5-氮杂胞苷诱导培养骨髓间充质干细胞的膜离子通道的改变及其机理研究
发布时间：2019-05-11 08:34
【摘要】： 目的:了解5-氮杂胞苷诱导培养的骨髓间充质干细胞向心肌样细胞分化过程中的膜离子通道的改变并探讨其机理。 方法:按文献方法获得和培养Sprague-Dawley大鼠骨髓间充质(Mesenchymal stem cells, MSCs)干细胞,培养干细胞至第2周时部分骨髓MSCs以10μmol/L 5-氮杂胞苷(5-Azacytidine,5-Aza)诱导后再培养4周,以全细胞膜片钳技术检测诱导培养第1、2、3、4周和未诱导培养第6周MSCs的膜电流性质以及电流密度的大小。电流检测时各周随机封测30例MSCs,其中10例用含钠检测液检测以便于研究细胞表达的所有电流种类,20例以无钠检测液检测以便于研究其表达的K+电流密度的大小。同时免疫组织化学分别检测诱导与非诱导培养MSCs心肌特异性T型肌钙蛋白(Cardiac troponin T , cTnT)和缝隙连接蛋白(Gap junction protein 43,Cx43)的表达。 结果: ⑴电流检测结果: ①未诱导和诱导后各周MSCs均检测到延迟整流钾电流(Delayed rectifier K+ current, IkDR)、瞬时外向钾电流(Transient outward K+ current, Ito)和内向整流钾电流(Inward rectifier K+ current, Ikir)单独或复合存在,表达不均一,各周三种K+电流检出率相近。 ②未诱导培养6周和诱导后第1周MSCs没有钠电流(Sodium current, INa)及钙电流(Calcium current, ICa)表达,而在诱导第2周开始各周均有INa和ICa单独或复合表达。 ③诱导后第1周MSCs三种K+电流强度与未诱导组比较无明显差异,而从诱导第2周起开始增强(p0.05),至第4周时进一步增强(p0.01)。 ④诱导培养组MSCs三种K+电流均随培养时间延长逐渐增强(p0.05)。 ⑵免疫组化检测结果:未诱导培养MSCs的cTnT和Cx43无表达,而诱导培养4周两者结果均有表达。 结论: ⑴5-Aza诱导可促使部分MSCs分化为表达IkDR、Ito、Ikir、INa和ICa的心肌样细胞。 ⑵5-Aza诱导可促早期MSCs膜钾离子通道成熟而使分化过程中的IkDR、Ito和Ikir增强,促钠、钙离子通道生成或更趋成熟使INa和ICa可表达。
[Abstract]:Aim: to investigate the changes of membrane ion channels in cultured bone marrow mesenchymal stem cells (BMSCs) induced by 5-azacytidine (5-azacytidine) during differentiation into cardiomyocyte-like cells and to explore its mechanism. Methods: bone marrow mesenchymal (Mesenchymal stem cells, MSCs) stem cells from Sprague-Dawley rats were obtained and cultured according to the methods of literature. Some of the stem cells were induced by 10 渭 mol / L 5-azacytidine (5-Aza) for 4 weeks at the second week of culture, and some of the stem cells were induced by 5 渭 mol / L 5-azacytidine (5-Aza) for 4 weeks. Whole cell patch clamp technique was used to detect the current properties and current density of MSCs at the 1st, 2nd, 3rd, 4th week and 6th week of uninduced culture. In order to study all kinds of current expressed in cells, 10 cases were detected with sodium-containing detection solution, and 20 cases were detected with sodium-free detection solution to study the K current density of MSCs,. At the same time, the expression of myocardial specific T-type troponin (Cardiac troponin T, cTnT) and gap junction protein (Gap junction protein 43, Cx43) in induced and uninduced MSCs were detected by immunohistochemistry. Results: 1 the results of current detection: 1 the transient outward potassium current (Transient outward K current, of delayed rectifier potassium current (Delayed rectifier K current, IkDR), was detected in MSCs at all weeks after induction and induction. Ito) and inward rectifier potassium current (Inward rectifier K current, Ikir) were present alone or in combination, and the expression was uneven, and the detection rates of three K currents were similar in each week. (2) there was no expression of sodium current (Sodium current, INa) and calcium current (Calcium current, ICa) in MSCs after 6 weeks of uninduced culture and 1 week after induction, but INa and ICa were expressed alone or in combination at the beginning of the second week of induction. 3There was no significant difference in the three K current intensities of MSCs between the first week after induction and the uninduced group, but increased from the second week of induction (p0.05) to the 4th week (p0.01). (4) the three K currents of MSCs in the induced culture group increased gradually with the prolongation of the culture time (p0.05). 2 Immunohistochemistry: there was no expression of cTnT and Cx43 in uninduced cultured MSCs, but both of them were expressed in culture for 4 weeks. Conclusion: 15-Aza induction can induce some MSCs to differentiate into cardiomyocytes expressing IkDR,Ito,Ikir,INa and ICa. 25-Aza induction can promote the maturation of potassium channels in early MSCs membranes, enhance IkDR,Ito and Ikir during differentiation, and promote the formation or maturation of sodium and calcium channels to make INa and ICa express.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329.25
