本文選題：維生素D + DNA損傷�。� 參考：《蘇州大學(xué)》2012年碩士論文

【摘要】：目的：探討維生素D對環(huán)磷酰胺和苯致DNA損傷的保護作用。 方法：(1) CHL細(xì)胞(中國倉鼠肺成纖維細(xì)胞)在含1,25(OH)2D3濃度為0、10、20、50、100nM的培養(yǎng)液培養(yǎng)后加入S9混合液和終濃度為50μg/ml的環(huán)磷酰胺進行處理。染色體畸變實驗觀察CHL細(xì)胞染色體畸變情況。免疫熒光法檢測細(xì)胞中γ-H2AX焦點形成。流式細(xì)胞儀測定CHL細(xì)胞ROS水平。(2)雄性ICR小鼠隨機分為5組，其中四組小鼠給予0、1000、5000、10000IU維生素D3總體積為0.1ml的茶油注射液，共兩周，每周一次，最后一次注射后給予40mg/kg環(huán)磷酰胺腹腔注射。流式細(xì)胞儀自動化檢測方法分析小鼠骨髓嗜多染紅細(xì)胞微核率。彗星實驗檢測小鼠淋巴細(xì)胞損傷情況。免疫熒光法檢測骨髓細(xì)胞γ-H2AX焦點形成。流式細(xì)胞儀測定骨髓細(xì)胞內(nèi)ROS水平。(3) ICR小鼠隨機分為正常對照組、苯染毒組和VD+苯處理組。VD+苯處理組在染苯處理前提前一周給予維生素D3注射，另外兩組給予等量注射用茶油。在第2次維生素D3注射后對苯染毒組和VD+苯處理組小鼠染苯處理。流式細(xì)胞儀檢測小鼠骨髓嗜多染紅細(xì)胞微核率。彗星實驗檢測小鼠外周血淋巴細(xì)胞損傷情況。ROS熒光探針流式細(xì)胞儀檢測小鼠骨髓細(xì)胞活性氧水平。 結(jié)果：(1)體外試驗顯示給予環(huán)磷酰胺處理以后CHL細(xì)胞畸變率顯著升高，且細(xì)胞內(nèi)γ-H2AX焦點數(shù)目升高明顯，說明環(huán)磷酰胺引起CHL細(xì)胞DNA嚴(yán)重?fù)p傷。染色體畸變分析和γ-H2AX檢測結(jié)果顯示1,25(OH)2D3處理后CHL細(xì)胞DNA損傷有顯著改善。(2)體內(nèi)試驗顯示環(huán)磷酰胺處理小鼠嗜多染紅細(xì)胞微核率和彗星實驗各指標(biāo)以及骨髓細(xì)胞γ-H2AX焦點數(shù)目顯著上升，DNA損傷嚴(yán)重。維生素D處理組小鼠嗜多染紅細(xì)胞微核率下降明顯，，與單純環(huán)磷酰胺處理組相比，彗星實驗的尾部DNA百分含量、彗尾長度和Olive尾距三者均明顯下降，γ-H2AX檢測結(jié)果顯示維生素D3可以減輕DNA雙鍵斷裂程度。(3)與正常對照組相比，小鼠骨髓嗜多染紅細(xì)胞微核率和彗星實驗各指標(biāo)顯示苯染毒組DNA損傷嚴(yán)重，骨髓細(xì)胞ROS水平顯著升高。經(jīng)維生素D處理后小鼠微核率和彗星實驗各指標(biāo)明顯下降，DNA損傷情況有明顯改善，VD+苯處理組小鼠骨髓細(xì)胞ROS水平顯著低于苯染毒組。 結(jié)論：(1)活性維生素D可以減少環(huán)磷酰胺致CHL細(xì)胞的染色體畸變率，這種保護作用與其減少γ-H2AX的陽性表達有關(guān)。(2)維生素D可以減少環(huán)磷酰胺致小鼠骨髓嗜多染紅細(xì)胞微核率以及外周血淋巴細(xì)胞DNA的損傷效應(yīng)。這種保護作用與其可以減少嚴(yán)重DNA雙鏈斷裂的細(xì)胞比例有關(guān)。(3)維生素D可以降低由于苯暴露引起的小鼠骨髓嗜多染紅細(xì)胞的微核率，并且減少了外周血淋巴細(xì)胞的慧星尾長和Olive尾距。說明維生素D對苯引起的DNA損傷有一定的保護作用，這可能與其降低細(xì)胞內(nèi)ROS水平有關(guān)。
[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 緯 -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 緯-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 緯 -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 緯 -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 緯 -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 緯 -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 緯 -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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R114

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相關(guān)期刊論文 前3條

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