经心脏逆行置管灌注法在小鼠原代肝细胞分离中的应用.pdf

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1、川大学学‘医学版JSichuanUniv(MedSciEdi)2014；45(1)：’138一l41dEdi·技术与方法·经心脏逆行置管灌注法在小鼠原代肝细胞分离中的应用余招焱，白燕南，张涛，吴泓，曾勇△四川大学华西医院肝胆胰外科(成都610041)【摘要】目的观察采用经心脏逆行置管灌注肝脏的方法分离小鼠原代肝细胞的效果。方法对传统的分离原代肝细胞方法(Seglen两步胶原酶灌注法)进行改良，即从小鼠右心房的裂VI处经肝上下腔静脉向下方置管，并以输液器滴注含体积分数1O胎牛血清的胶原酶灌注液进行肝脏灌注。评价此法与传统方法的置管成功率及所

2、得肝原代细胞的数量及活力。结果改良组总的置管操作成功率高达95，且在一次置管成功率上改良组高于传统组(94．7VS．68．8，P<0．05)。改良组小鼠肝脏灌注均匀，肝细胞产量高达1．07×10／g小鼠体质量，细胞活力平均为92．16，两者均高于传统组(分别为0．99×10／g小鼠体质量及86．44％，P<0．05或P<0．01)。结论经心脏逆行置管灌注法分离小鼠原代肝细胞简便易学，重复性高，且能保证分离所得的肝细胞产量和质量。【关键词】原代肝细胞胶原酶灌注法逆行置管法IsolationofMousePrimaryHepatocytesb

3、yRetrogradeLiverPerfusionwithCatheterizationviaHeartyUZhao—yan。BAIYan—nan，ZHANGTao，WUHong，ZENGYong．DepartmentoJHepato—Biliary—PancreaticSurgery，WestChinaHospital，SichuanUniversity，Chengdu610041，China△Correspondingauthor，E—mai1：zengyong@medmail．corn．cn[Abstract]ObjectiveTo

4、exploretheeffectsofretrogradeliverperfusionwithcatheterizationviaheartontheisolationofprimarymousehepatocytes．MethodsInordertomoreefficientlyisolateprimarymousehepatocytes，weimprovedthetraditionaltWO—stepcollagenaseperfusionmethod．Theliverperfusioncatheterwasinsertedthrou

5、ghrightatriumandsuprahepaticvenacava，andtheperfusionvelocitywascontrolledbythedripinfusionwithcollagenaseperfusatecontaining10offetalcalfserum．ResultsTotalSUCCESSrateofcatheterizationintheimprovedgroupwasashighas95，andthesuccessrateoffirstattemptintheimprovedgroupwassigni

6、ficantlyhigherthanthatinthetraditionalgroup(94．7％VS．68．8)．LiverperfusionintheimprovedgroupwassymmetricalwiththehighyieldofhepatocytesuptO1．07×10。pergramofmouseweightand92．16ofaveragecellvitality，whichwerehigherthanthoseinthetraditionalgroup．ConclusionTheretrogradeliverper

7、fusionthroughtheheartisasimpleandeasy—tO—learnmethodtOisolatemouseprimaryhepatocytes，whichalsocouldguaranteethesatisfactoryyieldandvitalityofprimaryhepatocytes．[Keywords]PrimaryhepatocytesCollagenaseperfusionmethodRetrogradecathetermethod原代肝细胞是体外研究肝脏药物毒性、细胞相力较差等。此外还有先采用原位

8、灌注去除肝内血液互作用及代谢等肝脏功能的有力工具_1]；且应用肝而后采用机械酶消化使肝细胞分离两者相结合的方细胞移植和生物人工肝支持系统治疗急性肝衰竭和式l6]，这仍然存在由于胶原酶不能充分接