【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理。 方法:按文獻方法獲得和培養(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。電流檢測時各周隨機封測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的K+電流密度的大小。同時免疫組織化學(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。 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)單獨或復(fù)合存在,表達不均一,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,而在誘導(dǎo)第2周開始各周均有INa和ICa單獨或復(fù)合表達。 ③誘導(dǎo)后第1周MSCs三種K+電流強度與未誘導(dǎo)組比較無明顯差異,而從誘導(dǎo)第2周起開始增強(p0.05),至第4周時進一步增強(p0.01)。 ④誘導(dǎo)培養(yǎng)組MSCs三種K+電流均隨培養(yǎng)時間延長逐漸增強(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,而誘導(dǎo)培養(yǎng)4周兩者結(jié)果均有表達。 結(jié)論: ⑴5-Aza誘導(dǎo)可促使部分MSCs分化為表達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,促鈉、鈣離子通道生成或更趨成熟使INa和ICa可表達。
[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

【參考文獻】

相關(guān)期刊論文 前10條

1 呂鐵偉,田杰,鄧兵,朱靜,陳沅;NKx2.5、GATA-4基因表達對骨髓間充質(zhì)干細(xì)胞分化為心肌細(xì)胞的影響[J];基礎(chǔ)醫(yī)學(xué)與臨床;2005年01期

2 王金菊;田杰;呂鐵偉;張小飛;朱靜;李揚;陳沅;;大鼠MSCs誘導(dǎo)分化為心肌樣細(xì)胞時離子通道基因的表達[J];基礎(chǔ)醫(yī)學(xué)與臨床;2007年01期

3 沈興;余更生;田杰;白永紅;朱靜;;擴張型心肌病兔電重構(gòu)機制的初步探討[J];臨床心血管病雜志;2007年07期

4 李瑩,葛均波,史劍慧,于曉,虞勇,章毅,馬軍,張少衡,趙嫣,沈建穎,宿燕剛,王克強,周專;骨髓源性心肌細(xì)胞瞬間外向鉀電流狀態(tài)的研究[J];中國臨床醫(yī)學(xué);2004年02期

5 沈興;田杰;余更生;白永虹;朱靜;劉官信;陳沅;;骨髓間充質(zhì)干細(xì)胞移植對擴張型心肌病兔心功能及結(jié)構(gòu)重構(gòu)的影響[J];實用兒科臨床雜志;2008年01期

6 Bartosz Balana;Cecilia Nicolett;Ihor Zahanich;Eva M Graf;Torsten Christ;Sabine Boxberger;;5-Azacytidine induces changes in electrophysiological properties of human mesenchymal stem cells[J];Cell Research;2006年12期

7 吳鐵軍;湯服民;蔡振杰;俞世強;周和平;;5-氮胞苷體外誘導(dǎo)骨髓間質(zhì)干細(xì)胞分化為心肌細(xì)胞的實驗研究[J];中國心血管病研究雜志;2006年01期

8 農(nóng)耀明;宋治遠;尹戴佳佳;陳鵬慧;;大鼠骨髓間質(zhì)干細(xì)胞誘導(dǎo)分化為心肌樣細(xì)胞膜電流的研究[J];中國病理生理雜志;2007年09期

9 農(nóng)耀明;宋治遠;尹戴佳佳;陳鵬慧;;大鼠骨髓間充質(zhì)干細(xì)胞培養(yǎng)一周后的細(xì)胞膜電流[J];中國心臟起搏與心電生理雜志;2006年04期

10 張文,田杰,江德勤,張蕾,朱靜,陳沅;骨髓間充質(zhì)干細(xì)胞體外分化為心肌樣細(xì)胞相關(guān)調(diào)控基因的時序表達[J];中華心血管病雜志;2004年11期

，

本文編號：2474347