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目的 探讨大鼠脂肪来源干细胞(adipose-derived stem cells,ADSCs)对紫外线照射造成的软骨细胞DNA损伤的修复作用.方法 取3～4周龄SD大鼠(体质量100 ～ 120 g)腹股沟脂肪,利用Ⅰ型胶原酶消化法体外分离培养ADSCs并传代;取第3代细胞采用流式细胞仪检测其表面相关标记,行成骨及成脂诱导鉴定其多向分化潜能.另取SD大鼠关节软骨,利用酶消化法分离培养软骨细胞并传代,并行甲苯胺蓝染色鉴定.取第3代软骨细胞,采用40 J/m2剂量紫外线照射;照射后细胞分别以正常培养基培养(照射组)、以含ADSCs培养上清的培养基培养(ADSCs上清组)或与ADSCs共培养(ADSCs组)24 h.以不进行紫外线照射的正常第3代软骨细胞作为对照(对照组).采用MTS法检测细胞增殖情况,免疫荧光染色、Western blot法检测软骨细胞DNA损伤标志蛋白磷酸化组蛋白2A变异体(phosphorylated histone family 2A variant,γH2AX)的表达情况.结果 经流式细胞仪检测及成骨、成脂诱导鉴定,所培养细胞为ADSCs.对照组、照射组及ADSCs上清组吸光度(A)值分别为2.20±0.10、1.34±0.04、1.57±0.06,照射组及ADSCs上清组显著低于对照组,照射组显著低于ADSCs上清组,比较差异均有统计学意义(P＜0.05).免疫荧光染色示,照射组、ADSCs上清组及ADSCs组γH2AX蛋白荧光强度明显高于对照组,但ADSCs上清组及ADSCs组γH2AX蛋白荧光强度明显弱于照射组,ADSCs组和ADSCs上清组则无明显区别.Western blot法检测示,照射组、ADSCs上清组及ADSCs组γH2AX蛋白相对表达量均明显高于对照组,但ADSCs上清组及ADSCs组显著低于照射组,差异均有统计学意义(P＜0.05);ADSCs组和ADSCs上清组比较差异无统计学意义(P＞0.05).结论 大鼠ADSCs有助于恢复紫外线照射后的软骨细胞的增殖,降低DNA损伤标志蛋白γH2AX的表达,对紫外线照射所致软骨细胞损伤具有修复作用.“,”Objective To explore the DNA repair effect of rat adipose-derived stem cells (ADSCs) on chond-rocytes exposed to ultraviolet (UV) radiation.Methods ADSCs were isolated and cultured from the inguinal adipose tissue of Sprague Dawley rat by digestion with collagenase type Ⅰ.ADSCs cell phenotype was assayed with flow cytometry.Multiple differentiation capability of ADSCs at passage 3 was identified with osteogenic and adipogenic induction.The chondrocytes were obtained from rat articular cartilage by digestion with collagenase type Ⅱ and were identified with toluidine blue staining.The chondrocytes at passage 3 were irradiated with 40 J/m2 UV and cultured with normal medium (irradiated group),and medium containing the ADSCs supernatant (ADSCs supernatant group) or ADSCs was used for co-culture (ADSCs group) for 24 hours;no irradiation chondrocytes served as control group.The cell proliferation was estimated by MTS method.The expression of phosphorylated histone family 2A variant (γH2AX) was detected by immunofluorescence and Western blot.Results ADSCs presented CD29(+),CD44(+),CD106(-),and CD34(-);and results of the alizarin red staining and oil red O staining were positive after osteogenic and adipogenic induction.Cell proliferation assay demonstrated the absorbance (A) values were 2.20±0.10 (control group),1.34±0.04 (irradiated group),and 1.57±0.06 (ADSCs supernatant group),showing significant difference between groups (P＜0.05).Immunofluorescence and Western blot showed that the γH2AX protein expression was significantly increased in irradiated group,ADSCs supernatant group,and ADSCs group when compared with control group (P＜0.05),and the expression was significantly decreased in ADSCs supernatant group and ADSCs group when compared with irradiated group (P＜0.05),but no significant difference was found between ADSCs supernatant group and ADSCs group (P＞0.05).Conclusion ADSCs can increase the cell proliferation and down-regulate the γH2AX protein expression of irradiated cells,indicating ADSCs contribute to the repair of irradiated chondrocyte.