本文關鍵詞： 心肌干細胞 分離培養(yǎng) 分選純化 生物學特性 誘導分化　出處：《南京中醫(yī)藥大學》2007年博士論文　論文類型：學位論文

【摘要】：目的：心肌梗死最終導致心臟擴大、心力衰竭和生存率下降，治療心肌梗死的常用方法為溶栓、經皮冠狀動脈成形術(PTCA)、冠狀動脈旁路移植術(CABG)和心臟移植術，前三種治療僅能改善心肌缺血，不能挽救壞死心肌，心臟移植雖可替換終末期心臟，但因供體缺乏及免疫排斥反應，臨床應用受到制約。目前，干細胞移植是治療缺血性心臟病的一種新方法，但隨著干細胞研究的深入，胚胎干細胞和成體干細胞移植治療缺血性心臟病尚存在許多爭議，因此人們正在尋找“最好”的干細胞去重建受損心肌和改善心臟功能。本研究擬建立體外分離、培養(yǎng)、純化小鼠成體心肌干細胞的方法，鑒定心肌干細胞表面標記，探討其生物學特性，研究心肌干細胞誘導分化及中藥干預作用。 方法：(1)應用機械剪切和酶消化法從C57小鼠心臟中分離出單細胞懸液，置于DMEM／ham’s F12、10％FBS、2％B27、10ng／mlEGF、20ng／mlbFGF、10ng／ml CT-1、10ng／mlLIF培養(yǎng)液中原代培養(yǎng)，傳代培養(yǎng)后體外擴增，相差顯微鏡下觀察細胞形態(tài)和生長。(2)應用免疫磁珠技術從傳代培養(yǎng)的細胞中分選、純化出sca-1~+、c-kit~+及sca-1~+／c-kit~+三種細胞，為增加分選純度，免疫磁珠法分選1～2次，，流式細胞儀檢測細胞分選純度。(3)免疫熒光顯微鏡、激光共聚焦顯微鏡、流式細胞儀檢測細胞表面sca-1、c-kit、sca-1-c-kit、CD34、lineage抗原，通過細胞表面抗原標記物鑒定干細胞；流式細胞儀測定sca-1~+、c-kit~+及sca-1~+／c-kit~+細胞周期；體外培養(yǎng)擴增sea-1~+、c-kit~+及sca-1~+／c-kit~+細胞，記數(shù)并描繪細胞生長曲線，研究細胞生物學特性。(4)對sca-1+、c-kit+及sca-1+／c-kit+細胞進行分組誘導，三七總甙組(終末濃度為100μl／ml)、5-氮胞苷(5-aza)組(終末濃度為10μmol／L)、二甲亞楓(DMSO)組(終末濃度為0.8％)分別誘導分化1h、24h、24h，并設立空白對照紐。免疫細胞化學法檢測細胞是否表達心肌特異性轉錄因子GATA-4、Nkx2.5，證實中藥對心肌干細胞的誘導分化作用。 結果：(1)相差顯微鏡下觀察從C57小鼠心臟組織中分離的細胞，可見散在體積小、圓形、折光性強的細胞，原代培養(yǎng)時生長緩慢，傳代培養(yǎng)后容易生長，并保持未分化狀態(tài)。(2)免疫磁珠法從傳代培養(yǎng)的細胞中分選、純化出sca-1~+、c-kit~+及sca-1~+c-kit~+三種細胞，流式細胞術檢測分選純度分別達87.4％、87.8％及90％以上，傳代培養(yǎng)時保持未分化狀態(tài)。(3)流式細胞儀、激光共聚焦顯微鏡、免疫熒光顯微鏡檢測心肌干細胞表面抗原標記物，分別表達sca-1~+CD34~(low)lin~-、c-kit~+c D34~-lin~-、sca-1~+c-kit~+ CD34~(low)lin~-；流式細胞儀檢測心肌干細胞細胞周期，sca-1~+，c-kit~+，sca-1~+／c-kit~+細胞G_0／G_1期分別占71.60％、42.45％、60.44％。(4)免疫細胞化學法結合激光共聚焦顯微鏡檢測心肌特異性轉錄因子表達。三七總甙體外誘導sca-1~+、c-kit~+及sca-1~+c-kit~+三種心肌千細胞1h，心肌特異性轉錄因子GATA-4、Nkx2.5顯著表達，5-氮胞苷體外誘導sca-1~+和二甲亞楓體外誘導sca-1~+／c-kit~+ 24h，心肌特異性轉錄因子GATA-4呈弱陽性，不加誘導劑的空白對照組不表達心肌特異性轉錄因子GATA-4、Nkx2.5。 結論：(1)發(fā)現(xiàn)C57小鼠心臟組織分離的細胞中存在圓形、折光性強的小細胞，適合在DMEM／F12、10％FBS、2％B27、10ng／mlEGF、20ng／mlbFGF、10ng／ml CT-1、10ng／mlLIF培養(yǎng)體系中體外生長。(2)免疫磁珠法能分選純化高純度的sca-1~+、c-kit~+及sca-1~+ c-kit+三種細胞。(3)分選純化的細胞表達干細胞表面標記物，證實sca-1~+、c-kit~+及sca-1~+ c-kit~+為心肌干細胞，心肌干細胞具有自我更新、慢周期性等干細胞生物學特征。(4)中藥三七總皂甙能夠快速誘導心肌干細胞分化，顯著表達心肌早期特異性轉錄因子，5-氮胞苷、二甲亞楓體外誘導心肌干細胞分化不顯著，可能與作用時間短有關。上述實驗結果證實小鼠心臟組織中存在成體心肌干細胞，具有向心肌細胞分化能力，中藥三七總甙能夠誘導心肌干細胞分化。
[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.

【學位授予單位】：南京中醫(yī)藥大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R329

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