本文選題：小鼠肺巨噬細胞　切入點：氧化應(yīng)激　出處：《瀘州醫(yī)學(xué)院》2012年碩士論文

【摘要】：目的：目前國內(nèi)外對氧化應(yīng)激在脂多糖（Lippolysaccride,LPS）與自噬之間的相互作用及機制研究較少，本實驗通過觀察LPS誘導(dǎo)對小鼠肺巨噬細胞（Ana-1）損傷的影響，并從氧化應(yīng)激Ana-1細胞DNA損傷和自噬角度研究氧化應(yīng)激對LPS誘導(dǎo)對Ana-1細胞自噬的作用機制及其相關(guān)的分子機制。方法：1. LPS刺激Ana-1細胞模型的建立及氧化應(yīng)激下LPS誘導(dǎo)Ana-1細胞損傷模型的建立。LPS作用于Ana-1細胞， ROS熒光探針-DHE檢測Ana-1細胞內(nèi)活性氧族物質(zhì)（ReactiveOxygen Species,ROS）的表達情況，總SOD活性檢測試劑盒（WST法）檢測Ana-1細胞總超氧化物歧化酶（Superoxide Dismutase, SOD）的表達情況，彗星實驗試劑盒檢測Ana-1細胞DNA損傷的情況。氧化應(yīng)激作用于LPS誘導(dǎo)下的Ana-1細胞，ROS熒光探針-DHE檢測Ana-1細胞ROS的表達情況，總SOD活性檢測試劑盒（WST法）檢測Ana-1細胞總SOD的表達情況，通過彗星實驗試劑盒檢測Ana-1細胞DNA損傷的情況，比較LPS作用于Ana-1細胞與氧化應(yīng)激下LPS誘導(dǎo)的Ana-1細胞的指標(biāo)變化情況。2.LPS作用下的Ana-1細胞自噬現(xiàn)象和氧化應(yīng)激下LPS誘導(dǎo)的Ana-1細胞自噬現(xiàn)象。LPS濃度為1ug/ml刺激Ana-1細胞4h（LPS處理組），逆轉(zhuǎn)錄聚合酶鏈反應(yīng)（RT-PC R）法檢測自噬相關(guān)因子LC3-Ⅱ的mRNA表達水平變化，脂質(zhì)體Lipofectamine2000轉(zhuǎn)染質(zhì)粒GFP-LC3（green fluorescent protein-microtubule associated protein lightchain3，綠色熒光蛋白-微管相關(guān)蛋白1輕鏈3）和質(zhì)粒RFP-LC3（Redfluorescent protein-microtubule associated protein light chain3，紅色熒光蛋白-微管相關(guān)蛋白1輕鏈3）觀察LPS作用下的Ana-1細胞的自噬現(xiàn)象，免疫蛋白印記（Western Blot）法檢測自噬基因BECN1蛋白和LC3Ⅰ/Ⅱ蛋白的表達。氧化應(yīng)激下LPS誘導(dǎo)的Ana-1細胞（LPS+H_2O_2處理組，，外源性H_2O_2濃度為12.5uM刺激Ana-1細胞2h后，再加入LPS濃度為1ug/ml刺激Ana-1細胞4h），RT-PCR法檢測自噬相關(guān)因子LC3-Ⅱ的mRNA表達水平變化，脂質(zhì)體Lipofectamine2000轉(zhuǎn)染質(zhì)粒GFP-LC3和質(zhì)粒RFP-LC3觀察自噬現(xiàn)象，Western Blot法檢測自噬基因BECN1蛋白和LC3Ⅰ/Ⅱ蛋白表達。3.維生素C抗氧化應(yīng)激損傷處理后(維生素C濃度為2000ug/ml,刺激1h后加入濃度為12.5uM的外源性H_2O_2，刺激Ana-1細胞2h后，再加入濃度為1ug/ml的LPS刺激Ana-1細胞4h)觀察Ana-1細胞的自噬現(xiàn)象以及Western Blot法檢測自噬基因LC3Ⅰ/Ⅱ蛋白表達。4.對照組用PBS進行刺激。比較對照組、LPS處理組和LPS+H_2O_2處理組以及維生素C處理組兩兩間所測指標(biāo)的變化和差異。結(jié)果：1. LPS作用于Ana-1細胞，與對照組相比較，細胞內(nèi)ROS的表達濃度增高（P0.01）；總SOD的表達減少；彗星實驗檢測出Ana-1細胞DNA有損傷（P0.01）,提示LPS處理可誘導(dǎo)Ana-1細胞的氧化應(yīng)激損傷。氧化應(yīng)激下LPS誘導(dǎo)的Ana-1細胞內(nèi)的ROS的表達濃度較LPS處理組明顯增高，（P0.01）；總SOD的表達減少（P0.01）；彗星實驗檢測出Ana-1細胞DNA明顯損傷（P0.01）。2.共聚焦顯微鏡下觀察對照組、LPS處理組、LPS+H_2O_2處理組的ROS熒光強度和熒光數(shù)量發(fā)現(xiàn)：LPS處理組的ROS熒光強度和熒光數(shù)量與對照組相比較有所增加（P0.01）；LPS+H_2O_2處理組的ROS熒光強度和熒光數(shù)量較LPS處理組明顯增加（P0.01）。3.RT-PCR法檢測自噬相關(guān)因子LC3-Ⅱ的mRNA表達水平變化發(fā)現(xiàn)：LPS+H_2O_2處理組較LPS處理組的LC3-Ⅱ的mRNA表達水平量多；LPS處理組較對照組的LC3-Ⅱ的mRNA表達水平量多。4.