1.广西壮族自治区南宁市妇幼保健院 腺体外科，广西 南宁 530028;2.广西医科大学第五附属医院 2. 创面修复科 3. 病理科，广西 南宁 530022;3.广西壮族自治区柳州工人医院 整形美容科，广西 柳州 545005;4.广西壮族自治区人民医院 科研实验中心，广西 南宁 530021
1.Department of Gland Surgery, Nanning Maternal and Child Health Hospital, Nanning 530028, China;2.Department of Wound Repair 3. Department of Pathology, the Fifth Affiliated Hospital of Guangxi Medical University, Nanning 530022, China;3.Department of Plastic Surgery, Liuzhou Workers' Hospital, Liuzhou, Guangxi 545005, China;4.Laboratory Center, the People's Hospital of Guangxi Zhuang Autonomous Region, Nanning 530021, China
背景与目的 基质细胞衍生因子1α（SDF-1α）是一种定向诱导细胞迁移的趋化因子，研究显示，间充质干细胞（MSCs）在受损组织中可以沿着SDF-1梯度迁移到损伤部位并参与组织修复，然而目前尚缺乏SDF-1α诱导脂肪来源干细胞（ASCs）对糖尿病缺血下肢进行组织修复的体内研究。因此，本研究探讨SDF-1α促进大鼠脂肪来源干细胞（rASCs）向糖尿病大鼠缺血下肢肌肉组织迁移及对组织修复的影响。方法 取SD大鼠脂肪组织分离培养rASCs，行细胞形态观察，鉴定成脂、成软骨及成神经分化能力，并使用带绿色荧光蛋白（GFP）的腺病毒转染和标记rASCs。将大鼠用STZ法构建糖尿病模型，并结扎大鼠的右下肢股动脉造成下肢缺血后，随机分为两组，通过尾静脉向两组大鼠体内注射rASCs，其中一组在患肢中段部位肌肉处注射SDF-1α蛋白（SDF-1α+rASCs组），另一组则用同样方式注射等量磷酸盐缓冲溶液（rASCs组）。治疗后的第1、2周行大鼠双下肢血流量检测，计算及比较各组大鼠的缺血下肢-健侧下肢血流比值。在第4周时处死大鼠，取缺血部位的肌肉组织行HE染色，观察不同治疗方法组中肌肉组织的排列情况。以因子Ⅷ（FⅧ）作为微血管的标记，行免疫荧光染色，荧光显微镜下观察组织中FⅧ及GFP的分布情况。结果 所培养的细胞呈长梭形或多角形样生长，并可向脂肪、软骨、神经细胞多向分化，鉴定为rASCs。糖尿病下肢缺血大鼠下肢血流量检测结果显示，在治疗后第1周SDF-1α+rASCs组的缺血下肢-健侧下肢血流比值明显高于rASCs组（0.33±0.03 vs. 0.26±0.02，P=0.016），治疗后第2周可发现上述差异进一步扩大（0.60±0.02 vs. 0.47±0.01，P=0.050）。HE染色结果显示，在治疗后第4周SDF-1α+rASCs组大鼠的肌肉组织排列更为整齐。免疫荧光结果显示，SDF-1α+rASCs组的骨骼肌组织中rASCs的数量在治疗后第4周明显高于rASCs组（P<0.05），还能观察到红色荧光（FⅧ）与绿色荧光（rASCs）的重叠。结论 通过提高缺血部位的SDF-1α的浓度能够有效增加迁移至患处的rASCs数量，从而获得优化rASCs改善患肢血流灌注，促进肌肉组织修复的明显效果。此外，rASCs分化为血管内皮细胞可能是rASCs促进缺血肌肉修复的关键机制之一。
Background and Aims Stromal cell-derived factor-1α (SDF-1α) is a chemotactic factor that directs cell migration. Studies have shown that mesenchymal stem cells (MSCs) can migrate along the SDF-1 gradient to the site of tissue injury and participate in tissue repair. However, there is currently a lack of in vivo studies on the tissue repair of diabetic ischemic lower limbs using SDF-1α-induced adipose-derived stem cells (ASCs). Therefore, this study was conducted to investigate the effect of SDF-1α on the migration of rat adipose-derived stem cells (rASCs) to the muscle tissue of diabetic ischemic rat lower limbs and its impact on tissue repair.Methods The rASCs were isolated and cultured from the adipose tissue of SD rats. Cell morphology was observed, and the differentiation capacity towards adipocytes, chondrocytes, and neurons was evaluated. Then, the rASCs were transfected and labeled with green fluorescent protein (GFP) using adenovirus. Diabetes was induced in rats using the streptozotocin (STZ) method, and the right femoral artery of the rats was ligated to induce lower limb ischemia. The rats were randomly divided into two groups and injected with rASCs via the tail vein. The SDF-1α protein was injected into the midsection of the affected limb muscle in one group (SDF-1α+rASCs group), while the other group was injected with an equal amount of phosphate-buffered saline (rASCs group). Blood flow measurements of the rat lower limbs were conducted at weeks 1 and 2 after treatment, and the ischemic limb-to-contralateral limb blood flow ratio was calculated and compared. At week 4, the rats were euthanized, and muscle tissue from the ischemic region was subjected to HE staining to observe the arrangement of muscle tissue in different treatment groups. Immunofluorescent staining using factor Ⅷ (FⅧ) as a marker for microvessels was performed to observe the distribution of FⅧ and GFP in the tissues under a fluorescence microscope.Results The cultured cells exhibited spindle or multiangular growth and had the ability to differentiate into adipocytes, chondrocytes, and neurons, confirming their identity as rASCs. The blood flow measurements of the diabetic ischemic rat lower limbs showed that the ischemic limb-to-contralateral limb blood flow ratio was significantly higher in the SDF-1α+rASCs group than in the rASCs group at week 1 (0.33±0.03 vs. 0.26±0.02, P=0.016), and this difference further increased at week 2 (0.60±0.02 vs. 0.47±0.01, P=0.050). HE staining revealed a more orderly arrangement of muscle tissue in the SDF-1α+rASCs group at week 4. Immunofluorescent staining showed that the number of rASCs in the skeletal muscle tissue of the SDF-1α+rASCs group was significantly higher than that in the rASCs group at week 4 (P<0.05), and overlapping red fluorescence (FⅧ) and green fluorescence (rASCs) were observed.Conclusion Increasing the concentration of SDF-1α at the site of ischemia can effectively increase the number of rASCs migrating to the affected area, resulting in optimized blood flow perfusion and significant improvement in muscle tissue repair. In addition, the differentiation of rASCs into endothelial cells may be one of the key mechanisms.