论文实例：透明质酸在腹膜透析中作用的机理研究

1、论文实例：透明质酸在腹膜透析中作用的机理研究 作者简介：郭群英，女，1972年05月出生，1998年09月师从于中山大学叶任高教授，于20_年07月获博士学位。 摘要腹膜透析,特别是持续性不卧床腹膜透析（continuousambulatoryperitonealdialysis,capd）是慢性肾功能衰竭的主要治疗方法之一。超滤失败是目前导致透析不充分乃至腹膜透析失败的重要原因。如何预防腹膜超滤功能下降,有效地保护持续性腹膜透析中的腹膜功能,是腹膜透析研究的热点。目前临床尚无有效防止超滤失败的渗透压制剂或药物。透明质酸是一种由双糖单位n-乙酰葡糖胺和葡醛酸重复连接构成的多糖。我们以前的研究发

2、现,在急性大鼠腹膜透析模型中应用透明质酸可以有效地增加超滤量，增加尿素氮的清除率,减少蛋白质丢失，而且这种作用与其分子量和其在透析液中的浓度密切相关。透明质酸的作用机理目前还不清楚。有研究表明，腹膜表面可能存在一层由腹膜间皮细胞分泌的透明质酸、磷脂所形成的表面活性层，而且这一层可能在腹膜转运，特别是腹膜水转运中起很重要的作用。通过振动腹腔来破坏这一液体层可增高腹膜的通透性。我们推测,长期腹透过程中，腹透液的冲刷可使这一表面活性层结构破坏，腹膜失去其表面这一极为重要的疏水层后功能发生改变，对水通透性增加，腹腔液体重吸收增加，从而导致超滤功能下降。透析液中加入的大分子量透明质酸（外源性透明质酸）在

3、腹腔静水压作用下，与内源性透明质酸层一起在腹膜表面形成一层保护膜（restrictfiltercake）,保护其免受非生理性腹透液损害,并有降低腹膜通透性的作用,从而减少腹腔液体吸收,增加超滤量，维护腹膜功能。 虽然透明质酸在大鼠急性腹膜透析模型中显示了较好的效能，但其在大鼠慢性腹膜透析中的作用、持续性应用透明质酸后腹膜功能将如何变化尚不肯定。尽管国际腹透界对腹膜表面活性层有种种猜测，并通过对腹膜间皮细胞（mpcs）研究，间接证实了该层的存在，然而，至今仍未有形态学证据。 heldin的研究早已证实人间皮细胞(hmcs)能合成大量透明质酸（ha），新合成的ha在细胞外形成一环绕细胞的胞衣结构，

4、对细胞有保护作用。最近研究已证实哺乳动物有三种透明质酸合成酶基因has-1、has-2、has-3，这三个基因的产物透明质酸合成酶1(has-1)、has-2和has-3在氨基酸序列和分子结构特征上有很大的同源性。虽然酶学特征各有不同，这三种酶都可以在细胞膜内侧面催化透明质酸链合成，但他们催化合成的ha链的长度却各不相同。has1和has2蛋白催化合成大分子量的ha，has-3则催化合成较短的ha链。不同分子量的ha对细胞功能有不同的影响。不断延长的透明质酸链伸出细胞膜外，成为细胞基质（胞衣样结构）的骨架。哪一种透明质酸合成酶是人腹膜间皮细胞（hpmcs）透明质酸合成的关键酶？炎症因子、各种腹

5、膜透析液渗透压制剂对hpmcs透明质酸合成有什么影响？如前所述，透明质酸的作用与其分子量相关，不同分子量的外源性透明质酸对hpmcs内源性的透明质酸合成有什么影响？所有这些问题均未见报道。为探讨这些国内外尚未解决的问题，我们设计并进行了以下研究： 一透明质酸在大鼠慢性腹膜透析模型中的作用 为了解透明质酸在大鼠慢性腹膜透析中的作用及腹膜功能变化，进行以下研究：20只雄性sd大鼠随机分为3组。高糖组（n=8）和透明质酸组(n=6)每天分别腹腔内注射25ml的4.25葡萄糖腹膜透析液或含0.025透明质酸(分子量为1.6106道尔顿)的4.25葡萄糖腹膜透析液,共7天;正常对照组(n=6),不进行腹

6、腔注射。第8天进行4h腹膜功能实验并于不同时点留取血和腹透液样品进行分析。结果如下： 高糖组和透明质酸组的腹腔内容积(ipv)显著低于正常对照组(p值均0.05）。高糖组尿素钠总蛋白d/p值均显著高于其他两组(p均0.05）;对照组、l、il-1、tnf-组细胞培养上清液透明质酸（ha）的浓度均显著高于对照组(p值均0.05）,甘露醇组has-3mrna表达较对照组减弱(p0.05）。对照组、高糖组、低糖组、葡聚糖组、甘露醇组细胞培养上清液ha的浓度均显著高于对照组（p值均0.05）;高糖组ha浓度显著高于低糖组、葡聚糖组和甘露醇组（p0.05）。 因此与高浓度葡萄糖相比，葡聚糖虽增强hpmc

