维生素D对化学物致DNA损伤保护作用的实验研究

发布时间：2018-04-27 20:34

本文选题：维生素D + DNA损伤　；参考：《苏州大学》2012年硕士论文

【摘要】：目的：探讨维生素D对环磷酰胺和苯致DNA损伤的保护作用。方法：(1) CHL细胞(中国仓鼠肺成纤维细胞)在含1,25(OH)2D3浓度为0、10、20、50、100nM的培养液培养后加入S9混合液和终浓度为50μg/ml的环磷酰胺进行处理。染色体畸变实验观察CHL细胞染色体畸变情况。免疫荧光法检测细胞中γ-H2AX焦点形成。流式细胞仪测定CHL细胞ROS水平。(2)雄性ICR小鼠随机分为5组，其中四组小鼠给予0、1000、5000、10000IU维生素D3总体积为0.1ml的茶油注射液，共两周，每周一次，最后一次注射后给予40mg/kg环磷酰胺腹腔注射。流式细胞仪自动化检测方法分析小鼠骨髓嗜多染红细胞微核率。彗星实验检测小鼠淋巴细胞损伤情况。免疫荧光法检测骨髓细胞γ-H2AX焦点形成。流式细胞仪测定骨髓细胞内ROS水平。(3) ICR小鼠随机分为正常对照组、苯染毒组和VD+苯处理组。VD+苯处理组在染苯处理前提前一周给予维生素D3注射，另外两组给予等量注射用茶油。在第2次维生素D3注射后对苯染毒组和VD+苯处理组小鼠染苯处理。流式细胞仪检测小鼠骨髓嗜多染红细胞微核率。彗星实验检测小鼠外周血淋巴细胞损伤情况。ROS荧光探针流式细胞仪检测小鼠骨髓细胞活性氧水平。结果：(1)体外试验显示给予环磷酰胺处理以后CHL细胞畸变率显著升高，且细胞内γ-H2AX焦点数目升高明显，说明环磷酰胺引起CHL细胞DNA严重损伤。染色体畸变分析和γ-H2AX检测结果显示1,25(OH)2D3处理后CHL细胞DNA损伤有显著改善。(2)体内试验显示环磷酰胺处理小鼠嗜多染红细胞微核率和彗星实验各指标以及骨髓细胞γ-H2AX焦点数目显著上升，DNA损伤严重。维生素D处理组小鼠嗜多染红细胞微核率下降明显，，与单纯环磷酰胺处理组相比，彗星实验的尾部DNA百分含量、彗尾长度和Olive尾距三者均明显下降，γ-H2AX检测结果显示维生素D3可以减轻DNA双键断裂程度。(3)与正常对照组相比，小鼠骨髓嗜多染红细胞微核率和彗星实验各指标显示苯染毒组DNA损伤严重，骨髓细胞ROS水平显著升高。经维生素D处理后小鼠微核率和彗星实验各指标明显下降，DNA损伤情况有明显改善，VD+苯处理组小鼠骨髓细胞ROS水平显著低于苯染毒组。结论：(1)活性维生素D可以减少环磷酰胺致CHL细胞的染色体畸变率，这种保护作用与其减少γ-H2AX的阳性表达有关。(2)维生素D可以减少环磷酰胺致小鼠骨髓嗜多染红细胞微核率以及外周血淋巴细胞DNA的损伤效应。这种保护作用与其可以减少严重DNA双链断裂的细胞比例有关。(3)维生素D可以降低由于苯暴露引起的小鼠骨髓嗜多染红细胞的微核率，并且减少了外周血淋巴细胞的慧星尾长和Olive尾距。说明维生素D对苯引起的DNA损伤有一定的保护作用，这可能与其降低细胞内ROS水平有关。
[Abstract]:Objective: to investigate the protective effect of vitamin D on DNA damage induced by cyclophosphamide and benzene. Methods CHL cells (Chinese hamster lung fibroblasts) were treated with S9 mixed medium and cyclophosphamide with final concentration of 50 渭 g/ml after cultured in a culture medium containing a concentration of 1 / 25 OHH ~ (2 +) D _ (3) 0 ~ 10 ~ (10) O ~ (20) O ~ (50) N ~ (-1) N ~ (-1) and 50 渭 g/ml cyclophosphamide at the final concentration of 50 渭 g/ml. Chromosome aberration of CHL cells was observed by chromosome aberration experiment. The focal formation of 纬 -H2AX was detected by immunofluorescence assay. Male ICR mice were randomly divided into 5 groups by flow cytometry. Four of them were given tea oil injection with total volume of 0.1ml in the range of 10 000 IU and 10 000 IU of vitamin D 3 for two weeks, once a week, in which 4 groups of mice were given tea oil injection with a total volume of 10 000 IU of vitamin D 3. 40mg/kg cyclophosphamide was given intraperitoneally after the last injection. The micronucleus rate of mouse bone marrow polychromatic erythrocytes was analyzed by flow cytometry. Comet assay was used to detect lymphocyte damage in mice. The focal formation of bone marrow cells 纬-H 2 AX was detected by immunofluorescence. ROS levels in bone marrow cells were measured by flow cytometry) ICR mice were randomly divided into normal control group, benzene exposed group and VD benzene-treated group. Vitamin D _ 3 was injected one week before benzene treatment, and the other two groups were given the same amount of tea oil for injection. After