小鼠成体心肌干细胞分离、培养、纯化、生物学特性及诱导分化的实验研究

发布时间：2018-01-23 07:12

本文关键词： 心肌干细胞分离培养分选纯化生物学特性诱导分化　出处：《南京中医药大学》2007年博士论文　论文类型：学位论文

【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~(low)lin~-、c-kit~+c D34~-lin~-、sca-1~+c-kit~+ CD34~(low)lin~-；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G_0／G_1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。
[Abstract]:Objective: myocardial infarction leads to cardiac enlargement, heart failure and decreased survival, commonly used methods for the treatment of myocardial infarction after thrombolysis, percutaneous transluminal coronary angioplasty (PTCA), coronary artery bypass grafting (CABG) and heart transplantation, the former three kinds of treatment can only improve the myocardial ischemia, myocardial necrosis can save the heart. Although transplantation can replace the end-stage heart, but due to lack of donor and immune rejection, clinical application is restricted. At present, stem cell transplantation is a new method for the treatment of ischemic heart disease, but with the development of stem cell research, embryonic stem cells and adult stem cell transplantation for the treatment of ischemic heart disease is still there is a lot of controversy. So people are looking for the "best" stem cells to reconstruct the damaged myocardium and improve cardiac function. This study intends to establish in vitro isolation, culture, purification of mice into cells of myocardial stem method, identification of myocardial The surface markers of stem cells were used to investigate their biological characteristics, to study the induction of differentiation of myocardial stem cells and the intervention of traditional Chinese medicine.
Methods: (1) the application of mechanical shear and enzyme digestion from C57 mouse heart isolated single cell suspension in DMEM / Ham s F12,10%FBS, 2%B27,10ng / mlEGF, 20ng / mlbFGF, 10NG / ml CT-1,10ng / mlLIF medium primary culture, cultured in vitro, to observe cell morphology and phase difference growth under the microscope. (2) application of immunomagnetic beads sorting from cultured cells, purified sca-1~+, three c-kit~+ and sca-1~+ / c-kit~+ cells, in order to increase the purity separation, immunomagnetic cell sorting and 1~2 times, the purity of cell sorting by flow cytometry. (3) immunofluorescence microscopy, confocal laser scanning microscope detection of cell surface, Sca-1, flow cytometry, c-kit, sca-1-c-kit, CD34, lineage antigen, through cell surface antigen markers of stem cells; flow cytometric detection of sca-1~+, c-kit~+ and sca-1~ + / c-kit~+ cell cycle; body In vitro amplification of sea-1~+, c-kit~+ and sca-1~+ / c-kit~+ cell count and cell growth curve was drawn, study on the biological characteristics of cells. (4) of sca-1+, c-kit+ and sca-1+ / c-kit+ cells were induced by 37 of the total group, glycoside group (the final concentration of 100 L / ml), 5- cell (5-aza) group by nitrogen (the final concentration of 10 mol / L), two methyl sulfoxide (DMSO) group (final concentration 0.8%) were induced differentiation of 1H, 24h, 24h, and blank control group. Immunohistochemical method was used to detect cell expression of cardiac specific transcription factor GATA-4, Nkx2.5, confirmed the Chinese medicine on inducing the differentiation of cardiac stem cells.
Results: (1) were isolated from the cells in the C57 mice heart under phase contrast microscope, scattered in small, round, strong refraction cell, primary culture of slow growth, the subculture is easy to grow and maintain the undifferentiated state. (2) the separation of immunomagnetic beads from cultured in cells, the purified sca-1~+, three kinds of c-kit~+ and sca-1~+c-kit~+ cells, flow cytometry sorting purity reached 87.4%, more than 87.8% and 90%, when subcultured and maintained undifferentiated state. (3) by flow cytometry, confocal microscopy, cell surface antigen markers to detect myocardial stem immunofluorescence microscopy, respectively. The expression of sca-1~+CD34~ (low) lin~-, c-kit~+c D34~-lin~-, sca-1~+c-kit~+ CD34~ (low) lin~-; stem cell cycle, flow cytometric detection of myocardial sca-1~+, c-kit~+, sca-1~+ / c-kit~+ G_0 / G_1 phase cells accounted for 71.60%, 42.45%, 60.44%. (4) immunocytochemistry combined with confocal laser scanning microscope to detect the expression of cardiac specific transcription factor. Sca-1~+ induced 37 of total saponins in vitro, c-kit~+ and sca-1~+c-kit~+ three kinds of myocardial stem cells 1H, cardiac specific transcription factor GATA-4, Nkx2.5 significantly induced sca-1~+ / c-kit~+ expression, 24h 5- cells in vitro induced by sca-1~+ and nitrogen by two a sulfoxide in vitro, cardiac specific transcription factor GATA-4 was weakly positive, and blank control group did not induce expression of cardiac specific transcription factor GATA-4, Nkx2.5.
Conclusion: (1) found a circular heart tissue isolated from C57 mice cells, with strong refraction small cell, suitable for DMEM / F12,10%FBS, 2%B27,10ng / mlEGF, 20ng / mlbFGF, 10NG / ml CT-1,10ng / mlLIF in vitro growth system. (2) to separation and purification of high purity sca-1~+ immunomagnetic beads. Three kinds of c-kit~+ and sca-1~+ in c-kit+ cells. (3) separation and purification of cells expressing stem cell surface markers, sca-1~+ c-kit~+ and sca-1~+ c-kit~+ confirmed that cardiac stem cells, myocardial stem cell self-renewal, slow cycling stem cell biological characteristics. (4) Chinese medicine 37 saponins can rapidly induce cell differentiation of myocardium dry, marked expression of cardiac specific transcription factor, 5- azacytidine, myocardial stem cell differentiation induced by methyl sulfoxide was two in vitro, and the possible role in a short time. The deposit confirmed these results in heart tissue of mice The adult cardiac stem cells have the ability to differentiate into the centripetal muscle cells. The 37 total glucosides of the traditional Chinese medicine can induce the differentiation of myocardial stem cells.

【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R329

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