7、shas-2mrna表达，但对hpmcs的has-3mrna无影响，提示其主要促进大分子透明质酸的合成，因而更接近hpmcs的生理状态。这一作用与渗透压可能无关。 3不同分子量透明质酸（ha）对人腹膜间皮细胞（hpmcs）透明质酸合成酶的调节作用 分离培养的第2代人腹膜间皮细胞(hpmcs)消化后传入6孔培养板，将细胞随机分为3组（每组6个样本）：正常对照组（对照组）；高分子量透明质酸组（分子量为1，600，000道尔顿，浓度0.01,hha组）；低分子量透明质酸组（分子量为280，000道尔顿，浓度0.01,lha组）。继续培养24小时后，提取总rna,以rt-pcr法检测has-2mrna

8、和has-3mrna的表达;微粒排除法观察细胞基质的面积,结果如下: lha组has-2mrna及has-3mrna表达均较对照组增强（p值均0.05。hha组has-2mrna表达较对照组增强，而has-3mrna表达与对照组无显著差异。lha对has-3mrna表达增强作用是其对has-2mrna作用的4.8倍和1.025倍。 这提示lha和hha均促进hpmcshas-2mrna表达，lha对has-3mrna表达也有增强作用，而hha则没有这一作用。因此，从选择腹膜保护剂的角度分析，我们倾向于选择更具生物相容性hmw-ha。 综合以上结果，我们得出以下结论： 1.大鼠慢性腹膜透析模型中

9、多次重复使用透明质酸，可以防止由于长期使用以葡萄糖为渗透压制剂的腹膜透析液所引起的腹腔吸收率增加，增加超滤量，保护腹膜功能。 2.通过电镜研究证实(1)正常大鼠的腹膜表面覆盖了一层假膜结构（即腹膜表面活性层）。(2)这一表面活性层中含有透明质酸，并可能含磷脂。 3.has-2是hpmcs透明质酸合成和细胞细胞衣样结构形成的关键酶。正常生理状态下，人腹膜间皮细胞以催化合成大分子量透明质酸为主。 4.炎症因子l、tnf-、il-1均使hpmcs合成ha增加。由于其对has-3mrna表达的促进作用是主要的，故其主要促进低分子透明质酸的合成。 5.高浓度葡萄糖（90mmol/l），低浓度葡萄糖(30

10、mmol/l),葡聚糖(5)及90mmol/l甘露醇都促进人腹膜间皮细胞透明质酸合成。高浓度葡萄糖促进ha合成的作用最为明显，其同时促进has-2和has-3mrna的表达。与高浓度葡萄糖相比，葡聚糖促进ha合成的作用较弱，其对has-3mrna没有影响，主要促进大分子透明质酸的合成，因而更接近hpmcs的生理状态。这一作用与各组间渗透压差可能无关。 6.lha和hha均促进hpmcshas-2mrna表达，lha对has-3mrna表达也有增强作用，而hha则没有这一作用。因此，从选择腹膜保护剂的角度分析，我们倾向于选择更具生物相容性hmw-ha。 themechanismofhyaluro

11、naninperitonealdialysis peritonealdialysis(pd)isanestablishedformofrenalreplacementtherapy.ultrafiltrationfailureisanimportantreasonofinadequacyandfailureofpdandtheresearchtopreventthedecreaseofultrafiltrationandmaintaintheperitonealfunctioninlong-termperitonealdialysisisalwaysthehototofpd.hyalurona

12、nisalongpolysaccharidechainthatismadeupofrepeatingdisaccharideunitsofn-acetylglucosamineandglucuronicacid.wehaverecentlyshownthatadditionofhyaluronaninanacuteratmodelofpdcoulddecreasetheperitonealfluidaorptionrateandimprovethene tultrafiltrationvolume.thiseffectisbothconcentrationandmolecularweightd

13、ependent.themechanismofhyaluronanisnotclear.ithasbeensuosedlongagothattherewasasurfactantlayeronperitoneumandthislayermayplayimportantroleinperitonealtraort,eeciallyonperitonealfluidtraort.vibrationcandestroythislayerandincreasetheperitonealtraortrate.wesuectinlong-termperitonealdialysisthislayermay