共聚焦顯微鏡下觀察對照組、LPS處理組、LPS+H_2O_2處理組和維生素C處理組的GFP-LC3和RFP-LC3轉(zhuǎn)染發(fā)現(xiàn)：LPS處理組較對照組的自噬小體有所增加；LPS+H_2O_2處理組較LPS處理組的自噬小體在胞質(zhì)內(nèi)表達明顯增加；維生素C處理組較LPS+H_2O_2處理組的自噬小體有所減少。5.Western Blot法檢測自噬基因BECN1蛋白表達發(fā)現(xiàn)：LPS處理組較對照組的BECN1表達水平量增多（P0.01）；LPS+H_2O_2處理組較LPS處理組的BECN1表達水平量增多（P0.01）。Western Blot法檢測自噬基因LC3Ⅰ/Ⅱ蛋白發(fā)現(xiàn)：LPS處理組較對照組的LC3表達水平量增多（P0.01）；LPS+H_2O_2處理組較LPS處理組的LC3表達水平量增多（P0.01）；維生素C處理組較LPS+H_2O_2處理組的LC3表達水平量減少（P0.01）。結(jié)論：1.LPS可誘導(dǎo)小鼠肺巨噬細胞Ana-1的氧化應(yīng)激，導(dǎo)致細胞損傷。氧化應(yīng)激下LPS誘導(dǎo)小鼠肺巨噬細胞Ana-1的氧化應(yīng)激加強，加重細胞的DNA損傷。2.氧化應(yīng)激下LPS誘導(dǎo)小鼠肺巨噬細胞Ana-1的自噬加強,上調(diào)自噬相關(guān)蛋白BECN1和LC3Ⅰ/Ⅱ蛋白的表達，這與氧化應(yīng)激造成細胞DNA損傷有關(guān)。3.通過抗氧化劑維生素C處理發(fā)現(xiàn)，維生素C能降低氧化應(yīng)激下LPS誘導(dǎo)小鼠肺巨噬細胞Ana-1的GFP-LC3和RFP-LC3的轉(zhuǎn)染表達以及自噬基因LC3Ⅰ/Ⅱ蛋白，再次證明氧化應(yīng)激加強LPS誘導(dǎo)下小鼠肺巨噬細胞Ana-1的細胞損傷，從而加重細胞自噬現(xiàn)象。
[Abstract]:AIM : To investigate the interaction and mechanism of oxidative stress between lipopolysaccharide ( LPS ) and autophagy at home and abroad . The mechanism and molecular mechanism of oxidative stress on the autophagy of Ana - 1 cells induced by LPS from oxidative stress Ana - 1 cell DNA damage and autophagy were studied . The effects of LPS on the expression of total superoxide dismutase ( SOD ) in Ana - 1 cells were detected by MTT assay . The expression of total superoxide dismutase ( SOD ) in Ana - 1 cells was detected by MTT assay . Compared with the control group , the concentration of ROS in the cells was higher than that of the control group ( P0.01 ) .
the expression of total SOD decreased ;
The DNA of Ana - 1 cells was damaged by comet assay ( P0.01 ) . It was suggested that LPS treatment could induce oxidative stress injury in Ana - 1 cells . The expression of ROS in Ana - 1 cells induced by LPS was significantly higher than that in LPS - treated group ( P0.01 ) .
The expression of total SOD was decreased ( P0.01 ) .
The content of ROS fluorescence intensity and fluorescence in LPS treated group , LPS treated group , LPS + H _ 2O _ 2 treatment group were compared with that of control group ( P0.01 ) .
The levels of ROS fluorescence intensity and fluorescence in LPS + H _ 2O _ 2 treatment group were significantly higher than those in LPS - treated group ( P0.01 ) .
The expression of LC3 - 鈪

本文編號：1720184