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ID:33180743
大小:3.51 MB
页数:47页
时间:2019-02-21
《内皮祖细胞移植改善大鼠肝移植缺血再灌注损伤的研究》由会员上传分享,免费在线阅读,更多相关内容在学术论文-天天文库。
1、重庆医科大学硕士学位论文内皮祖细胞移植改善大鼠肝移植缺血再灌注损伤的研究姓名:袁智斌申请学位级别:硕士专业:外科学指导教师:涂兵201205重庆医科大学磺士研究生学位论文内皮祖细胞移植改善大鼠肝移植缺血再灌注损伤的研究摘要目的:成功提取SD大鼠骨髓单个核细胞,体外培养并诱导其分化为血管内皮祖细胞(EPCs);成功鉴定及染色EPCs,用以干细胞移植实验;成功建立大鼠原位肝移植(OLT)模型,观察对发生肝移植缺血再灌注损伤(IRI)的大鼠移植EPCs后,内皮祖细胞是否在大鼠肝血窦内发生归巢并观察EPCs移植对肝移
2、植后IRI相关指标的影响。方法:从同种SD大鼠股骨及胫骨骨髓中采用Percoll密度梯度离心法提取单个核细胞,二次贴壁后以选择性DMEM培养液诱导其分化,观察其形态学改变,对其进行免疫细胞化学染色鉴定和双荧光染色鉴定,并以红色荧光染料PKH26GL标记;将48只SD大鼠随机分为2组,采用改良Kamada袖套法建立大鼠原位肝移植模型,分组为0LT+EPC移植组12对和OLT对照组12对,EPC组肝移植受体大鼠关腹后经尾静脉输注EPCs,对照组经尾静脉输注生理盐水;两组均于术后6小时,24小时,48小时取血液标本
3、及肝组织标本,冰冻切片荧光观察EPCs归巢情况,免疫组化观察肝血管区的EPCs抗原表达;检测肝功能ALT及血清TNF.Q水平,HE观察肝组织病理损伤并评分。l【基金项目】重庆市卫生局科研项目支持(编号2008—2-220)重庆医科大学硬士研究生学位论文结果:成功提取大鼠单个核细胞并诱导培养为EPCs,形态学观察,免疫细胞化学染色和双荧光染色鉴定其为EPCs:在3个时间点中EPC组可用荧光显微镜观察到EPCs在肝血窦中的归巢,免疫组化可观察肝血管区有EPCs抗原阳性表达;在3个时间点中,EPC组较对照组肝脏损伤
4、较轻,肝功能改善明显,血清中炎症因子表达较低,具有统计学意义(p5、USIONINJURYOFRATAFTERLIⅦRGRAFTABSTRACTObjective:Successfullyisolatethebonemarrow珈IonomJclearcdlsforcultureandinduceittoEPCs,immunocytochemicalstainand(10ublefluorescencestaintoidentify;SuccessfullyusePKH26GLtosta证ⅡleEPCsforstemcelltransplantation;Successful6、lyestablishtheratOLTmodeltostudytheliverIRIandobservewhethertheEPCsCanell衙iI】舾ⅡlerathepaticsinusoidaftertheEPCtransplanting;Observethea腧t咄ofliv盯andliverfunctionaftertransplantation.Methods:UsedpercolldensitygradientcentrifugationtoisoJatemebonemarrowmononuc7、learcellsfromthefemoralandtibialbonema玎ow’thenculturedinselectiveDMEMmediumto砌uce;0bs州Ⅱ1emorphologicalchangesandtheexpressionofthecellm刮慨bvimmunocytochemistry,stainedwithDil.ac-LDLandFITC-UEA-1,andmarkedbytheredfluorescentdyePKH26GLforthefollowingexperiflle8、m;48femaleSDratswererandomlydividedintotwogroupsandusedthemodifiedKamadacufftechniquetoestablishtheratOLTmodels;OnegroupwasOLT+EPCstransplantationgroupandtheothercontrolgrouponlywithOLT,thentheEPCgroup
5、USIONINJURYOFRATAFTERLIⅦRGRAFTABSTRACTObjective:Successfullyisolatethebonemarrow珈IonomJclearcdlsforcultureandinduceittoEPCs,immunocytochemicalstainand(10ublefluorescencestaintoidentify;SuccessfullyusePKH26GLtosta证ⅡleEPCsforstemcelltransplantation;Successful
6、lyestablishtheratOLTmodeltostudytheliverIRIandobservewhethertheEPCsCanell衙iI】舾ⅡlerathepaticsinusoidaftertheEPCtransplanting;Observethea腧t咄ofliv盯andliverfunctionaftertransplantation.Methods:UsedpercolldensitygradientcentrifugationtoisoJatemebonemarrowmononuc
7、learcellsfromthefemoralandtibialbonema玎ow’thenculturedinselectiveDMEMmediumto砌uce;0bs州Ⅱ1emorphologicalchangesandtheexpressionofthecellm刮慨bvimmunocytochemistry,stainedwithDil.ac-LDLandFITC-UEA-1,andmarkedbytheredfluorescentdyePKH26GLforthefollowingexperiflle
8、m;48femaleSDratswererandomlydividedintotwogroupsandusedthemodifiedKamadacufftechniquetoestablishtheratOLTmodels;OnegroupwasOLT+EPCstransplantationgroupandtheothercontrolgrouponlywithOLT,thentheEPCgroup
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