14、bewashedoffandtheloofthishydrophobiclayermightleadtotheincreaseoftheperitonealfluidaorptionandthedecreaseofultrafiltration.theaddedexogenoushyaluronanmightaccumulateonperitonealsurfaceandformarestrictivefilter“cake”withendogenoushyaluronan,whichmightprotecttheperitoneumfromun-physiologicaldialysatea

15、ndimproveultrafitrationthroughdecreasingperitonealtiuehydraulicconductivity.althoughapreviousshortreportshowedthatrepeateduseofhyaluronanincreaseperitonealfluidremoval,thedetailedchangeinperitonealtraortcharacteristicsafterrepeateduseofhyaluronaninperitonealcavityisstillnotknow.inaddition,althoughth

16、eperitonealsurfactantlayerwasindirectlyestablishedbytheresearchofhumanperitonealmesothelialcells(hpmcs),therewasnotanymorphologicalevidenceofit. recentstudieshaveshownthatperitonealmesothelialcellscouldsynthesizelargeamountofhyaluronanandthenewlysynthesizedpolymermayformapericellularhyaluronancoatto

17、protectthecellsfromdamage.recently,threerelatedmammaliangeneshavebeenidentifiedashasynthases,designatedhas-1,has-2andhas-3,andtheenzymesofthesethreegenesdilayhomologyinmolecularstructureandaminoacidsequence.allofthemcancatalyzethesynthesisofhyaluronanattheierfaceoftheplasmamembrane.thenewlysynthesiz

18、edpolymerextrudedthroughthemembrane,whereitcanformpericellularcoatbybindingwithotherpolymersorhyaluronanbindingprotein.whichhyaluronansynthaseisthecriticalenzymetocatalyzehyaluronanandformpericellularcoats?howdoinflammationmediatorsandosmoticagentsmodulatehyaluronansynthasesexpreioninhpmcs?asweknow,

19、hyaluronanwithdifferentmolecularweighthasdifferentfunction.howdoeshyaluronanwithdifferentweightaffectthehyaluronansynthasesexpreion?inordertosolvetheproblemswedesignedthefollowingstudies. 1.hyaluronanpreservesthemembranetraortpropertiesinchronicperitonealdialysisanimalmodelofrats twentymalesdratsrec

20、eiveddailyipinjectionof25ml4.25glucosedialysissolutionwithout(hp,n=8)orwith0.025hyaluronan(ha,n=6)foroneweek,anothersixratsdidnotreceiveanyperitonealinjection.twenty-fourhoursafterthelastinjection,a4hdwellstudyusing25ml4.25glucosedialysissolutionwithipvolumemarkerandfrequentdialysateandbloodsampling

21、swasperformedineachrat. resultipvwassignificantlyhigherinthehagroupascomparedtothehpgroup(anovarepeatedmeasurement,p0.01).theperitonealfluidaorptionrate,ke,wassignificantlyincreasedinthehpgroupascomparedtotheothergrou.thedirectlymphaticfluidaorptionrate,keb,wassignificantlyhigherinthetwodailyinfusio

22、ngrou(hpandha)ascomparedtothecontrolgroup.however,thetiueaorptionrate,ket,wassignificantlyhigherinhpgroupascomparedwithhagroupandcontrolgroup. 2.ultrastructuralidentificationofanovelmembraneonperitonealsurfaceandelectrogoldmicroscopicinvestigation. normalratperitoneumwastakenfromfoursdrats.thetiuesw

23、ereimmediatelyfixedusingoneofthefollowingfixativesfor24hoursbeforeconventionaltiueproceing:(1)2.5glutaraldehydeand2polyformaldehyde(control);(2)2.5glutaraldehydeand0.5cetylpyridinumchloridetofixmainlytheglycosaminoglycan(gag);(3)2oso4tofixthephoshpolipids(ph1);(4)4oso4(ph2)and(5)3taicacid(taicacidgr

24、oup).afterpostfixiation,dehydration,embedmentandultrathinsection,thesectiowereoervedinahitachi-600tramiionelectronmicroscope.twelvenickelgrids(200mesh)weretouchedgentlyontoperitonealsurfaceofratsfor twosecondsandthenfixedasfollowing:formaldehydevaporfor5miairdriedwith40for30miwashedwithtfor15min3.th

25、enthegridswereusedtoinvestigatehyaluronanbyelectrogoldmicroscop:sixnickelgridswereincubatedwithha-biotin,theothersixwereincubatedwithtascontrol. resultinthecontrol,theperitonealmesotheliumwascoveredwithmicrovilliwhereasnosurfacelayerwasoerved.however,inthegaggroup,thereaearedtobeadiscontinuousamorph

