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

发布时间：2018-06-20 11:44

  本文选题：脂肪来源干细胞 + 软骨细胞　； 参考：《中国修复重建外科杂志》2017年05期

【摘要】：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/m~2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)24h。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(phosphorylatedhistone 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(P0.05)。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(P0.05);ADSCs组和ADSCs上清组比较差异无统计学意义(P0.05)。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。
[Abstract]:Objective to investigate the repair effect of adipose-derived stem cells (ADSCs) on chondrocyte DNA damage induced by ultraviolet radiation in rats. Methods the inguinal fat of SD rats aged 3 ~ 4 weeks (body mass 100 ~ 120 g) was isolated and cultured by type I collagenase digestion in vitro, and the surface related markers of ADSCs were detected by flow cytometry (FCM) in the third passage of SD rats. The multidirectional differentiation potential was identified by osteogenesis and adipogenic induction. The articular cartilage of SD rats was isolated and cultured by enzyme digestion, and identified by toluidine blue staining. The third passage of chondrocytes were irradiated with 40 J / m ~ (2) dose of ultraviolet radiation. After irradiation, the cells were cultured in normal medium (irradiation group, culture medium containing ADSCs supernatant) or co-cultured with ADSCs for 24 hours. The normal third passage chondrocytes without ultraviolet radiation were used as control group (control group). The expression of phosphorylated histone family 2A variant (phosphorylatedhistone family 2A variant, 纬 H 2AX) in chondrocytes was detected by MTS and immunofluorescence staining. Results the cultured cells were identified as ADSCs by flow cytometry and osteogenesis. The absorbance (A) values of the control group, the irradiation group and the ADSCs supernatant group were 2.20 卤0.10 卤1.34 卤0.04 卤1.57 卤0.06, respectively, which were significantly lower in the irradiation group and the ADSCs supernatant group than in the ADSCs supernatant group, and the difference was statistically significant (P 0.05). Immunofluorescence staining showed that the fluorescence intensity of 纬 -H2AX protein in ADSCs supernatant group and ADSCs group was significantly higher than that in control group, but the fluorescence intensity of 纬 -H2AX protein in ADSCs supernatant group and ADSCs group was significantly lower than that in ADSCs supernatant group and ADSCs supernatant group. The relative expression of 纬 H2AX protein in ADSCs supernatant group and ADSCs group was significantly higher than that in control group, but the expression of 纬 H2AX protein in ADSCs supernatant group and ADSCs group was significantly lower than that in irradiation group. There was no significant difference between ADSCs supernatant group and ADSCs supernatant group. Conclusion ADSCs can restore the proliferation of chondrocytes after ultraviolet irradiation, decrease the expression of DNA damage marker 纬 H _ 2AX, and repair the damage of chondrocytes induced by ultraviolet radiation.
【作者单位】： 广州市红十字会医院暨南大学医学院附属广州红十字会医院创伤外科研究所;广州市红十字会医院暨南大学医学院附属广州红十字会医院烧伤整形科;
【基金】：国家自然科学基金资助项目(81272222) 广州市卫生局一般引导项目(20131A011038) 广州市科技计划项目(201508020262)~~
【分类号】：R68

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