翻译心脉佳超滤膜提取物对过氧化氢诱导的人平滑肌细胞损伤的保护作用研究Word文档格式.docx
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Results:
H2O2significantlypromotedtheproliferationofhumanaorticsmoothmusclecells,whiletheultra-filtrationextractfromXMJcouldsignificantlyimprovethemorphologicalchangesoftheinjuredHASMC,decreasetheexpressionofICAM-1、VCAM-1、IL-1、IL-6、NF-κBandincreasetheexpressionofMMP-2、TIMP-2.
结论心脉佳超滤膜提取物能有效发挥抗炎、抗氧化的作用,显著抑制HASMC增殖、迁移,在动脉粥样硬化疾病的治疗方面具有重要价值。
Conclusion:
XMJhasobviousanti-inflammatoryeffectandantioxidantability,whichcouldsignificantlyinhibittheproliferationandmigrationofHASMX,ithasimportantvalueforatherosclerosistreatment.
【关键词】XMJ、人主动脉平滑肌细胞、增殖、迁移、炎症
Keywords:
XMJ,humanaorticsmoothmusclecells,proliferation,migration,inflammation
动脉粥样硬化(atherosclerosis,AS)是一慢性炎症过程,“氧化应激”产生的活性氧自由基(reactiveoxygenspecies,ROS)如过氧化氢(H2O2)等,以配体形式与血管内皮细胞及平滑肌细胞胞核受体结合,或直接充当胞内信号分子参与并调节多种基因表达产物,如白细胞介素(interleukin,IL)、细胞间细胞黏附分子(intercellularcelladhesionmolecule,ICAM)、血管细胞间黏附分子(vascularcelladhesionmolecule,VCAM)等增强单核细胞对血管内膜的粘附、迁移,在AS发病早期起着关键作用[1-2]。
AS由多种细胞及炎症递质参与[3],其中在刺激因子作用下血管中膜的平滑肌细胞(vascularsmoothmusclecells,VSMC)增殖、表型转变、向内膜迁移以及形成肌源性泡沫细胞,在AS形成的早期、中期、晚期以及血管狭窄性疾病的发病机制中都起着关键性的作用,是AS病变的重要病理学特征之一[4]。
ASisachronicinflammationcourse,ROSproducedinoxidativestress,suchasH2O2andsoforth,bindstothenuclearreceptorofvascularendothelialcellsandsmoothmusclecellsasligand,orregulateskindsofgeneexpressionassignalingmoleculedirectly,suchas(interleukin,intercellularcelladhesionmolecule,vascularcelladhesionmolecule),itcouldenhancetheadhesionandmigrationofMonocytestotunicaintima,andhasvitalimportanceintheearlyperiodofAS.KindsofcellsandinflammatorymediatorsparticipateintheoccurrenceofAS,particularlytheVSMCoftunicamedia,whichformsSmoothMuscleCell-derivedFoamCells,aftertheproliferation,phenotypictransition,migrationtointimaundertheeffectofStimulatingFactor,playsasignificantpartnotonlyintheearly,middleandadvancedstageofAS,butalsointhePathogenesisofvascularstenosisdiseases,itbelongstoimportantPathologicalfeaturesofAS
本实验研究的中药组方-XMJ(XMJ,专利号:
ZL201010536001.x,现有胶囊制剂)由以下原料按照重量份数配制而成:
功能红曲粉10-35份、葛根黄酮粉1-10份、大豆异黄酮粉1-8份、竹叶黄酮粉1-8份、白藜芦醇粉1-8份、山楂粉1-6份、天麻粉1-6份、黑木耳粉1-30份、海马粉0.1-0.2份、虾青素粉0.008-0.04份,薄荷脑粉0.1-0.3份和抗性淀粉20-50份。
ChineseMedicineCompound(XMJ,patentnumber:
ZL201010536001.x,Existingcapsulepreparation)inthisexperimenthasfollowingingredients:
FunctionalRedKojicRicepowder10-35%
KudzuFlavonoidspowder1-10%
Soybeanisoflavonepowder1-8%
BambooLeafFlavonepowder1-8%
Resveratrolpowder1-8%
Hawthornpowder1-6%
Gastrodiapowder1-6%
Auriculariaauriculapowder1-30%
Hippocampuspowder0.1-0.2%
Astaxanthinpowder0.008-0.04%
Mentholpowder0.1-0.3%
ResistantStarch20-50%
该保健食品含大量天然强抗氧化剂,在理论上还有增强免疫、延缓衰老的作用。
自愿者试食,几个月后AS性心脑血管疾病的症状减轻,血脂下降、血压缓慢恢复正常、不易疲劳、睡眠改善等,整体健康状况明显改善,取得令人满意的效果,但其具体的抗AS机制还不太明确,因此我们用H2O2损伤HASMC模拟AS中VSMC受损模型,从XMJ对受损HASMC的影响方面探讨其起保护性作用的具体机制。
Lotsofnaturalantioxidantwerecontainedinthishealthfood,withenhancingimmunityandanti-agingbenefitstheoretically.Volunteerswhoexperimentedwiththishealthfood,alleviatedASCardiovascularandcerebrovasculardiseases,reducedbloodfat,returnedtonormalbloodpressureslowly,feltenergeticandsleptwellafterseveralmonths.Althoughtheeffectwassatisfactory,theconcreteanti-ASmechanismwasnotobvious.
