RNA干扰用于支气管上皮细胞5-脂氧合酶对苯并（a）芘活化及DNA损伤研究
发布时间：2018-12-11 22:15
【摘要】：[目的]研究人支气管上皮细胞(HBE)中苯并(a)芘氧化活化与DNA损伤的关系,进一步证实LOX是P450氧化代谢的替代途径,也为LOXsiRNA可能通过抑制LOX发挥防治化学物毒性作用的新线索提供有力证据。 [方法]基因沉默实验：通过AgeI和EcoRI酶切质粒并用T4Ligase和shRNA Oligo片段连接,构建pAJ-U6-shRNA-CMV-GFP重组体,通过慢病毒载体系统,进行共转染293T细胞最后得到病毒浓缩液来感染目的细胞,之后通过PCR和Western-blot的方法检测基因沉默的效果。 细胞实验：以不同浓度的苯并(a)芘对HBE细胞和经5-LOXsiRNA处理后的细胞组染毒,通过MTT实验检测苯并(a)芘对两组细胞存活率的影响；用Western-blot印记法检测各组5-LOX蛋白表达的情况；同时用单细胞凝胶电泳实验及流式细胞计数检测各组的DNA损伤情况。最后检测苯并(a)芘染毒后经过5-LOXsiRNA和化学抑制剂处理组对细胞的毒性和DNA损伤,并比较化学抑制剂和5-LOXsiRNA的细胞组抑制效果的差异。 [结果]通过PCR鉴定转化子和阳性克隆后的测序以及病毒感染HBE细胞后通过PCR和Western-blot的方法检测,说明沉默5-LOX基因位点是较为成功。苯并(a)芘可以使HBE细胞内5-LOX蛋白表达增加,AA-861对5-LOX蛋白表达基本没有影响；苯并(a)芘可以对HBE产生明显细胞毒作用和DNA损伤,经5-LOXsiRNA,5-LOX抑制剂AA-861处理后的细胞组可以抑制苯并(a)芘毒性和DNA损伤,且5-LOXsiRNA细胞组抑制效果最为显著。 [结论]5-LOX在HBE细胞内的蛋白表达可以经苯并(a)芘调节,特异性5-LOX抑制剂AA-861和5-LOXsiRNA都可抑制苯并(a)芘对细胞的毒性和DNA损伤,推测B(a)P可能是主要通过5-LOX介导的氧化活化生成亲电子物质,与DNA结合导致其损伤；这种DNA损伤可能与B(a)P的致癌作用有关,而利用RNA干扰技术抑制5-LOX可有效的阻止这一现象的发生发展。
[Abstract]:[objective] to study the relationship between the oxidative activation of benzo (a) pyrene and DNA damage in human bronchial epithelial cells (HBE), and to confirm that LOX is an alternative pathway to P450 oxidative metabolism. It also provides strong evidence for new clues that LOXsiRNA may play a role in the prevention and treatment of chemical toxicity by inhibiting LOX. [methods] Gene silencing experiment: plasmids were digested by AgeI and EcoRI and ligated with T4Ligase and shRNA Oligo fragments to construct pAJ-U6-shRNA-CMV-GFP recombinant. After cotransfection of 293T cells, the virus concentrate was obtained to infect the target cells, and then the effect of gene silencing was detected by PCR and Western-blot. Cell experiment: HBE cells and cells treated with 5-LOXsiRNA were exposed to benzo (a) pyrene at different concentrations. The effect of benzo (a) pyrene on the survival rate of HBE cells was detected by MTT assay. The expression of 5-LOX protein was detected by Western-blot imprinting assay and DNA damage was detected by single cell gel electrophoresis and flow cytometry. Finally, the cytotoxicity and DNA damage of benzo (a) pyrene treated with 5-LOXsiRNA and chemical inhibitor group were detected, and the difference of inhibition effect between chemical inhibitor group and 5-LOXsiRNA group was compared. [results] the sequence of transformants and positive clones were identified by PCR and the PCR and Western-blot methods were used to detect the viral infection of HBE cells. The results showed that the silencing of 5-LOX gene loci was more successful. Benzo (a) pyrene could increase the expression of 5-LOX protein in HBE cells, but AA-861 had no effect on the expression of 5-LOX protein. Benzo (a) pyrene could induce cytotoxicity and DNA damage to HBE. The cell group treated with 5-LOXsiRNA-5-LOX inhibitor AA-861 could inhibit benzo (a) pyrene toxicity and DNA damage. The inhibitory effect of 5-LOXsiRNA cell group was the most significant. [conclusion] the protein expression of 5-LOX in HBE cells can be regulated by benzo (a) pyrene. Both AA-861 and 5-LOXsiRNA, specific 5-LOX inhibitors, can inhibit the cytotoxicity and DNA damage induced by benzo (a) pyrene. It is speculated that B (a) P may be induced by 5-LOX mediated oxidative activation to form electron-lipophilic substances, which can be damaged by binding to DNA. This kind of DNA damage may be related to the carcinogenesis of B (a) P, and the suppression of 5-LOX by RNA interference technique can effectively prevent the occurrence and development of this phenomenon.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114
本文编号：2373316
[Abstract]:[objective] to study the relationship between the oxidative activation of benzo (a) pyrene and DNA damage in human bronchial epithelial cells (HBE), and to confirm that LOX is an alternative pathway to P450 oxidative metabolism. It also provides strong evidence for new clues that LOXsiRNA may play a role in the prevention and treatment of chemical toxicity by inhibiting LOX. [methods] Gene silencing experiment: plasmids were digested by AgeI and EcoRI and ligated with T4Ligase and shRNA Oligo fragments to construct pAJ-U6-shRNA-CMV-GFP recombinant. After cotransfection of 293T cells, the virus concentrate was obtained to infect the target cells, and then the effect of gene silencing was detected by PCR and Western-blot. Cell experiment: HBE cells and cells treated with 5-LOXsiRNA were exposed to benzo (a) pyrene at different concentrations. The effect of benzo (a) pyrene on the survival rate of HBE cells was detected by MTT assay. The expression of 5-LOX protein was detected by Western-blot imprinting assay and DNA damage was detected by single cell gel electrophoresis and flow cytometry. Finally, the cytotoxicity and DNA damage of benzo (a) pyrene treated with 5-LOXsiRNA and chemical inhibitor group were detected, and the difference of inhibition effect between chemical inhibitor group and 5-LOXsiRNA group was compared. [results] the sequence of transformants and positive clones were identified by PCR and the PCR and Western-blot methods were used to detect the viral infection of HBE cells. The results showed that the silencing of 5-LOX gene loci was more successful. Benzo (a) pyrene could increase the expression of 5-LOX protein in HBE cells, but AA-861 had no effect on the expression of 5-LOX protein. Benzo (a) pyrene could induce cytotoxicity and DNA damage to HBE. The cell group treated with 5-LOXsiRNA-5-LOX inhibitor AA-861 could inhibit benzo (a) pyrene toxicity and DNA damage. The inhibitory effect of 5-LOXsiRNA cell group was the most significant. [conclusion] the protein expression of 5-LOX in HBE cells can be regulated by benzo (a) pyrene. Both AA-861 and 5-LOXsiRNA, specific 5-LOX inhibitors, can inhibit the cytotoxicity and DNA damage induced by benzo (a) pyrene. It is speculated that B (a) P may be induced by 5-LOX mediated oxidative activation to form electron-lipophilic substances, which can be damaged by binding to DNA. This kind of DNA damage may be related to the carcinogenesis of B (a) P, and the suppression of 5-LOX by RNA interference technique can effectively prevent the occurrence and development of this phenomenon.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114
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