【作者】 娄彬；

【导师】 施广飞；

【作者基本信息】 南京中医药大学 ， 中西医结合临床， 2007， 博士

【摘要】 目的：心肌梗死最终导致心脏扩大、心力衰竭和生存率下降，治疗心肌梗死的常用方法为溶栓、经皮冠状动脉成形术（PTCA）、冠状动脉旁路移植术（CABG）和心脏移植术，前三种治疗仅能改善心肌缺血，不能挽救坏死心肌，心脏移植虽可替换终末期心脏，但因供体缺乏及免疫排斥反应，临床应用受到制约。目前，干细胞移植是治疗缺血性心脏病的一种新方法，但随着干细胞研究的深入，胚胎干细胞和成体干细胞移植治疗缺血性心脏病尚存在许多争议，因此人们正在寻找“最好”的干细胞去重建受损心肌和改善心脏功能。本研究拟建立体外分离、培养、纯化小鼠成体心肌干细胞的方法，鉴定心肌干细胞表面标记，探讨其生物学特性，研究心肌干细胞诱导分化及中药干预作用。方法：（1）应用机械剪切和酶消化法从C57小鼠心脏中分离出单细胞悬液，置于DMEM／ham’s F12、10％FBS、2％B27、10ng／mlEGF、20ng／mlbFGF、10ng／ml CT-1、10ng／mlLIF培养液中原代培养，传代培养后体外扩增，相差显微镜下观察细胞形态和生长。（2）应用免疫磁珠技术从传代培养的细胞中分选、纯化出sca-1⁺、c-kit⁺及sca-1⁺／c-kit⁺三种细胞，为增加分选纯度，免疫磁珠法分选1～2次，流式细胞仪检测细胞分选纯度。（3）免疫荧光显微镜、激光共聚焦显微镜、流式细胞仪检测细胞表面sca-1、c-kit、sca-1-c-kit、CD34、lineage抗原，通过细胞表面抗原标记物鉴定干细胞；流式细胞仪测定sca-1⁺、c-kit⁺及sca-1⁺／c-kit⁺细胞周期；体外培养扩增sea-1⁺、c-kit⁺及sca-1⁺／c-kit⁺细胞，记数并描绘细胞生长曲线，研究细胞生物学特性。（4）对sca-1+、c-kit+及sca-1+／c-kit+细胞进行分组诱导，三七总甙组（终末浓度为100μl／ml）、5-氮胞苷（5-aza）组（终末浓度为10μmol／L）、二甲亚枫（DMSO）组（终末浓度为0.8％）分别诱导分化1h、24h、24h，并设立空白对照纽。免疫细胞化学法检测细胞是否表达心肌特异性转录因子GATA-4、Nkx2.5，证实中药对心肌干细胞的诱导分化作用。结果：（1）相差显微镜下观察从C57小鼠心脏组织中分离的细胞，可见散在体积小、圆形、折光性强的细胞，原代培养时生长缓慢，传代培养后容易生长，并保持未分化状态。（2）免疫磁珠法从传代培养的细胞中分选、纯化出sca-1⁺、c-kit⁺及sca-1⁺c-kit⁺三种细胞，流式细胞术检测分选纯度分别达87.4％、87.8％及90％以上，传代培养时保持未分化状态。（3）流式细胞仪、激光共聚焦显微镜、免疫荧光显微镜检测心肌干细胞表面抗原标记物，分别表达sca-1⁺CD34^lowlin^-、c-kit⁺c D34^-lin^-、sca-1⁺c-kit⁺ CD34^lowlin^-；流式细胞仪检测心肌干细胞细胞周期，sca-1⁺，c-kit⁺，sca-1⁺／c-kit⁺细胞G₀／G₁期分别占71.60％、42.45％、60.44％。（4）免疫细胞化学法结合激光共聚焦显微镜检测心肌特异性转录因子表达。三七总甙体外诱导sca-1⁺、c-kit⁺及sca-1⁺c-kit⁺三种心肌千细胞1h，心肌特异性转录因子GATA-4、Nkx2.5显著表达，5-氮胞苷体外诱导sca-1⁺和二甲亚枫体外诱导sca-1⁺／c-kit⁺ 24h，心肌特异性转录因子GATA-4呈弱阳性，不加诱导剂的空白对照组不表达心肌特异性转录因子GATA-4、Nkx2.5。结论：（1）发现C57小鼠心脏组织分离的细胞中存在圆形、折光性强的小细胞，适合在DMEM／F12、10％FBS、2％B27、10ng／mlEGF、20ng／mlbFGF、10ng／ml CT-1、10ng／mlLIF培养体系中体外生长。（2）免疫磁珠法能分选纯化高纯度的sca-1⁺、c-kit⁺及sca-1⁺ c-kit+三种细胞。（3）分选纯化的细胞表达干细胞表面标记物，证实sca-1⁺、c-kit⁺及sca-1⁺ c-kit⁺为心肌干细胞，心肌干细胞具有自我更新、慢周期性等干细胞生物学特征。（4）中药三七总皂甙能够快速诱导心肌干细胞分化，显著表达心肌早期特异性转录因子，5-氮胞苷、二甲亚枫体外诱导心肌干细胞分化不显著，可能与作用时间短有关。上述实验结果证实小鼠心脏组织中存在成体心肌干细胞，具有向心肌细胞分化能力，中药三七总甙能够诱导心肌干细胞分化。更多还原

【Abstract】 Objective Myocardial infarction induces cardiac dilatation, heart failure and decreasedsurvival rate eventually. The therapeutic methods of myocardial infarction includethrombolysis, percutaneous transluminal coronary angioplasty（PTCA）,coronary arterybypass graft （CABG）,and heart transplantation. Although PTCA and CABG can improveischemia, they can not survive necrotic myocardium. Heart transplanttation can replaceterminal stage failing heart, but insufficient donators and immune reaction have limitedclinical application. Stem cells transplantation therapy may be a new method to treatischemic heart disease. With progress of stem cell research, many questions remainunanswered in this new field. Scientists have been looking for the best stem cell to repairdamaged heart and improve cardiac function. The aim of investigation was to establish amethod for isolating, culturing, purifying adult cardiac stem cells in vitro from mouse,identify the cell surface markers, explore their biological characteristics and theirdifferentiated probability induced by traditional Chinese medicine.Metheds （1）Hearts of C57 mouse were dissociated into single cells suspension bymechanical and enzymatic method, and then were cultured in Dulbecco’s modifiedEagle’s medium and Ham’s F12 （DMEM/ham’s F12） supplemented with 10%FBS,2%B27, 10ng/mlEGF, 20ng/mlbFGF, 10ng/ml CT-1,10ng/mlLIF for expanding in vitro.Proliferation of the cells were observed under the phase contrast microscope.（2）Enrichment of sca-1⁺, c-kit⁺ and sca-1⁺/c-kit⁺ cells was achieved by sorting using themagnetic activated cell sorting （MACS） system. To increase the purity of the cells,magnetic sorting was performed one or twice. The percentage of sca-1⁺,c-kit⁺ andsca-1⁺/c-kit⁺ cells was analyzed by the flow cytometer （FCM）. （3）The cell surface antigens of sca-1, c-kit, sca-1/c-kit, CD34 and lineage mixture （CD3-e, CD-11b, CD45R,Ly-6C/G; TER-119） were observed under laser confocal microscope and analyzed byflow cytometer to identify cardiac stem cells. In order to explore the cardiac stem cellbiological characteristics, we measured cardiac stem cell cycle by the flow cytometer（FCM）, counted cardiac stem cells under phase contrast microscope after culturing andexpanding in vitro, respectively. The cell proliferation curves have been depictedsuccessfully. （4） Sca-1⁺,c-kit⁺ and sca-1⁺/c-kit⁺ cells were indused by Panax notoginoside（100μL/ml）, 5-azacitidine（5-aza, 10μmol/L）, dimethyl sulfoxide minimum （DMSO, 0.8%）for 1 hour, 24 hours and 24 hours. Meanwhile, a blank control group was set to comparethe effect. Immunocytochemical analysis showed the results that sca-1⁺,c-kit⁺ andsca-1⁺/c-kit⁺ cells could express the genes of cardiac transcription factors includingGATA-4 and Nkx2.5.Results （1）Round, bright and small ceils from mouse could be observed under thephase contrast microscope,which proliferated slowly in primary culture, but theyproliferated easily and kept undifferentiation in subculture. （2） Three kinds of cellslabelled with sca-1, c-kit, sca-1/c-kit were sorted by the magnetic activated cell sorting（MACS） system. Flow cytometric analysis revealed that sca-1,c-kit and sca-1/c-kitpositive cells were enriched to 87.4%, 87.8% and 90%, respectively. These cells couldkeep undifferentiation in subculture. （3） The cell surface antigens tested by the flowcytometer （FCM）, laser confocal microscopy and immunofluorescence microscope weresca-1⁺CD34^lowlin^-, c-kit⁺CD34^-lin^-, sca-1⁺c-kit⁺CD34^lowlin^-, respectively. The percentageof G₀/G₁ in sca-1⁺, c-kit⁺ and sca-1⁺/c-kit⁺ analyzed by the flow cytometer （FCM） was71.60%, 42.45%, and 60.44%, respectively. After treatment with Panax notoginoside for1 hour, 5-azacitidine for 24 hours and DMSO for 24 hours, the expression of GATA-4and Nkx2.5 in sca-1⁺,c-kit⁺ and sca-1⁺-c-kit⁺ cells were notably positive in Panaxnotoginoside group,while in 5-aza group and in DMSO group only GATA-4 wereexpressed slightly positive, the expressions of GATA-4 and Nkx2.5 were not detected inblank control group.Conclusion （1）We found round, bright and small cells from adult C57 mouse heart.These cells can proliferate in Dulbecco’s modified Eagle’s medium and Ham’s F12（DMEM/ham’s F 12） supplemented with 10%FBS, 2%B27, 10ng/mlEGF, 20ng/mlbFGF,10ng/ml CT-1,10ng/mlLIF （2） The sca-1⁺,c-kit⁺ and sca-1⁺/c-kit⁺ positive cells can be sorted by magnetic activated cell sorting （MACS） system. （3） Sortting cells can expressstem cell markers and have biology characteristics of stem cell. （4） When treated withPanax notoginoside, cardiac stem cells highly expressed genes of cardiac transcriptionfactors in vitro for a short time. When treated with 5-aza and DMSO, cardiac stem cellsslightly expressed genes of cardiac transcription factors. These results suggest that adultcardiac stem cells exist in the mouse heart which might have the potential ofdifferenciating into cardiomyocytes in vitro. Panax notoginoside can inducedifferentiation of cardiac stem cells into the cells with cardiac characteristics.更多还原