1材料
1.1细胞HASMC细胞株(美国ACTT公司)。
1.2药品XMJ原料药(中国北京同仁堂有限责任公司);
洛伐他汀、H2O2(美国Sigma公司);
脂必妥(成都地奥九泓制药厂)。
1.3试剂1640培养基、胎牛血清(美国GIBCO公司);
瑞-吉染液(长沙丽欣生物有限公司);
碳酸二乙酯、XTT、PMS(diethypyrocarbonate,DEPC)(美国Sigma公司);
胰蛋白酶、乙二胺四乙酸、乙醇等为国产分析纯。
1.4试剂盒MDA、SOD检测试剂盒(南京建成生物工程研究所);
IL-1、IL-6、ICAM-1、VCAM-1、MMP-2、TIMP-2、NF-кB的ELISA检测试剂盒(美国R&
D公司)。
1.5仪器0.01-0.05μm陶瓷膜、0.02μm微孔滤膜(南京米格过滤材料有限公司);
肽核酸(PNA)中空纤维超滤膜(外径:
0.5-2.0mm,内径:
0.3-1.4mm,截留分子量:
10万)、针头式滤器(北京九鼎高科过滤设备有限公司);
CO2培养箱、WX系列离心机、ELX-800酶标仪(美国Bio-Tek公司);
倒置显微镜(日本OLYMPUS公司)。
1Materials
1.1CellsHASMCCellLines(AmericanACTTCorporation)
1.2DrugsXMJCrudeDrugs(BeijingTongRenTangCo.,Ltd.)
Iovastatin
H2O2(AmericanSigmaCorporation);
Zhibituo(ChengduDioJiuHongPharmaceuticalIndustryco.,Ltd.)
1.3Preparations
1640CultureMedium,
Fetalcalfserum(AmericanGIBCOCorporation);
Ray-Geedyeliquor(ChangshaLiXinBio-technologyCo.,Ltd.)
Diethylcarbonate,
XTT,
PMS(diethypyrocarbonate,DEPC)(AmericanSigmaCorporation),
Trypsin,
Ethylenediaminetetraceticacid,
ethanol
1.4Kit:
MDA
SODDetectionKit(NanjingJianChengBioengineeringInstitute)
ELISADetectionKitsofIL-1、IL-6、ICAM-1、VCAM-1、MMP-2、TIMP-2、NF-кB(AmericanR&
DCorporation)
1.5ExperimentalApparatus:
0.01-0.05μmceramicmembrane
0.02μmMicroporousmembranes(NanjingMiGeFilterMaterialCo,.Ltd.)、
Peptidenucleicacid(PNA)Hollowfiberultrafiltrationmembrane(outerdiameter:
:
0.5-2.0mm,innerdiameter:
0.3-1.4mm,rejectionmolecularweight:
100,000)
Needle-typefilter(BeijingJiuDingHi-technologyFilterCo,.Ltd.)
