114HBsAg-positivepregnantwomenandtheirnewbornsPBMCHBVinfectionstatus-畢業(yè)論文翻譯

1、114 HBsAg-positive pregnant women andtheir newborns PBMC HBV infection statusTo write thesis net:Author: Wang Xiao-jun WANGSu-ping core Business Feng YongliangKeywordsperipheralbloodmononuclearcells,hepatitisBvirus,insituhybridizationAbstractObjective:Tounderstandthe HBsAgpositivepregnantwomenandthe

2、irnewbornsPBMCHBVinfectionstatus.Methods:Insituhybridization(ISH)was detected114cases of pregnant women and newborns of HBV DNA inPBMCsmear.Results:114casesofHBsAgpositivepregnantwomenandtheirnewbornsPBMCHBVDNAdetectionratewas 28.07%(32/114and32.46%(37/114PBMC HBV infection in HBsAg positive pregnan

3、t womenand their newborns between the existence of consistency(Kappa=0.52, P<0.01.Conclusion:confirmedandPBMC HBV infection in HBsAg positive pregnant women and their newborns between the existence of consistency.Keywords peripheral blood mononuclear cells,hepatitisBvirus,insituhybridizationA

4、bstract1Objective:To observethestatusofHBVinfectioninperipheralbloodmononuclearcells(PBMC)fromtheHBsAgpositivemothersandtheirneonates.Methods:Insitu hybridization(ISH)wasusedtodetect HBV DNAinPBMC smear from 114 HBsAgpositive mothers and theirsnewborns.Results:ThepositiveratesofthePBMCHBVinfection o

5、f the mothers and their neonates were 28.07%and 32.46%, respectively. There was an agreement of theHBVinfectionstatusbetweenthemothersandtheirnewborns(Kappa=0.52,P<0.01).Conclusion:TheresultsuggeststhattheHBV infectionstatusfromthemothers agrees with that from their newborns.Keywords: periphe

6、ral blood mononuclear cells;hepatitisB virus;in situhybridizationHepatitisB virus(hepatitis B virus, HBV intrauterine infection is the cause ofchronicHBsAgcarryingimportantreasonHBVintrauterinetransmissionofHBV intrauterineinfectionmechanismis complexanddiverseclinicalhepatitisBvaccine(hepatitisB va

7、ccine,HBVacandcombinationofhepatitis B immune globulin (hepatitis B immune globulin,200IU ofHBIG can notcompletelyblocked (intrauterine2transmissionofHBVmaternal-fetalintrauterinetransmissionstudy1confirmedinHBsAg-positivepregnantwomenandtheir newbornsperipheralbloodmononuclearcells(peripheralbloodm

8、ononuclearcells,PBMC could be detected in HBV replication intermediates(covalentlyclosedcircularcccDNA.HBVinfectedPBMC,affect the humoral and cellular immune status, leading topersistentHBV infectionorrecurrentinfections.ShiXiaohong2 hadnewbornHBsAgpositive pregnantwomen,newbornsofHBVinfectionfor re

9、search.Tofurther understand the condition of pregnant women andtheir newbornsPBMCHBVinfectionusinginsituhybridization (ISH technology study, the results reportedbelow.1 objects andmethods1.1 The objectofstudyHBsAgpositivepregnantwomenandtheirnewbornsinTaiyuanhospitalforinfectiousdiseases,obstetrics

10、and gynecology to July 2003 to November 2004forprenatalcareandchildbirthforthestudywerecollectedfrompregnantwomenbeforedeliverycubitalveinandbirthwithin24h notfemoralveininjectionof3hepatitisB vaccine,EDTA anticoagulant,anti-clottingat4 to save backup.1.2 laboratory testing1.2.1 PBMC the smearprepar

11、ationanticoagulantplusthe sameamountFicolllymphocyte separation medium, 2000r/min centrifugal 30min, draw the monocytes layer to another test tube, add 2mL of Hank s balanced salt solution, 1000 r / min for 10min, washed 2 times repeat, after washing the cells with30 LHank s solution into the cell s

12、uspension; take 5 10L appliedby APES (3-aminopropyl-3ethoxy-silyl-offpreventiontreated carrierslide,fixed in70% ethanol,dried naturally, 4°C and stored.1.2.2HBVSgeneprobePreparationofrecombinant plasmid DNA (1)PCP10HBV DNA extraction:lessons 1.0 1.5 mLrecoveryPCP10plasmidculturemedium in a 1.5

