【摘要】：[目的]研究人支氣管上皮細(xì)胞(HBE)中苯并(a)芘氧化活化與DNA損傷的關(guān)系,進(jìn)一步證實(shí)LOX是P450氧化代謝的替代途徑,也為L(zhǎng)OXsiRNA可能通過(guò)抑制LOX發(fā)揮防治化學(xué)物毒性作用的新線索提供有力證據(jù)。 [方法]基因沉默實(shí)驗(yàn)：通過(guò)AgeI和EcoRI酶切質(zhì)粒并用T4Ligase和shRNA Oligo片段連接,構(gòu)建pAJ-U6-shRNA-CMV-GFP重組體,通過(guò)慢病毒載體系統(tǒng),進(jìn)行共轉(zhuǎn)染293T細(xì)胞最后得到病毒濃縮液來(lái)感染目的細(xì)胞,之后通過(guò)PCR和Western-blot的方法檢測(cè)基因沉默的效果。 細(xì)胞實(shí)驗(yàn)：以不同濃度的苯并(a)芘對(duì)HBE細(xì)胞和經(jīng)5-LOXsiRNA處理后的細(xì)胞組染毒,通過(guò)MTT實(shí)驗(yàn)檢測(cè)苯并(a)芘對(duì)兩組細(xì)胞存活率的影響；用Western-blot印記法檢測(cè)各組5-LOX蛋白表達(dá)的情況；同時(shí)用單細(xì)胞凝膠電泳實(shí)驗(yàn)及流式細(xì)胞計(jì)數(shù)檢測(cè)各組的DNA損傷情況。最后檢測(cè)苯并(a)芘染毒后經(jīng)過(guò)5-LOXsiRNA和化學(xué)抑制劑處理組對(duì)細(xì)胞的毒性和DNA損傷,并比較化學(xué)抑制劑和5-LOXsiRNA的細(xì)胞組抑制效果的差異。 [結(jié)果]通過(guò)PCR鑒定轉(zhuǎn)化子和陽(yáng)性克隆后的測(cè)序以及病毒感染HBE細(xì)胞后通過(guò)PCR和Western-blot的方法檢測(cè),說(shuō)明沉默5-LOX基因位點(diǎn)是較為成功。苯并(a)芘可以使HBE細(xì)胞內(nèi)5-LOX蛋白表達(dá)增加,AA-861對(duì)5-LOX蛋白表達(dá)基本沒(méi)有影響；苯并(a)芘可以對(duì)HBE產(chǎn)生明顯細(xì)胞毒作用和DNA損傷,經(jīng)5-LOXsiRNA,5-LOX抑制劑AA-861處理后的細(xì)胞組可以抑制苯并(a)芘毒性和DNA損傷,且5-LOXsiRNA細(xì)胞組抑制效果最為顯著。 [結(jié)論]5-LOX在HBE細(xì)胞內(nèi)的蛋白表達(dá)可以經(jīng)苯并(a)芘調(diào)節(jié),特異性5-LOX抑制劑AA-861和5-LOXsiRNA都可抑制苯并(a)芘對(duì)細(xì)胞的毒性和DNA損傷,推測(cè)B(a)P可能是主要通過(guò)5-LOX介導(dǎo)的氧化活化生成親電子物質(zhì),與DNA結(jié)合導(dǎo)致其損傷；這種DNA損傷可能與B(a)P的致癌作用有關(guān),而利用RNA干擾技術(shù)抑制5-LOX可有效的阻止這一現(xiàn)象的發(fā)生發(fā)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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R114

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