间充质干细胞与淋巴管内皮祖细胞联合移植对小鼠后肢淋巴水肿的影响

Effect of combined transplantation of mesenchymal stem cells and lymphatic endothelial progenitor cells on hindlimb lymphedema in mice

摘要:

目的 研究间充质干细胞（MSCs）与淋巴管内皮祖细胞（LEPCs）联合移植对小鼠后肢淋巴水肿的影响，为淋巴水肿疾病的细胞疗法提供新思路。方法 从C57BL/6小鼠骨髓中分离并培养MSCs，培养至3～4代，使用流式细胞术鉴定表型，使用成脂、成骨及成软骨分化实验鉴定多系分化潜能；从小鼠骨髓中分离单个核细胞后扩增培养，通过磁珠分选筛选出CD34⁺VEGFR-3⁺细胞，使用免疫荧光染色鉴定细胞表面标志物、内皮潜能，成管实验鉴定淋巴管分化能力；通过手术方式复制后肢淋巴水肿模型，将MSCs与LEPCs注射于淋巴水肿部位，测量足垫厚度，观察水肿变化，通过免疫组织化学染色观察淋巴管面积及数量变化，通过Western blotting检测淋巴管内皮透明质酸受体-1（LYVE-1）蛋白表达。对照组、MSCs组、LEPCs组和MSCs+LEPCs组各组间进行比较。结果 ①分离并培养的MSCs呈长梭形，流式细胞术结果显示CD29、CD44和Sca1阳性表达，表达率分别为99.80%、99.70%和96.48%；CD45和CD11b阴性表达，表达率分别为0.04%和0.03%，符合间充质干细胞形态学特征及表型特征。成脂诱导分化可见脂滴形成，成骨诱导分化可见钙盐沉淀形成的“骨结节”，成软骨诱导分化可见软骨球形成，以上诱导分化实验验证了其具有三系分化能力。②分离并培养的LEPCs呈“铺路石”样，通过免疫荧光染色鉴定LEPCs细胞特异性表面抗原表达，VEGFR-3、CD34和CD133表达阳性，符合LEPCs细胞形态学特征及特异性标志物特征。Dil-Ac-LDL和FITC-UEA-1双荧光染色可见细胞质摄取Dil-Ac-LDL后呈现红色荧光，细胞膜结合FITC-UEA-1后显示绿色荧光，证明其具有内皮潜能。LYVE-1荧光染色阳性证明其具有向淋巴内皮细胞分化能力。成管实验可见小管形成，证明其具有分化为淋巴管的能力。③通过手术方式成功复制稳定的小鼠后肢淋巴水肿模型，术后第1天开始出现水肿，水肿至少持续4周，至第7天左右达到最大程度，足垫厚度为（3.984±0.171）mm。单纯手术组与非手术组第1、4、7、10、13、16、19、22、25、28、31天足垫厚度比较，结果 a.不同时间点足垫厚度比较，差异有统计学意义（P <0.05）；b.单纯手术组与非手术组间足垫厚度比较，差异有统计学意义（P <0.05）；c.两组足垫厚度变化趋势比较，差异有统计学意义（P <0.05）。术后第7和31天，单纯手术组足垫厚度均大于非手术组（P <0.05）。④对照组、MSCs组、LEPCs组和MSCs+LEPCs组第1、4、7、10、13、16、19天足垫厚度比较，结果 a.不同时间点足垫厚度比较，差异有统计学意义（P <0.05）；b.4组足垫厚度比较，差异无统计学意义（P >0.05）；c.4组足垫厚度变化趋势比较，差异有统计学意义（P <0.05）。第19天，MSCs组、LEPCs组、MSCs+LEPCs组足垫厚度均小于对照组（P <0.05），且MSCs+LEPCs组足垫厚度小于LEPCs组和MSCs组（P <0.05）。MSCs+LEPCs组的淋巴管面积小于MSCs组、LEPCs组、对照组（P < 0.05），LYVE-1蛋白相对表达量高于MSCs组、LEPCs组、对照组（P < 0.05）。结论 MSCs、LEPCs移植可以改善淋巴水肿，且两者联合移植改善淋巴水肿效果优于单一移植。

Abstract:

