骨髓间充质干细胞体外诱导培养后的电生理特性

背景:骨髓间充质干细胞在宿主心肌中能够生长,并可在心肌环境诱导下分化,改善心脏功能,但目前骨髓干细胞分化过程中生物学特性尚不甚清楚.目的:观察经5-氮胞苷诱导培养后,骨髓间充质干细胞体外缝隙连接蛋白43的表达及电生理特性变化.设计、时间及地点:细胞学体外对照观察,于2007-07/2009-02在河北省人民医院心脏中心完成.材料;2月龄雄性冀中自猪24只,购自河北医科大学实验动物中心.方法:无菌条件下抽取猪双侧股骨骨髓,Percoll密度梯度法体外分离培养骨髓间充质干细胞,传至第2代后,加入10μmol/L的5-氮胞苷,24 h后更换为不含诱导剂的DMEM培养基继续培养,分别于诱导后1,2,3周进行指标检测;并设立未经5-氮胞苷诱导的对照组.实验猪抽取骨髓后麻醉,取心室肌,组织块酶消化法分离培养正常心室肌细胞.主要观察指标:ABC法免疫细胞化学染色检测缝隙连接蛋白43的表达,以膜片钳全细胞记录模式测定Ito电流密度及动作电位.结果:5-氮胞苷诱导后1,2周,部分骨髓间充质干细胞表达缝隙连接蛋白43,呈核周散在分布的棕黄色颗粒;诱导后3 周缝隙连接蛋白43阳性率为95%,细胞密度大的区域出现类似正常心肌的闰盘结构.在+80 mV时与正常心室肌细胞比较,未诱导对照组及诱导1,2周的骨髓间充质干细胞k电流密度均明显降低(P<0.05),诱导3周的骨髓间充质干细胞Ito电流密度升高至正常心室肌细胞水平(P>0.05).5-氮胞苷诱导1,2周的骨髓间充质干细胞未能检测到动作电位,诱导3周后可检测到动作电位,与正常心室肌细胞相似.结论:骨髓间充质干细胞经5-氮胞苷体外诱导3周后,与正常心肌具有相似的缝隙连接蛋白43表达能力及电生理特性.

Electrophysiological characteristics of bone marrow stem cells following in vitro induction

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) can grow in host myocardium, differentiate under myocardial condition, improve cardiac function. However, biological characteristics of BMSC differentiation are still unclear presently.OBJECTIVE: To study the expression and electrophysiological characteristics of BMSC/n vitro connexin-43 following 5-azacitidine (5-aza) treatment.DESIGN, TIME AND SETTING: The cytological in vitro controlled study was perormed at the Heart Center, Hebei Provincial People's Hospital from July 2007 to February 2009.MATERIALS: A total of 24 male pigs aged 2 months were purchased from Exparimental Animal Center, Hebei Medical University.METHODS: Bilateral femoral bone marrow was obtained from pigs under sterile condition. BMSCs were harvested by Percoll density gradient in vitro. At passage 2, BMSCs were treated with 10 μmol/L 5-aza, and incubated in DMeM without inductor 24 hours later. Indices were measured I, 2, 3 weeks following induction. A control group was set up, which was not treated with 5-azacitidine. Following bone marrow extraction, experimental pigs were anesthetized to obtain ventricular muscle. Normal ventricular muscle cells were isolated and cultured by tissue block enzyme digestion method.MAIN OUTCOME MEASURES: Expression of connexin-43 was measured by immunohistochemical staining (ABC method). Ito current density and action potential were determined by patch clamp technique.RESULTS: At 1 and 2 weeks following 5-aza induction, some BMSCs were positive for connexin-43, with the presence of brown particles surrounding nuclei. At 3 weeks, positive rate of connexin-43 was 95%. The area with large cell density was presented with similar structure to normal myocardium. At +80 mV, compared with normal myocardial cells, Ito current density was significantly reduced in BMSCs following 1 and 2 weeks and in the control group (P < 0.05). Ito current density was significantly increased to a normal levels in BMSCs 3 weeks following induction (P > 0.05). No action potential was detected in BMSCs following 1 and 2 weeks of 5-aza, and action potential could be determined 3 weeks following induction, which was identical to normal myocardial cells.CONCLUSION: Through induced by 5-aza for three weeks, BMSCs have the similar expression of connexin-43 and electrophysiological characteristics as normal myocardium.