辛硫磷对大鼠骨髓间充质干细胞DNA损伤、细胞凋亡及P53蛋白表达的影响

目的研究辛硫磷对大鼠骨髓间充质干细胞(BMSCs)DNA的损伤作用及其对氧化损伤、细胞凋亡和P53蛋白表达的影响。方法 Percoll离心法分离培养大鼠BMSCs,正常传代。取第3代BMSCs,调整细胞密度为1.0×106/瓶,当细胞至亚融合状态,分别以0(对照)、0.2、2和20μg/L的辛硫磷浓度染毒24h。采用MTT法检测BMSCs的存活率,分光光度比色法检测BMSCs超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性和丙二醛(MDA)含量,单细胞凝胶电泳检测BMSCs的DNA损伤,流式细胞术检测大鼠骨髓间充质干细胞凋亡率,Western blotting检测BMSCs的P53蛋白表达水平。结果与对照组相比,0.2～20μg/L辛硫磷染毒24h,可诱发大鼠BMSCs的DNA损伤,且具有剂量-效应关系;各染毒组大鼠BMSCs的存活率、SOD和CAT活性均显著下降(P<0.05),细胞凋亡率和MDA含量均显著升高(P<0.05);辛硫磷染毒可以诱导大鼠BMSCs P53蛋白表达水平的增加(P<0.05)。结论辛硫磷可诱导大鼠BMSCs氧化损伤、DNA损伤、细胞凋亡和P53蛋白表达,且具有剂量-效应关系。

Effects on DNA damage and apoptosis and P53 protein expression induced by phoxim in rat bone marrow stem cells

Objective To study the DNA damage and oxidative damage and apoptosis and P53 protein expression in rat bone marrow stem cells cultured in vitro induced by phoxim.Methods Rat bone marrow stem cells of P3 cultured in vitro were treated with phoxim of different concentrations(0,0.2,2 and 20μg/L)for 24 h after 48h cultured.The total activity of SOD、CAT and MDA content,survival rate in cell were detected by spectrophotometry,and the DNA damage were detected by single cell gel electrophoresis.The cell apoptosis were detected by flow cytometry,and the P53 protein expression were detected by Western blot.Results With the 0.2 and 20μg/L of phoxim concentrations treated for 24h in culture media,the total activity of SOD and CAT were significantly decreased compared with the controls(P<0.05).With the increase of phoxim concentration in culture media,the MDA contents,DNA damage,apoptosis rates and P53 protein expression of rat bone marrow stem cells was increased significantly compared with the controls(P<0.05).Conclusion Phoxim can increase the rates of DNA damage,oxidative damage and induce apoptosis and expression of P53 in rat bone marrow stem cells,and there exists a rise tendency with the increase concentration of phoxim.