26、ouslayercoveringthemesothelialcells.intheph1group,acontinuousamorphouslayercoveredtheperitonealsurfaceanditsthicknedependedontheabundanceofmicrovilli.thislayerwasmuchbetterpreservedintheph2groupwiththickneupto10m.inthetaicacidgroup,notonlythisstagnantlayercouldbeeasilyseen,wecouldbutalsooervetherewe

27、realotoflamellarbodiesinthelayerandiidethemesothelialcells.theimprintshowedperitonealsurfacelayerwithlatticesstructure.hapositivesignalcouldbeoervedintheexperimentalgroup,whereasinthecontrolgroupnogoldenparticlewasoerved. 3.hyaluronansynthase(has)2ecificantiseeoligonucleotidesinhibittheformationofpe

28、ricellularcoatinhumanperitonealmesothelialcells(hpmcs) asaprerequisitestudy,theexpreionofeachhasmrnainculturedhpmcswasmeasuredbyreversetracription-polymerasechainreaction(rt-pcr).onlyhas-2andhas-3mrnaexpreionweredetected,thelevelofhas-2mrnaexpreionwasabout10foldhigherthanthatofhas-3.has-2ecificantis

29、eeoligonucleotideswerethentrafectedintoculturedhpmcs.after0,8,48and24hoursthehpmcswereoervedusingparticleexclusionaayandtheirhas-2mrnaexpreionwasdetectedagain.theresultsshowedthathas-2mrnaexpreionwasmostlyinhibitedatthe24hourasmeasuredbyrt-pcr,andthediameteroftheextracellularcoatalmostdisaearcomplet

30、ely.seeandreversehas-2showednoeffect. 4.theimpactofinflammationonhyaluronansynthesesandaemblyinhumanperitonealmesothelialcells(hpmcs) hpmcswereculturedandstimulatedwithl,il-1bortnf-a.differenthasynthesizinggeneshas-2(whichsynthesizehighmolecularweighthyaluronan),andhas3(whichsynthesizelowmolecularwe

31、ighthyaluronan),weredetectedbyrt-pcr.thepericellularhyaluronancoatwasalsovisualizedbyparticleexclusionaay.theconcentrationofhyaluronaniculturedmediumwasmeasuredbyhyaluronanradio-immunoaaykits. resultl,il-1bortnf-aallstimulatedhas-2andhas-3mrnaexpreion.theeffectsoftheseinflammatorymediatorsonhas3expr

32、eionwasmuchhigherascomparedtotheireffectsonhas-2expreion,eeciallywiththestimulationofil-1bwhichincreasedthehas-3mrnaby19times.however,thesizesofthepericelluarhyaluronancoatinhpmcsdidnotchangesignificantlyafterl,il-1bortnf-astimulation.theconcentrationofhyaluronanininflammationmediatorsgrouweresignif

33、icantlyhigherthanthecontrolgroup. 5.theeffectofosmoticagentsonhyaluronansynthasesexpreioninhumanperitonealmesothelialcells culturedhpmcswerestimulatedwith90mmol/lglucose(hg),30mmol/lglucose(lg),5polyglucose(pg)and90mmol/lmaitol(mol).extracellularcellcoatwasoervedusingparticleexclusionaay,andtheexpre

34、ionofhyaluronansynthase2and3(has-2andhas-3)mrnainhpmcswasmeasuredbyreversetracription-polymerasechainreaction(rt-pcr).theconcentrationofhyaluronaniculturedmediumwasmeasuredbyhyaluronanradio-immunoaaykits. resulthas-2mrnaexpreioninthefourexperimentgrouwassignificantlyhigherthanthatofcontrolgroup.howe

35、ver,onlyhgenhancedhas-3mrnaexpreion.thesizesoftheextracelluarcoatinthefourgrouwerenotsignificantdifferentwiththatofcontrol.inaddition,hyaluronanconcentrationwassignificantlyhigherinthefourgrouascomparedwithcontrolgroup.thehyaluronanconcentrationinhggroupwasmuchhigherthantheotherthreegrou. 6.theeffec

36、tofhyaluronanwithdifferentmolecularweightonhyaluronansynthasesexpreioninhpmcs culturedhpmcswerestimulatedwith0.01lowmolecularweighthyaluronan (280,000da,lha)and0.01highmolecularweighthyaluronan(1,600,000da,hha).extracellularcellcoatwasoervedusingparticleexclusionaay,andtheexpreionofhyaluronansynthase2and3(has2andhas3)mrnainhpmcswasmeasuredbyreversetracription-polymerasechainreaction(rt-pcr). resultlhaandhhastimulatedhas-2mrnaexpreion,however,onlylhaenhancehas-3mrnaexpreion.thesizesoftheextracelluarcoatinthetwogrouwerenotsignificantdifferentwiththatofcontrol. inconclusion: