【摘要】： 目的:了解5-氮雜胞苷誘導(dǎo)培養(yǎng)的骨髓間充質(zhì)干細(xì)胞向心肌樣細(xì)胞分化過程中的膜離子通道的改變并探討其機(jī)理。 方法:按文獻(xiàn)方法獲得和培養(yǎng)Sprague-Dawley大鼠骨髓間充質(zhì)(Mesenchymal stem cells, MSCs)干細(xì)胞,培養(yǎng)干細(xì)胞至第2周時(shí)部分骨髓MSCs以10μmol/L 5-氮雜胞苷(5-Azacytidine,5-Aza)誘導(dǎo)后再培養(yǎng)4周,以全細(xì)胞膜片鉗技術(shù)檢測誘導(dǎo)培養(yǎng)第1、2、3、4周和未誘導(dǎo)培養(yǎng)第6周MSCs的膜電流性質(zhì)以及電流密度的大小。電流檢測時(shí)各周隨機(jī)封測30例MSCs,其中10例用含鈉檢測液檢測以便于研究細(xì)胞表達(dá)的所有電流種類,20例以無鈉檢測液檢測以便于研究其表達(dá)的K+電流密度的大小。同時(shí)免疫組織化學(xué)分別檢測誘導(dǎo)與非誘導(dǎo)培養(yǎng)MSCs心肌特異性T型肌鈣蛋白(Cardiac troponin T , cTnT)和縫隙連接蛋白(Gap junction protein 43,Cx43)的表達(dá)。 結(jié)果: ⑴電流檢測結(jié)果: ①未誘導(dǎo)和誘導(dǎo)后各周MSCs均檢測到延遲整流鉀電流(Delayed rectifier K+ current, IkDR)、瞬時(shí)外向鉀電流(Transient outward K+ current, Ito)和內(nèi)向整流鉀電流(Inward rectifier K+ current, Ikir)單獨(dú)或復(fù)合存在,表達(dá)不均一,各周三種K+電流檢出率相近。 ②未誘導(dǎo)培養(yǎng)6周和誘導(dǎo)后第1周MSCs沒有鈉電流(Sodium current, INa)及鈣電流(Calcium current, ICa)表達(dá),而在誘導(dǎo)第2周開始各周均有INa和ICa單獨(dú)或復(fù)合表達(dá)。 ③誘導(dǎo)后第1周MSCs三種K+電流強(qiáng)度與未誘導(dǎo)組比較無明顯差異,而從誘導(dǎo)第2周起開始增強(qiáng)(p0.05),至第4周時(shí)進(jìn)一步增強(qiáng)(p0.01)。 ④誘導(dǎo)培養(yǎng)組MSCs三種K+電流均隨培養(yǎng)時(shí)間延長逐漸增強(qiáng)(p0.05)。 ⑵免疫組化檢測結(jié)果:未誘導(dǎo)培養(yǎng)MSCs的cTnT和Cx43無表達(dá),而誘導(dǎo)培養(yǎng)4周兩者結(jié)果均有表達(dá)。 結(jié)論: ⑴5-Aza誘導(dǎo)可促使部分MSCs分化為表達(dá)IkDR、Ito、Ikir、INa和ICa的心肌樣細(xì)胞。 ⑵5-Aza誘導(dǎo)可促早期MSCs膜鉀離子通道成熟而使分化過程中的IkDR、Ito和Ikir增強(qiáng),促鈉、鈣離子通道生成或更趨成熟使INa和ICa可表達(dá)。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329.25

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