【作者】 黄永坚；

【导师】 周播江；

【作者基本信息】 遵义医学院 ， 人体解剖学， 2009， 硕士

【摘要】 背景SP(side population)细胞是近年来人们关注的一群成体干细胞。1996年Goodell等首先发现于骨髓,并称之为SP细胞(中文称之为边缘群或侧群细胞)。现已发现,SP细胞在成体组织中广泛存在。来源于心脏的SP细胞(CSP)在体外与心肌细胞共培养条件下能向心肌细胞分化。直接回输体内实验也证实CSP细胞能够形成心肌样细胞,参与损伤心肌的修复。然而成体心脏组织中,CSP细胞含量较少,不能满足临床治疗的需要。因此本课题进一步探讨CSP细胞通过体外培养进行扩增的可能性及其扩增细胞参与心肌重建的能力。目的:1.观察CSP细胞能否体外培养扩增。2.扩增的CSP是否具有干细胞特性。3.探讨移植体外扩增CSP细胞在梗死心肌重建中的作用。方法:1.采用多次胰酶消化法从SD新生大鼠心脏制备心肌细胞悬液,通过流式细胞分选分离获取CSP细胞。用DMEM+10%FBS培养CSP细胞;通过RT-PCR检测Bcrpl、c-kit、MDR1、GATA4、β-MHC等标志,对扩增的CSP细胞进行鉴定;2.采用左冠状动脉前降支结扎法建立鼠AM1动物模型,并随机分为两组;细胞移植组即刻移植Dil标记的CSP细胞;对照组不移植CSP细胞。3.分别于建模成功后第3、7、14、28天,进行心脏超声检查及组织取材,组织冰冻切片通过HE染色和免疫荧光染色,观察移植的CSP细胞参与梗死心肌重建的情况。结果:1.每10只新生鼠的心脏组织,经分选可获得3—24×10~4个CSP细胞。2.CSP细胞体外培养,呈小三角形、多角形、圆形、半圆形或不规则形等贴壁生长,增殖活跃;CSP细胞反复传代培养后,仍具有很强的增殖能力。3.RT-PCR检测结果显示:原代CSP细胞表达Bcrp1和MDR1,低表达c-Kit,不表达GATA4和β-MHC。体外扩增的CSP细胞随着培养时间的延长和传代次数的增加,逐渐表达心肌标志心肌早期转录因子GATA4和成熟标志β-MHC,但同时持续高表达c-Kit和MDR1,而Bcrp1在逐渐减弱或消失。4.心脏超声检测:细胞移植组第28天的LVEF值(LVEF:71.1833±13.17553%)与对照组第28天的LVEF值(LVEF:46.2600±14.53801%)相比,统计学分析显示两者差异有统计学意义(P＜0.05)。5.组织学观察:冰冻切片HE染色光镜观察显示:各标本均出现左室前壁心肌坏死区,心梗区室壁变薄.证实心梗建模成功。免疫荧光共聚焦显微镜观察发现在CSP细胞移植后第7、14、28天,在各心梗区内及周围均发现有Dil标记的CSP细胞嵌入并表达心肌早期标志GATA4和成熟标志MHC。结论:1.CSP细胞能够在体外扩增培养并反复传代,且传代CSP细胞仍具有很强的增殖能力。2.扩增的CSP细胞随着传代培养的时间延长,逐渐向心肌细胞分化,但仍具有心肌干细胞特性。3.扩增的CSP细胞在体内能够向心肌细胞分化,参与梗死心肌的再生修复。4.扩增的CSP细胞心梗移植后能有效提高左室射血分数,改善心功能。更多还原

【Abstract】 Background:In recent years,the people has taked most attentions to the side population cells which are a group of cells from adult stem cells.It was the first time to be found in bone marrow by Goodell’s and was called as side population cells in 1996.Now,Side population cells have been shown to exist in various types of adult tissue.Cardiac side population cells(CSP_s) isolated from heart can differentiate into cardiac myocytes by co-cultured with the myocardial cells in vitro.It’s also to be identificated that CSP_s can differentiate into cardiac-like cells after being transplantated in vivo and involved in repair of myocardial injury.However,it is unable to meet the needs of clinical treatment because the quantitys of CSP_s is fewer in adult heart tissue.Therefore,it is necessary to further explored that CSP_s have the potential of amplification after being cultured in vitro,and the ability in reparation of myocardial injury.Objective:1.To observe whether the CSP_s have the potential of amplification after being cultured in vitro.2.To investigate whether the amplified CSP_s have the property of stem cell.3.To investigate the treatment effects of the transplantation of CSP_s in the early myocardial infarction remodeling.Methods:1.The suspension of myocardial cell was prepared by digested on times with trypsin from postnatal rat hearts,and then CSP_s were sorted out from the suspension by fluorescence-activated cell sorting based on the ability to efflux Hoechst 33342 dye.And CSP_s were cultured in vitro by using DMEM +10%FBS. 2.The expression of Bcrp1、c-kit、MDR1、GATA4 andβ-MHC in cultured CSP_s were detected by RT-PCR.3.Adult rat myocardial infarct model was set up by ligation of left Anterior descending coronary artery after breast- opening,and CSP_s labeled by using CM-DiI were transplanted into the periphery and central of infracting myocardium in experimental rat respectively and were not transplanted in control rat.4.To evaluated the cardiac function Echocardiography exams were performed respectively at 3、7、14and 28day after surgery operation.5.HE stain of the cardiac frozen sections were performed to evaluate the myocardial infaction.To survey differentiation of labeled CSP_s into myocardial cell by confocal microscopy,immunofluorescence stain of GATA4 and MHC were performed in the cardiac frozen sections.Result:1.3-24×10~4 CSPs were sorted out from 10 neonatal rat heart tissue by fluorescence-activated cell sorting.2.cultured CSPs adherently grew as a small triangle,polygon,round, semi-circular or irregular-shaped,and showed a strong proliferative capacity.3.RT-PCR test results show that:the uncultured CSP_s expressed Bcrp1 and MDR1 and did not express GATA4 and bata-MHC.C-kit was poorly or not expressed,cultured CSPs gradually express c-kit,GATA4 and bata-MHC,and meanwhile remain to express Bcrp1 and MDR1.4.echocardiography detection showed that CSPs transplantation can elevate the left ventricular ejection fraction and improve cardiac function.Compared with the control group(LVEF:46.2600±14.53801%),LVEF of the cell transplantation group(LVEF:71.1833±13.17553%) increased on 28th day(P＜0.05).5.Myocardial necrosis of left ventricular anterior wall and thinning myocardial wall confirmed the success of myocardial infarction model.Labeled cells expressing GATA4 and MHC were found in the region of myocardial infarction and its periphery on the 7th,14th and 28th day after implanting CSPs labeled CM-DiI.Conclusion:1.CSPs can proliferate when cultured in vitro and proliferated CSPs remain to possess characteristics of stem cell2.During culture,CSPs gradually differentiated into myocardial cells,but still remain property of stem cells3.Proliferated CSPs in vitro can differentiate into cardiac-like cells after being transplantated into heart,participating in the regeneration of infracted myocardium.4.Proliferated CSPs can elevate the left ventricular ejection fraction and improve cardiac function after being transplantated into heart更多还原