移植Toll样受体3处理脐带间充质干细胞对小鼠狼疮性肾炎的治疗效果及其机制

目的 探讨Toll样受体3（TLR3）激活对人脐带间充质干细胞（hUC-MSCs）移植治疗狼疮性肾炎（LN）的效果，并初步阐明其作用机制。 方法 培养hUC-MSCs并鉴定，10 mg·L^－1聚肌胞苷酸处理hUC-MSCs。45只MRL/Lpr雌性小鼠随机分为模型组、hUC-MSCs组和TLR3+hUC-MSCs组，另取15只C57BL/6C雌性小鼠作为对照组；小鼠尾静脉注射hUC-MSCs或聚肌胞苷酸处理的hUC-MSCs。给药后，每2周测定1次小鼠24 h尿蛋白水平；8周后采血并取肾组织，酶联免疫吸附测定（ELISA）法检测各组小鼠血清中抗双链DNA（dsDNA）抗体水平和肾组织中肿瘤坏死因子α（TNF?α）、白细胞介素1β（IL-1β）、白细胞介素6（IL-6）和白细胞介素17（IL-17）水平。HE染色和PAS染色观察各组小鼠肾组织病理形态表现，免疫荧光染色检测各组小鼠肾组织中IgG和补体C3沉积。Western blotting法检测各组小鼠肾组织中蛋白激酶B（Akt）、哺乳动物雷帕霉素靶蛋白（mTOR）和自噬相关蛋白表达水平。 结果 hUC-MSCs具有典型的间充质干细胞特征。与对照组比较，模型组小鼠血清中抗dsDNA抗体水平明显升高（P<0.05），肾组织中TNF?α、IL-1β、IL-6和IL-17水平明显升高（P<0.05），肾组织中出现肾小球系膜细胞增生、肾小管萎缩坏死和炎症细胞浸润现象，肾组织中IgG和C3沉积明显（P<0.05），肾组织中Akt和mTOR蛋白表达水平明显升高（P<0.05），而微管相关蛋白1轻链3Ⅱ（LC3Ⅱ）和酵母ATG6的同系物（Beclin1）蛋白表达水平明显降低（P<0.05）。与模型组比较，给药4周时TLR3+hUC-MSCs组小鼠24 h尿蛋白水平降低（P<0.05），6和8周时hUC-MSCs组和TLR3+hUC-MSCs组小鼠24 h尿蛋白水平均明显降低（P<0.05），血清中抗dsDNA抗体水平降低（P<0.05），肾组织中TNF?α、IL-1β、IL-6和IL-17水平明显降低（P<0.05），肾组织病变有一定程度的改善，肾组织中IgG和C3沉积减少（P<0.05），且TLR3+hUC-MSCs组小鼠肾组织改善更为明显；hUC-MSCs组和TLR3+hUC-MSCs组小鼠肾组织中Akt和mTOR蛋白表达水平明显降低（P<0.05），LC3Ⅱ和Beclin1蛋白表达水平明显升高（P<0.05）。与hUC-MSCs组比较，TLR3+hUC-MSCs组小鼠肾组织中LC3Ⅱ和Beclin1蛋白表达水平明显升高（P<0.05）。 结论 TLR3能够增强hUC-MSCs移植治疗小鼠LN的作用，可抑制肾组织炎症反应并调节细胞自噬水平。

Therapeutic effect of Toll-like receptor 3-treated umbilical cord mesenchymal stem cells transplantation on lupus nephritis in mice and its mechanism

Objective To investigate the efficency of Toll-like receptor 3 （TLR3） activation in the human umbilical cord mesenchymal stem cells（hUC-MSCs） transplantation for the treatment of lupus nephritis （LN）， and to preliminariy clarify its mechanism. Methods The hUC-MSCs were cultured and identified， and treated with 10 mg·L^－1 polyinosinic acid. A total of 45 MRL/Lpr female mice were randomly divided into model group， hUC-MSCs group， and TLR3+hUC-MSCs group，and another 15 C57BL/6C female mice were selected as control group；the mice were the injected with hUC-MSCs or polyinosinic acid-treated hUC-MSCs via tail vein. After administration， the levels of 24 h urine protein of the mice were measured every two weeks； the blood sample and kidney tissue were collected after 8 weeks，and the levels of anti-double stranded DNA（anti-dsDNA） antibody in serum and the levels of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， interleukin-6（IL-6）， and interleukin-17（IL-17） in kidney tissue of the mice in various groups were detected by enzyme linked immunosorbent assay（ELISA） method. HE staining and PAS staining were used to observe the pathomorphology of kidney tissue； immunofluorescence staining was used to detect the deposition of IgG and complement C3 in kidney tissue. Western blotting method was used to detect the expression levels of protein kinase B （Akt）， mammalian target of rapamycin （mTOR） and autophagy-related proteins in kidney tissue of the mice in various groups. Results The hUC-MSCs had typical mesenchymal stem cell characteristics. Compared with control group， the level of anti-dsDNA antibody in serum of the mice in model group was significantly increased （P<0.05），the levels of TNF-α， IL-1β， IL-6 and IL-17 in kidney tissue were significantly increased （P<0.05）， and there were glomerular mesangial cell proliferation， renal tubular atrophy and necrosis， and inflammatory cell infiltration in kidney tissue； IgG and C3 deposition in glomerulus was obvious （P<0.05）， and the expression levels of Akt and mTOR proteins in kidney tissue were significantly increased （P<0.05），while the expression levels of microtubule-assaciated protein 1 light chain 3 Ⅱ（LC3Ⅱ） and Beclin1 proteins were significantly decreased （P<0.05）. Compared with model group， the level of 24 h urine protein of the mice in TLR3+hUC-MSCs group was significantly reduced at 4 weeks of administration（P<0.05）， and the levels of 24 h urine protein of the mice in hUC-MSCs group and TLR3+hUC-MSCs group were significantly decreased at 6 and 8 weeks of administration（P<0.05）；and the levels of anti-dsDNA antibody in serum of the mice in hUC-MSCs group and TLR3+hUC-MSCs group were significantly decreased （P<0.05）；the levels of TNF-α， IL-1β， IL-6 and IL-17 in kidney tissue were significantly decreased （P<0.05）， the lesion of kidney tissue was improved to a certain extent， the depositions of IgG and C3 in kidney tissue were reduced （P<0.05）， and the improvement effect in TLR3+hUC-MSCs group was more significant； the expression levels of Akt and mTOR proteins in kidney tissue of the mice in hUC-MSCs group and TLR3+hUC-MSCs group were significantly decreased （P<0.05）， and the expression levels of LC3Ⅱ and Beclin1 proteins were significantly increased （P<0.05）. Compared with hUC-MSCs group， the expression levels of LC3Ⅱ and Beclin1 proteins in kidney tissue of the mice in TLR3+hUC-MSCs group were significantly increased （P<0.05）. Conclusion TLR3 can enhance the effect of hUC-MSCs transplantation in the treatment of LN in the mice， and inhibit kidney tissue inflammation and regulate the level of autophagy.