成年小鼠心脏干细胞的分离、培养及其离子通道特性

发布时间：2018-10-08 15:38

【摘要】： 长期以来人们认为成年哺乳动物心脏处于后有丝分裂期,即心脏是终末分化器官,不具备自我更新的能力。心脏内有丝分裂的发现,使人们认识到心脏内有可能存在着干细胞。新近的一些实验结果证实了心脏干细胞的存在,这些发现立即受到生物医学界的高度关注。现在已经从成年人和其他哺乳动物心脏中分离得到了心脏干细胞。通过细胞移植或是原位激活,心脏干细胞都有能力再生肌细胞,并能改善心脏功能。然而各家报道的心脏干细胞分离培养的方法不尽相同。关于心脏干细胞在分化前的离子通道特性至今未见报道。本实验致力于建立一种有效的、重复性好的成年小鼠心脏干细胞的分离、培养和鉴定方法,并在此基础上研究心脏干细胞在分化前存在的离子通道的类型及其特性。第一部分成年小鼠心脏干细胞的分离、培养和鉴定成年小鼠心脏干细胞的分离采用Langendorff法灌流小鼠心脏,用胶原酶酶解法制备单个心肌细胞,差速离心法将较大的细胞(如心室肌细胞)和较小的细胞分离开。取用较小细胞,用c-kit抗体孵育后,然后用免疫磁珠分离法分离心脏干细胞。结果显示,分离出的细胞数约104个,细胞形态为圆形,镜下观察较亮,每个细胞上带有一个或者多个磁珠,细胞直径较小,约在4～10μm之间。成年小鼠心脏干细胞的培养分离的细胞先在有血清培养基条件下培养48小时以除去贴壁杂细胞,然后将悬浮细胞转移到另一新培养皿中,在改良神经干细胞培养基条件下继续培养。结果显示,在培养两天后,有少量细胞开始贴壁,培养五天后,较多细胞开始贴壁生长,七到十天后,形成小克隆,并可持续生长。成年小鼠心脏干细胞的鉴定新鲜分离的原代细胞和传代培养后的细胞采用免疫荧光法鉴定其表型,在激光扫描共聚焦显微镜下观察荧光。结果显示,原代细胞和传代细胞表面都带有绿色荧光,证明其表型为c-kit,心脏干细胞传代培养后其表型没有发生变化。此结果说明,传代后细胞保持了干细胞特性,没有发生分化。第二部分成年小鼠心脏干细胞离子通道特性培养的小鼠CSCs在贴壁生长形成较大克隆之后,用0.25%胰蛋白酶消化,当大部分细胞回缩变圆之后,用血清终止消化。细胞一部分用于传代,一部分保存用于膜片钳实验。应用全细胞膜片钳记录方法,在电压钳模式下记录离子通道电流。结果显示,未分化的成年小鼠心脏干细胞上存在离子电流。我们记录到两种类型的电流,一种为外向电流,激活速度较慢,另一种为内向电流。大部分的心脏干细胞上所记录到的电流为外向电流,激活速度缓慢,呈电压依赖性,具有非失活特性。此外向电流对TEA敏感,能被TEA可逆性的抑制,该电流为延迟整流钾电流(IKDR)。结论:(1)采用胶原酶酶解,免疫磁珠分离法分离,能够分离出较纯的,数目较多的心脏干细胞;(2)分离出的心脏干细胞具有再生和克隆能力;(3)新鲜分离的心脏干细胞具有c-kit表型,传代培养后c-kit表型依然存在;(4)未分化的成年小鼠心脏干细胞上存在延迟整流钾电流(IKDR)。
[Abstract]:It has long been seen that the heart of the adult mammalian heart is in the post-mitotic phase, i.e. the heart is the terminal-differentiated organ and does not have the ability to self-update. The discovery of mitosis in the heart makes it possible to recognize that there may be stem cells in the heart. Recent experimental results confirm the presence of cardiac stem cells, which are now highly concerned by the biomedical community. The heart has now been isolated from the heart of adults and other mammals Stem cells. Cell transplantation or in situ activation, cardiac stem cells have the ability to regenerate muscle cells, and can improve heart dirty. However, the methods of separating and culturing cardiac stem cells in various reports do not To do the same. Ion channel characteristics prior to differentiation of heart stem cells have not yet been This experiment is devoted to the establishment of an effective and reproducible method for the isolation, culture and identification of cardiac stem cells in adult mice, and on the basis of which the types of ion channels present before differentiation of cardiac stem cells are studied. characterized in that the first part is divided into annual mouse heart stem cells Isolation, culture and identification of cardiac stem cells in adult mice were isolated by Langendorff method, and the hearts of mice were perfused with collagenase. The preparation of a single myocardial cell by the enzymatic method, the differential centrifugation method takes larger cells (e.g., ventricular myocytes). Cells are separated from smaller cells. Smaller cells are taken, incubated with c-kit antibodies, and then immunomagnetic The results show that the number of isolated cells is about 104, the morphology of the cells is round, the observation is bright under the mirror, one or more magnetic beads are provided on each cell, and the diameter of the cells is small. The cells cultured and isolated from adult mouse heart stem cells are first cultured in serum medium for 48 hours to remove adherent heteroatoms, and then suspended cells are transferred to another culture dish, The results showed that after two days of incubation, a small number of cells began to adhere, after five days of incubation, more cells began to adhere to the wall and seven to ten. After days, small clones were formed and allowed to grow continuously. The identification of fresh isolated primary and post-infected cells in adult mouse heart stem cells was identified by immunofluorescence Phenotyping, fluorescence was observed under a laser scanning confocal microscope. The results showed that both primary and passaged cells had green fluorescence to demonstrate that their phenotype was c-kit, There was no change in the phenotype of cardiac stem cells. Post-generation cells retain stem cell characteristics without differentiation In the second part of adult mouse cardiac stem cell ion channel characteristics, CSCs were cultured on adherent growth with 0. 25% after the formation of larger clones. Trypsin digestion, after most of the cells shrink back to a circle, use serum terminate the digestion. A portion of the cell is used for passage, and a portion of the cells are stored for an oral test. recording the ion channel electricity in the forceps mode by using the whole cell method of recording and recording. Flow. The results showed that there was an ionic current in the undifferentiated adult mouse heart stem cells. We recorded two types Current, an outward current, slower activation, and the other is the inward current. The current recorded on most of the heart stem cells For external current, the activation speed is slow, voltage dependent, and has non-deactivation characteristic. This external current is sensitive to TEA and can be Conclusion: (1) collagenase enzymatic hydrolysis and immunomagnetic bead separation can be used to separate and separate more pure and larger number of heart stem cells; (2) isolated cardiac stem cells. There was regeneration and cloning ability; (3) fresh isolated cardiac stem cells had c-kit phenotype, and c-kit phenotype was still present after decapsulation; (
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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