13、mL centrifuge tube, 4°C 14000 r / mincentrifugation30 s toabsorbthe culturemedium,thebacterial pellet as dry as possible, and the bacterial pelletresuspendedin0.5mLSTE 4 ° C 14000r /mincentrifugation1min,aspirate supernatant,thebacterial4pellet as dry as possible, the bacterial pellet was

14、resuspended severe shock in ice-cold solution I added 200L freshlypreparedsolutionII(tightlynozzlequicklyreversed5 times,nottoshock, placedonicefor3min,add 150L of ice-cold solution III (cover tight nozzle tubeinvertedgently shock 10 s, placed on icefor 35 min,4 under14000r/mincentrifugation10min,th

15、esupernatant was transferred to another 1.5 mL centrifugetube after the tube plus 1 times the supernatant volume ofphenol:chloroform(1:1), mixingshock,4°C14000 r /min centrifugation 2 min, the supernatant was transferredtoanother1.5mLcentrifugetube,plus2timesthesupernatantvolumeof95%ethanolands

16、hakenmixed,allowedtostandatroomtemperaturefor2min,14000r/mincentrifuged10 min,carefullyaspiratethesupernatant,centrifugetubesupsidedownonthefilterpaper, so thatall of the liquid as faras possible outflowattachedtothe wall, and then the filterpaperdivisibledroplets, and then adding 70% ethanol 1mL, 4

17、°C under14000 r / min for 5 min to wash the DNA precipitate, thencarefully aspirate the supernatant, centrifuge tubes upsidedown on the filter paper, so that all of the liquid as far as5possibleoutflowwithafilter paperdivisibleattachedtothe wall of the droplets, air dried for 10 min, 30LTE buff

18、ersolution, -20 (HBV S gene double-stranded DNA bareprobepreparationandmarking:HBVSgenewasamplifiedbyPCR primersselectedfromtheconservedsequenceofthe HBVS gene promoterandterminatornear 3 (189 689 nt, by ShanghaiSangonTechnologyCo.,synthesis,amplifiedfragmentlengthof500bp,primer sequences:HBVSP1 5 C

19、CT GTC CGT GTTACAGGC3(upstreamprimerHBV SP2 5GGCATC AGTAAACTG AGC3(downstreamprimerExtractionofHBVDNAas a template,asprimersP1, P2,andthetotalreactionvolumeof50l,thespecificreactionsystemincluding:10× Buffer5 L, 0.025mol/L MgCl24 L,0.01mol / L dNTP1L, HBV SP1 1 L, HBV SP2 1 L Taq DNApolymerase

20、0.2 L Template DNA 5L, sterile water, set thevolumeto50L,2dropsofsterileparaffinoilcap,instantaneouscentrifugationforDNAAmplification,placedin a PCR thermalcyclerandcycleparametersasfollows: 94 degeneration 300s, denaturation at 94°Cfor 50 s, annealing at 55°C for 50 s, 72°C extension

21、 506s, 35 cyclesof 72° Cfinalextension300s, taketheamplified product 10L sample buffer and mix in a 1.2%agaroseelectrophoresisgel(containing0.5mg/LEB)5v/cm, 20 min later observed under UV light appears near501 bp, compared with DNAmarker orange-red one withpositiveornegative.ultravioletlamprecy

22、clingpositiveelectrophoreticgelblock,theDNApurificationkit,isolated and purified, recycling the fragment of the HBV Sregion,smallsampleUV-visiblespectrophotometerquantitative,wasdissolvedinsterilewatertoapproximately100 300 mg / L to obtaintheHBV Sregion double-stranded DNA bare probe, reference Roc

23、heDIG High Prime DNA Labeling and Detection Starter Kitmanual probe labeling, 20.Links to free papers Download Center 1.2.3 situhybridization (1 steps: cell routine into thewater for 5 min, 0.01 mol / LPBS rinsed 3 min× 2,0.2 mol/ L HCl at room temperature digestion 15 min, 0.01 mol / LPBS rins

24、eterminationof3 min× 3,10mg /L proteaseK37 digestion 20 min, 0.2% glycine, 0.01 mol / L PBS at room temperature for 10 min, 0.01 mol / L PBS rinsed 3min× 2 gradedethanol(80%,85%,90%,95%,100%,7100% 5 min each dehydrated, dried naturally, and hybridization solution (containing 800 mg / L lab