CO2CultivationBox
WX-centrifuge
ELX-80EnzymeLabellingInstrument(AmericanBio-TecCorporation)
InvertedMicroscope(JapanOLYMPUSCompany)
2方法
2.1XMJ超滤膜提取物制备①10%(质量-体积百分比)XMJ水煎剂的制备:
精确称取XMJ原料药1000g,加入6000mL水浸泡2h→用1000W微波炉加热至水沸腾后开始计时,中火煎煮1h后,将药液倾出,然后用四层纱布过滤药液→XMJ药渣内再加水6000mL,重复上一步骤→分别合并两次的水煎滤液,趁热用无菌脱脂药棉过滤→分别将滤过的水煎液加热浓缩至10000mL,冷却至20℃后分装入2L塑料桶内,立即送新乡医学院药物研究所进行膜分离处理;
②应用超滤技术精制XMJ水煎剂:
陶瓷膜微滤10%的XMJ浓缩液,以便除去药液中的蛋白、细菌、柔质、果胶等大分子物质(技术参数:
压力0.5Kpa/m3、温度25℃、流量100L/h·
m2)→PNA中空纤维超滤膜XMJ水煎液,最后分离成10万分子量以下滤液5000mL(技术参数:
压力5Kg/m3、温度25℃、流量100L/h·
m2);
③XMJ浓缩超滤液:
将5000mL浓缩至1000mL,相当于每mL药液含XMJ量为1g→将超滤浓缩液喷干成粉末,高压灭菌后,用1640培养基配制成1200mg/mL的母液,贴标签置4℃冰箱储藏备用。
2Methods
2.1Ultra-filtrationMembraneExtractsPreparationsforXMJ
①10%(quality/volume)WaterDecoctionPreparationsforXMJ:
PutXMJcrudedrugs(1000g)intothecontainerwithinwater(6000mL)completelyfor2hours.→Heatitupinthemicrowaveoven(1000w),thenstarttimingafterthewaterisboiled.Afterdecoctingitinamediumheatfor1hour,pourouttheliquidmedicinewithdregsofdecoctionleftinthe,thenusethefour-gauzetofilteritandgetonetypeofliquidmedicinefiltered.→Add6000mLwaterinthecontaineragain,andthenrepeatthemethodsasthesameasthelaststepandgetanothertypeofliquidmedicinealsofiltered.→Emergethesetwotypesofliquidmedicine,usesterileabsorbentcottonwooltofilterthembeforetheygetcold.→Theseliquidmedicinefilteredcondenseintohighliquidmedicine(10000mL),putthemintotheplasticcask,aftertheyarecooledto20℃,andthensendthemtoXinxiangMedicalInstituteofMaterialMedicalfortheprocessofmembraneseparation;
②ApplyUltra-filtrationTechnologytorefiningXMJwaterdecoction:
theceramicmembranemicro-filters10%ofXMJinordertoremoveprotein,bacteria,softquality,pectinandothermacromolecularsubstancesfromliquidmedicine(technicalparameters:
pressureof0.5Kpa/m3,temperatureof25℃,flowrateof100L/h·
m2).→PNAhollowfiberultrafiltrationmembraneXMJwaterdecoctionisfinallyseparatedintoa100,000molecularsoastoget5000mLfiltrate(technicalparameters:
pressureKg/m3,temperature25℃,flowrateof100L/h•m2);
③XMJconcentratedultra-filtrate:
put5000mLfiltratetocondense1000mLconcentratedultra-filtrate,whichisequivalenttothepermLtheliquidmedicinecontaining1gXMJ→maketheultra-filtrationconcentratespraydriedintopowder,andthenthepowerismadeinto1200mg/mLmotherliquorwithDMEMmediumafteritisautoclaved,finallythemotherliquorislabeledandstoredintherefrigerator(4℃)inpreparationforusage.
2.2HASMC培养HASMC置于5%CO2培养箱(饱和湿度;
37℃),用含10%胎牛血清的1640培养基(含终浓度青霉素100U/mL,链霉素100U/mL,pH7.4)培养,当细胞达到80%-90%融合时用0.125%胰蛋白酶和0.01%乙二胺四乙酸按照1:
1的体积比配制的消化液进行消化,2~3天传代一次,实验用第5-10代的细胞。
2.2HASMCculture
PlaceHASMCintoa5%CO2incubator(saturatedhumidity:
37℃)tocultureitbyusing1640mediumcontaining10%fetalbovineserum(penicillin,100U/mL;
streptomycin,100U/mL;
pH7.4)).whenthecellsreach80%-90%confluences,digestingthembymeansof0.125%trypsinand0.01%ethylenediaminetetraaceticacidinaH1:
1volumeratiotoformulatedigestivejuices.Thewholeprocessneed2-3days,anditoftentakethecellsfromthe5th-8thgenerationintoexperiment.
2.3实验分组及处理取生长良好的HASMC,按4×
104/mL的密度接种,培养24小时后加对应终浓度的药物,再培养24小时后做后续实验。
实验分8组,①正常对照组:
加入等体积PBS(pH7.4)缓冲液;
②药媒对照组:
50mg/L的XMJ;
③模型组:
200μmol/L的H2O2;
④脂必妥组:
200μmol/L的H2O2+50mg/L脂必妥;
⑤洛伐他汀组:
200μmol/L的H2O2+1μmol/L洛伐他汀;
⑥低剂量XMJ组:
200μmol/L的H2O2+25mg/L的XMJ;
⑦中剂量XMJ组:
200μmol/L的H2O2+50mg/L的XMJ;
⑥高剂量XMJ组:
200μmol/L的H2O2+100mg/L的XMJ。
2.3Experimentalgroupsanddealing
TakingHASMCthatgrowwell,andseedingthematadensityof4×
104/ml.Culturethemabout