中国修复重建外科杂志

中国修复重建外科杂志

大鼠脂肪来源干细胞对紫外线造成的软骨细胞 DNA 损伤的修复作用研究

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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 (irradiated group), and the ADSCs supernatant was added (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 expre-ssion 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.

关键词: 脂肪来源干细胞; 软骨细胞; 辐射; 修复; 大鼠

Key words: Adipose-derived stem cells; chondrocyte; radiation; repair; rat

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