状细胞明显增多，黏膜上皮细胞未见变性、坏死；UC-MSCs+miR-NC组可见杯状细胞数量无明显变化，黏膜上皮细胞未见变性、坏死；UC-MSCs组可见空泡杯状细胞数量轻微增多，黏膜上皮细胞未见变性、坏死；提示miR-1-5p可能与UC-MSCs在促进杯状细胞增殖及减少肠黏膜上皮细胞变性、坏死方面具有协同作用。但miR-NC修饰后可能影响了UC-MSCs促进杯状细胞增殖的作用，见图5。"
2.5 Western blot 法检测 MRL/lpr小鼠肾组织中JAK2/STAT3通路蛋白及其磷酸化水平 各组MRL/lpr小鼠肾组织中p-JAK2/JAK2的相对表达水平，见图6。与对照组相比，UC-MSCs+miR-1-5p 组p-JAK2/JAK2的相对表达水平显著下降(P < 0.05)，表明miR-1-5p修饰的UC-MSCs能够下调JAK2 (Y1007+Y1008)位点的磷酸化水平；UC-MSCs+miR-NC 组、UC-MSCs组与对照组比较差异无显著性意义(P > 0.05)，但UC-MSCs+miR-NC 组、UC-MSCs组对p-JAK2蛋白的表达具有下调趋势，提示miR-1-5p 可能促进了UC-MSCs 对p-JAK2蛋白表达的调节作用；PBS组与对照组比较差异无显著性意义(P > 0.05)。"
各组MRL/lpr小鼠肾组织中p-STAT3(s727)/STAT3的相对表达水平，见图7。与对照组相比，UC-MSCs+miR-1-5p组和UC-MSCs组p-STAT3(s727)/STAT3的相对表达水平显著下降(P < 0.05)，表明UC-MSCs+miR-1-5p组和UC-MSCs组均能下调STAT3(s727)位点的磷酸化水平，但miR-1-5p并未加强UC-MSCs对MRL/lpr小鼠肾组织中STAT3(s727)位点磷酸化的抑制作用；UC-MSCs+miR-NC组与对照组比较差异无显著性意义(P > 0.05)，表明miR-NC修饰后可能影响了UC-MSCs对STAT3(s727)位点磷酸化蛋白表达的调节作用；PBS组与对照组比较差异无显著性意义(P > 0.05)。 "
各组MRL/lpr小鼠肾组织中p-STAT3(Y705)/STAT3的相对表达水平，见图8。与对照组STAT3(Y705)位点磷酸化水平相比，UC-MSCs+miR-1-5p组、UC-MSCs+miR-NC组、UC-MSCs 组、PBS组差异无显著性意义(P > 0.05)，提示miR-1-5p修饰的UC-MSCs、miR-NC修饰的UC-MSCs和UC-MSCs对MRL/lpr小鼠肾组织中STAT3(Y705)位点磷酸化水平均无调节作用。 "
2.6 Western blot 法检测 MRL/lpr小鼠肾组织中白细胞介素18蛋白表达水平 各组MRL/lpr小鼠肾组织中白细胞介素18的相对表达水平，见图9。与对照组相比，UC-MSCs+miR-1-5p组、UC-MSCs+miR-NC组白细胞介素18的相对表达水平下降(P < 0.05)、UC-MSCs 组白细胞介素18的相对表达水平显著下降(P < 0.01)；PBS组无显著变化(P > 0.05)。与PBS组相比，UC-MSCs 组白细胞介素18的相对表达水平下降(P < 0.05)；上述研究结果表明miR-1-5p修饰的UC-MSCs、miR-NC修饰的UC-MSCs和UC-MSCs均能在蛋白水平上对MRL/lpr小鼠肾组织中白细胞介素18的表达起下调作用，但miR-1-5p并未加强UC-MSCs组对白细胞介素18表达的下调作用。"
2.7 生物相容性 利用miR-1-5p基因修饰人UC-MSCs治疗MRL/lpr模型小鼠治疗期间，小鼠均未出现基本生命体征异常、急性免疫排斥反应等。"
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