摘要
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月龄雄性冀中白猪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,24h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+80mV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Ito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。
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 in vitro connexin-43 following 5-azacitidine （5-aza） treatment. DESIGN, TIME AND SE＇n＇ING： The cytological in vitro controlled study was performed 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 Experimental 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 1, 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 veotricular 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 potentia~ 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 e ectrophysiolog ca characteristics as normal myocardium.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第27期5261-5264,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