25、eled HBVDNA probe, with the first 42 pre hot 30 min), 25L /tablet drops in PBMC smear on plus a coverslip, denaturedat 95 at 100°C for 10 min, -70°C ice quenched for 5min,thenincubatedat42 for 20h, washing,washedsuccessivelywith2xSSC,1xSSC, 0.5xSSC vibratorwashing and rinsing, the blocking

26、 solution was 37°C andincubatedfor30 min,addantigrounddigoxigeninalkaline phosphatase labeled antibody (1:500,30L / chip37 incubatedfor2.5h,0.01mol/ LPBS rinsingfor3minx3NBT/BCIP(30 L/tablet),darkcolorfor5h,washedwithdistilledwaterfor10min,thrownpiece,nuclearfastred 1droplininginto3min3min;dist

27、illedwater rinse, and transparent, was washed successively with75%,85%,95%ethanolfor2mineach,of absoluteethanol for 3 min× 2, xylene I, xylene for 2 min each,mounted with neutral gum , microscopic photography. (2)control settings: (1) positive control, PCR confirmed thattheHBVDNApositivePBMC ce

28、llsmears, negativecontrol,unlabeledHBVS genebareprobeREF, PBSblankcontrol.pinkcellsappeardarkpurplesignal8determinedtobepositive,andthesawpinkcellsdetermined to be negative.1.3StatisticalStatisticalanalysiswasperformed using SPSS 12.0, consistency checking.2 Results114 cases of pregnant women andthe

29、ir newborns in PBMC HBV infection rates were 28.07%(32/114and32.46%(37/114),23 cases of 32 casesPBMC-positivepregnantwomen,newbornsPBMCHBVDNA-positive,82casesofPBMCnegativepregnantwomen14 casesofnewbornsPBMCHBVDNA-positivepregnantwomenPBMCHBVinfectionandneonatalPBMC HBV infection between consistency

30、 (Kappa = 0.52,Z =5.62, P<0.01,Table1Table 1 HBsAg-positivepregnantwomenandnewborntherelationshipofthechild PBMC HBV infection3 DiscussionAfter the 1980s, scholars have foundfromextrahepatictissuesandcellsofpatientswithhepatitisB, HBVpresenceofexpression in PBMCHBVinfection is relatively high

31、.PBMC including lymphocytes,monocytes, and NK collection of cells and other immune9activityofwhitebloodcells,thebody simmuneresponse to shoulder important immune function. alter itssecretoryfunctionofHBVinfectionPBMC,affectthebody s cells and humoral immunity. , PBMC HBV clearance of hepatitis B vir

32、us infection continued to carryas well as immune tolerance ending will play an important role in this paper ISH were detected HBV DNA in PBMC of pregnant women and newborns, confirmed PBMC as HBV misprision place.PBMC HBV infection testing to pregnant women PBMC more 4, the literature neonatal PBMC

33、HBV infection relatively small infection rates vary due todifferences in research methods and the object. Poovorawan 5 findings show HBsAg single positiveHBsAg,HBeAgdouble-positivepregnantwomenPBMCHBVdetectionrateof 69.2%(9/13)and94.7%(18/19),respectively, in their study of 34 cases of newborn child

34、renPBMCwerenotdetectedHBVDNAdomesticWANGSu-pinget al 2in situPCR andISH methodfoundinserum HBV DNA-positive and-negative neonatal PBMC ofHBV DNA detection rate was 49.09% (55/93 and 10.53%10(4/38 Shi Xiaohong 2 selective PCR, found in HBsAg positive pregnant women, newborns PBMC HBV DNA detection ra

35、te was 23.84% (36/151), similar to the resultsof this paper, further confirmed the ISH detection PBMC HBV DNA is stable and reliable.Inthispaper, theISHdetected114casesofmaternaland child HBsAg-positivepregnantwomeninPBMC HBVinfectionrate was 28.07%(32/114neonatalPBMCHBVinfectionratewas32.46%(37/114,theinfectionofbothconsistency(Kappa =0.52,Z = 5.62, P<0.01,usingPCRtechnologywithprevioussimilarstudies6 concludedunanimouslypromptdetectionofneonatalPBMCHBVinfectionimportantinthestudyofHBV intrauteri