肿瘤抑制基因pten过表达对体外培养的人气道平滑肌细胞增殖的影响

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1、硕士学位论文withmaturemethodsinourlaboratory．Theforthtoeighthgenerationsofairwaysmoothmusclecellswereusedforexperiments．Anti·smoothmuscleaactinmonoclonalantibodyisusedfortheidentificationofairwaysmoothmusclecells．2．Plasmidscarryingthefull—lengthofwild-typePTENcDNA(p—WT-PTEN)andtheempty

2、plasmid(P-EGFP)weretransformedintoEscherichiacoliDH5atoamplify,ScreenwithAmpicillin，selectpositiveclones，culturebacterium，andthenextractplasmidswithomegaendotoxin—freeplasmidextractionkit．Doublerestrictivedigestion，andgenesequencingwereusedtoidentifiedtherightnessofplasmid．Largea

3、mountofplasmidwasextractedandreservedunder-20℃circumstance．3．Experimentgrouping：cellsweredividedintothreegroups：p-WT-PTENtransfectedgroup(GroupA)，p-EGFPtransfectedgroup(GroupB)，andnon-transfectedgroup(GroupC)．CellsweretransfectedwithLipofectamin2000accordingtoits’procedure．Transf

4、ectionresultswereobservedunderinvertedfluorescencemicroscope．4．MTS／PMSmethodwasusedtodetecttheproliferationofHASMCs．5．CellcycleprogressofPI-stainingcellswasdetectedbyflowcytometry．6．TotalRNAofeachgroupwasaxtractedwithTrizol，theexpressionlevelsofPTENmRNAwasdetectedbyRT-PCRmethod．7

5、．HASMCsculturedin6-cultureplatesweretransfectedastheabove—mentionedmethod．After48hoursoftransfection，PTENproteinexpressionwasdetectedwithimmunohistochemistrySPmethod，andwasobservedandphotographedunderinvertedmicroscope．8．Dataareexpressedasmean±SEM．Statisticalcomparisonswereperfor

6、medusingone-wayANOVA(LSD)．StatisticalsignificancewassetatP

7、repositiveimmunostainingfora--actin．Passedtothe4thgeneration，theASMCspurityisabout95％．2．AfterdigestedbyXbaIandHind1II，agarosegelelectrophoresisofp—W-P11ENshowtwobands，themolecularweightofeachis5．4kband1．3kbrespectively，thesamesizeasthepGZdXZandtheinsertedPTENeDNA．Extractedplasmid

8、sweredeliveredtoBeijingNuosaiCompanyfors