【作者】 张莹；

【导师】 林海龙；

【作者基本信息】 大连医科大学 ， 心内科学， 2013， 硕士

【摘要】 研究背景：传统观点认为,成年哺乳动物的心脏是一个“静止”器官,不具备完全修复损伤再生的能力。研究表明,成熟机体的心脏组织中,存在具有高度自我更新能力和特异性心肌分化潜能的成体心肌干细胞(Cardiac stem cells CSCs),其能够分化为心肌细胞、内皮细胞及平滑肌细胞等心脏结构细胞。成体心肌干细胞是目前被认为最有希望以完全心肌再生应用于缺血性心脏病及其他终末期心脏病替代治疗的干细胞类型,具有很大的临床应用前景。因而掌握有效的分离培养CSCs方法,无疑是非常重要的。研究目的：建立稳定和有效的CSCs分离和培养方法以及对体外培养的CSCs进行扩增和鉴定其表面标记。研究方法：1组织取材：成人心脏组织标本来自行心脏外科手术患者1例(48岁)和冠心病患者行心肌活检1例(65岁)。心脏手术中取右心耳部心肌组织22g。通过心肌活检取右室间隔心肌组织21.9g。新生鼠(48小时)置于75%酒精内消毒3秒,无菌操作下取出整个心脏称重10mg。成体小鼠心脏组织来自昆明小鼠(6-7周),小鼠断颈处死后无菌操作下取出心脏,称重15mg。2细胞分离和原代细胞培养：取出的心脏组织均立即置于预先加入D-PBS液的6cm培养皿中,外膜组织全部剥离,将剥离的心肌组织剪碎成1-2mmm3小块后,放入0.2%Ⅱ型胶原酶消化,37℃,20分钟,反复消化2次。离心后弃上清,经70及40um滤器过滤,离心后的细胞放入培养皿,加入培养液DMEM/F12含10%FBS,青霉素,链霉素,bFGF1:10.3细胞传代：8-10天左右,细胞增殖至约占培养皿80%可进行传代。吸取并弃去培养皿中的培养液,D-PBS洗2次,TrypLETM完全消化约5 min,加入等量DMEM/F12终止消化,1500 r/min,离心5 min,1mlDMEM/F12重悬,计数,适量细胞数传代。4细胞鉴定：细胞传代时,取5×105细胞,PBS冲洗两次,标记CD29、CD90、CD105、CD45、Stro-1和HLA-DR抗体后,行流式细胞检测。取1×106细胞,采用TaKaRa RNA PCR Kit,提取RNA后,行逆转录反应及PCR扩增。检测干细胞因子Nanog、Oct-4、Sox-2、Rex-1的表达。研究结果：通过本研究方法成功分离出成体心肌干细胞,细胞放入培养皿后,大约1-2天后可贴壁生长,生长较好者一般7-9天可以长满6cm培养皿,可以进行传代。细胞既可见单个细胞生长,亦可见成团生长。经传代,可在体外有效增值达到细胞移植所需的数量。经过RT-PCR,流式细胞仪鉴定,表达胚胎干细胞表面抗原Nanog、Oct-4、Sox-2、Rex-1；间充质干细胞表面抗原CD29、CD90、CD105；不表达造血干细胞表面抗原CD45和人免疫抗原HLA-DR。结论：本研究建立了一种稳定有效的体外培养心肌干细胞的方法,尤其是可通过心肌活检这一微创技术获取少量心肌组织,可以有效的在体外培养扩增,大约1个月后可增值达到细胞移植所需要的细胞数量。本研究还将进一步探讨CSCs的体外分化和心梗模型体内细胞追踪分化,为以后心肌干细胞移植的临床应用奠定重要基础。更多还原

【Abstract】 Background:The heart has been traditionally regarded as terminally differentiated organ that adapt to increased work and compensate for disease exclusively through hypertrophy.Recent studies showed that intrinsic cardiac stem cells exist in the mammalian heart which have potent proliferation and differentiation to cardiac cells,endothelial cells and smooth muscle cells.Recent evidence suggested that cardiac stem cells have been considered to have most potential clinical application to treat patients with heart failure.Aims:This present study describes the isolation and preliminary characterization of cells from the adult human and murine heart.Methods:1.tissue samples:human tissue was derived from right atrial belonging to patients undergoing heart surgery and right septum by biopsy.2.cell isolation and primary cell culture:isolated myocardial tissue was cut into 1 to 2 mm3 pieces, washed with Ca2+-Mg2+-free phosphate-buffered solution and digested two times for 20 minutes at 37℃ with 0.2% or 0.4% collagenase Ⅱ.The obtained cells were cultured with medium supplemented with 10%FBS.3 secondary cell culture:CSCs were passaged every 8-10 days.4 idenfication:hCSCs were labeled with the following antibodies: CD29、CD90、CD105、CD45、Stro-land HLA-DR. Cell events were collected by FACS Calibur flow cytometer and data were analyzed by Cell Quest.Total RNA was exacted from cells using TaKaRa RNA PCR Kit and RT-PCR was performed with a SuperScript Ⅲ First Strand Synthesis Systerm.Results:The cells obstained became loosely adherent after seeded in dish about 1-2 days later,and became confluent about 7-9 days later. They can expand in vitro to reach enough cell number for cell transplantation.FACS analysis revealed that hCSCs did not express the hematopoietic progenitor cell -specific surface antigens:CD45 and HLA-DR, while they were positive for typical mesenchymal stem cells surface antigens: CD29、CD90、CD105 .RT-PCR showed that hCSCs expressed Rexl, Nanog, Sox2 and Oct4, suggesting that hCSCs express the embryonic stem cell markers and contain the mesenchymal cell-like population.Conculsions:We developed a stable and efficient method to isolate and expand CSCs successfully.More importantly, our data confirmed that it is possible to isolate cells from very small fragments of human myocardium and expand these cells in vitro in a month to reach numbers that would be appropriate for in vivo transplantation in patients.更多还原