iNOS在EGCG促A549細胞凋亡中的作用.pdf

1、青島大學碩士學位論文摘要目的：目前研究顯示表沒食子兒茶素一3一沒食子酸酯(epigallocatechin一3一gallate，EGCG)具有抗炎、抗氧化和抗腫瘤活性。但是尚不清楚對炎癥的作用和其作用機理，本實驗選擇人肺腺癌上皮細胞株A549細胞，觀察表沒食子兒茶素沒食子酸酯(EGcG)對A549細胞的增殖、凋亡和誘導型一氧化氮合酶(iNOS)蛋白和mRNA表達的影響。方法：體外培養(yǎng)和擴增A549細胞，選擇對數(shù)生長期的細胞，隨機分為對照

2、組和EGCG處理組。MTT方法檢測EGCG對A549增殖活性的影響、并篩選最顯著作用濃度；應用流式細胞儀檢測最強作用濃度的EGCG孵育對A549細胞凋亡作用；應用免疫酶組化染色法、蛋白質(zhì)印跡定量分析法與Realtime—PCR方法檢測EGCG組中iNOS蛋白和mRNA的表達情況，對比分析作用其特點。實驗結果：結果顯示，與對照組相比，在50’400mg／L濃度范圍內(nèi)EGCG可以顯著抑制A549細胞的增殖活性，其中200mg／L的EGCG抑

3、制作用最強；其差異具有顯著性(F=100035，q=343“6000，PO05)。EGCG處理可以顯著提高A549細胞的早期和晚期凋亡率，尤其晚期凋亡和總凋亡百分比率增加更明顯，與對照組比較有差異顯著性(x2=6523，P005)。與對照組比較，EGCG抑制iNOS蛋白與mRNA的表達，其表達量較對照組顯著降低。其差異有顯著性(t=94，pO05)、(t=57，p005)。并可見EGCG促進A549細胞的胞質(zhì)和胞核固縮、體積變小，這些固

4、縮的小的A549細胞中iNOS蛋白表達呈增高狀態(tài)。實驗結論：EGCG抑制A549細胞的增殖、促進其凋亡作用可能與下調(diào)iNOS蛋白與mRNA的表達量有關。碩士研究生任妍(組織學與胚胎學)指導老師劉曉萍教授青島大學碩士學位論文Abstract0bjectivesToinvestigatetheeffectofinducibleNOS(iNOS)inEGCGpromotingapoptosisofA549cellsMaterialsandMe

5、thods1MaterialsHumanlungadenocarcinomaA549cells(MedicalCenterofAffiliatedHospitalofQingdaoUniversityMedicalCollege)RPMI1640andfetalbovineserum(Hyclone)RibbitantihumaniNOSpolycloneantibody(Abeam)AnnexinVFITC／PIApoptosisAs

6、saySystem(BD，America)Steady—Glo⑧LuciferaseAssaySystem(Promega，America)2MethodsTheeffectofEGCGontheproliferationofA549cellsweredetectedbyMTTassayA549cellswereculturedinvitro，thecellsinlogarithmicgrowthphasewereselectedand

7、dividedinto2groupsacontrolgroupandEGCGgroupswhichadministratedwithnormalsalineandEGCGattheconcentrationof50mg，【，400mg／Lrespectivelyfor24hoursTheMTT(5mg／ml，20I_t1)wereapplifiedandthelightabsorptionvalueweredetectedusingal

8、lELISAplatereaderat570nmAllexperimentswereperformedinquintuplicate，乃ccellviabilityWascalculatedwithKarber乃皓apoptosiseffectofEGCGinA549cellsbyflowcytometryTheA549cellsinlogarithmicgrowthphaseat3x105cells／wellwereseededin6

9、wellplatesandculturedovernightThe80—90％confluentA549cellswerestarvedfor4hinserum—freeRPMI1640mediumbeforetreatedwithnormalsalineandEGCGattheconcentration200mg／Lrespectivelyfor24hat370C，thenthecellswerecollectedandstained

10、withPEand7AADandexaminedbyflowcytometryWithBeckmanCoulterCyAn9colorhi曲一speedanalyzerrheexpressionofiNOS，proteinsaswellastheirmRNA，wasdetected麗廿lImmtmocytochemicalstaining，westernblottingandRealtime—PCRanalysesrespectivel

11、y3ResultsTheresults，comparedwiththatincontrolgroup，showedthatEGCGsignificantlyinhibitedproliferatingactivityofA549cellsattheconcentrationof50mg，00mg／L(F=1000。35，q=343～6000，P005)TheroleofEGCGattheconcentrationof200mg／Lwas