Objective To investigate the effect of combined transplantation of mesenchymal stem cells (MSCs) and lymphatic endothelial progenitor cells (LEPCs) on hindlimb lymphoedema in mice, and to provide new insights into cellular therapies for lymphedema.Methods MSCs were isolated from the bone marrow of C57BL/6 mice and cultured for 3 to 4 passages. Phenotypes of MSCs were identified by flow cytometry, and their multilineage differentiation potential was assessed through adipogenic, osteogenic and chondrogenic differentiation assays. Single nucleated cells isolated from mouse bone marrow were expanded and cultured, and CD34⁺ VEGFR-3⁺ endothelial progenitor cells were screened by magnetic bead sorting. Cell surface markers and endothelial potential were identified by immunofluorescence staining. Lymphatic vessel differentiation capacity was identified by tube formation assay. The hindlimb lymphedema model was established by surgery. MSCs and LEPCs were injected into the site of lymphoedema, and changes in edema were observed by measuring the thickness of the footpad. Immunohistochemistry was used to observe the alterations in the area and number of lymphatic vessels. Western blot was used to observe the changes in the level of LYVE-1 protein. Comparisons were made among the control group, MSCs group, LEPCs group, and MSCs+LEPCs group.Results (1) The isolated and cultured MSCs exhibited a long, spindle-shaped morphology. Flow cytometry analysis revealed positive expression of CD29, CD44 and Sca1 with their expression rates being 99.80%, 99.70% and 96.48%, alongside negative expression of CD45 and CD11b with their expression rates being 0.04% and 0.03%, consistent with the morphological and phenotypic characteristics of MSCs. Adipogenic differentiation was evidenced by the formation of lipid droplets. Osteogenic differentiation could be indicated by the "bone nodules" formed by the precipitation of calcium salts. Chondrogenic differentiation could be supported by the formation of cartilage pellets. These induction experiments confirmed the tri-lineage differentiation potential of the cells. (2) The isolated and cultured LEPCs were shaped like "paving stones". Immunofluorescence staining confirmed the expression of surface markers specific to LEPCs, including VEGFR-3, CD34, and CD133. These findings were consistent with the morphological and phenotypic characteristics of LEPCs. Dual fluorescence staining of Dil-Ac-LDL and FITC-UEA-1 showed red fluorescence in the cytoplasm due to the uptake of Dil-Ac-LDL and green fluorescence on the cell membrane from FITC-UEA-1 binding, indicating the endothelial potential of the cells. Positive LYVE-1 fluorescence staining confirmed their capacity to differentiate into lymphatic endothelial cells. Furthermore, the tube formation assay showed they could differentiate into lymphatic vessels. (3) A stable hindlimb lymphedema mouse model was successfully established surgically. Edema appeared one day postoperatively, peaked at day 7 (footpad thickness: 3.984 ± 0.171 mm), and persisted for at least 4 weeks. A comparison was made in terms of the footpad thickness on days 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, and 31 between the surgery-only group and the non-surgery group, which demonstrated that the footpad thickness was different among the time points (P < 0.05) and between the groups (P < 0.05), and that the change trend of the footpad thickness was different between the two groups (P < 0.05). On days 7 and 31 postoperatively, the footpad thickness was greater in the surgery-only group than in the non-surgery group (P < 0.05). ④A comparison was made in terms of the footpad thickness on days 1, 4, 7, 10, 13, 16 and 19 in the control group, MSCs group, LEPCs group, and MSCs+LEPCs groups, which revealed that the footpad thickness was different among the time points (P < 0.05) but not among the groups (P > 0.05), and that the change trend of the footpad thickness was different among the four groups (P < 0.05). On day 19, the footpad thickness of the MSCs group, the LEPCs group, and the MSCs+LEPCs group was lower than that of the control group (P < 0.05), and the footpad thickness of the MSCs+LEPCs group was even lower than that of the LEPCs group and the MSCs group (P < 0.05). The area of lymphatic vessels in the MSCs+LEPCs group was smaller than that of the MSCs group, the LEPCs group, and the control group (P < 0.05). The relative expression of LYVE-1 protein in the MSCs+LEPCs group was higher than that of MSCs group, LEPCs group, and control group (P < 0.05).Conclusions Transplantation of MSCs and LEPCs improves lymphoedema, and combined transplantation of both is more effective than the single transplantation.