本文编号：2474347
[Abstract]:Aim: to investigate the changes of membrane ion channels in cultured bone marrow mesenchymal stem cells (BMSCs) induced by 5-azacytidine (5-azacytidine) during differentiation into cardiomyocyte-like cells and to explore its mechanism. Methods: bone marrow mesenchymal (Mesenchymal stem cells, MSCs) stem cells from Sprague-Dawley rats were obtained and cultured according to the methods of literature. Some of the stem cells were induced by 10 渭 mol / L 5-azacytidine (5-Aza) for 4 weeks at the second week of culture, and some of the stem cells were induced by 5 渭 mol / L 5-azacytidine (5-Aza) for 4 weeks. Whole cell patch clamp technique was used to detect the current properties and current density of MSCs at the 1st, 2nd, 3rd, 4th week and 6th week of uninduced culture. In order to study all kinds of current expressed in cells, 10 cases were detected with sodium-containing detection solution, and 20 cases were detected with sodium-free detection solution to study the K current density of MSCs,. At the same time, the expression of myocardial specific T-type troponin (Cardiac troponin T, cTnT) and gap junction protein (Gap junction protein 43, Cx43) in induced and uninduced MSCs were detected by immunohistochemistry. Results: 1 the results of current detection: 1 the transient outward potassium current (Transient outward K current, of delayed rectifier potassium current (Delayed rectifier K current, IkDR), was detected in MSCs at all weeks after induction and induction. Ito) and inward rectifier potassium current (Inward rectifier K current, Ikir) were present alone or in combination, and the expression was uneven, and the detection rates of three K currents were similar in each week. (2) there was no expression of sodium current (Sodium current, INa) and calcium current (Calcium current, ICa) in MSCs after 6 weeks of uninduced culture and 1 week after induction, but INa and ICa were expressed alone or in combination at the beginning of the second week of induction. 3There was no significant difference in the three K current intensities of MSCs between the first week after induction and the uninduced group, but increased from the second week of induction (p0.05) to the 4th week (p0.01). (4) the three K currents of MSCs in the induced culture group increased gradually with the prolongation of the culture time (p0.05). 2 Immunohistochemistry: there was no expression of cTnT and Cx43 in uninduced cultured MSCs, but both of them were expressed in culture for 4 weeks. Conclusion: 15-Aza induction can induce some MSCs to differentiate into cardiomyocytes expressing IkDR,Ito,Ikir,INa and ICa. 25-Aza induction can promote the maturation of potassium channels in early MSCs membranes, enhance IkDR,Ito and Ikir during differentiation, and promote the formation or maturation of sodium and calcium channels to make INa and ICa express.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329.25
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