the second injection of vitamin D _ 3, the mice were treated with benzene in benzene group and in VD group. The micronucleus rate of polychromatic erythrocytes in bone marrow of mice was detected by flow cytometry. Ros fluorescence probe flow cytometry was used to detect the level of reactive oxygen species (Ros) in bone marrow cells of mice. Results the in vitro test showed that the aberration rate of CHL cells was significantly increased and the number of 纬 -H2AX focal points increased significantly after cyclophosphamide treatment, indicating that cyclophosphamide induced severe DNA damage in CHL cells. The results of chromosome aberration analysis and 纬 -H2AX test showed that the DNA damage of CHL cells was significantly improved after treatment with 1t25OHH2D3.) in vivo tests, cyclophosphamide treatment showed that micronucleus rate of polychromatic erythrocytes, comet assay indexes and 纬 -H2AX focal cells of bone marrow cells in mice treated with cyclophosphamide were significantly improved. The number of points increased significantly and DNA damage was severe. The micronucleus rate of polychromatic erythrocytes in vitamin D treated mice decreased significantly, and the tail DNA content of comet assay was higher than that of cyclophosphamide alone. The length of comet tail and the tail distance of Olive decreased obviously. The results of 纬 -H2AX showed that vitamin D3 could reduce the breaking degree of DNA double bond. The micronucleus rate of bone marrow polychromatic erythrocytes and the indexes of comet assay showed that the DNA damage was serious and the ROS level of bone marrow cells was significantly increased in benzene-exposed group. The micronucleus rate and comet assay indexes of mice treated with vitamin D significantly decreased the DNA damage of bone marrow cells of mice treated with VDD-treated mice, and the level of ROS in bone marrow cells of mice treated with benzene was significantly lower than that of group treated with benzene. Conclusion Vitamin D can reduce chromosome aberration rate of CHL cells induced by cyclophosphamide. This protective effect is related to the decrease of 纬 -H2AX positive expression. Vitamin D can reduce micronucleus rate of bone marrow polychromatic erythrocytes and DNA damage of peripheral blood lymphocytes in mice induced by cyclophosphamide. This protective effect is related to the reduction of the proportion of cells with severe DNA double strand breaks.) Vitamin D can reduce the micronucleus rate of polychromatic erythrocytes in bone marrow of mice caused by benzene exposure. The comet tail length and Olive tail distance of peripheral blood lymphocytes were reduced. These results suggest that vitamin D has protective effect on DNA damage induced by benzene, which may be related to the decrease of intracellular ROS level.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

【参考文献】