摘要
目的探讨在大鼠脂肪干细胞(ADSCs)向心肌样细胞分化的一定时期内,通过干预NRG-1/ErbB通路是否可调控其向类窦房结样细胞或工作肌样细胞分化。方法分离培养大鼠ADSCs,免疫荧光测定细胞相关表面抗原鉴定其干细胞特性。取3代ADSCs加入含10μmol.L-15-氮胞苷(5-Aza)和10μg.L-1bFGF的培养基处理24 h后分3组向心肌样细胞诱导分化,包括对照组、AG1478组、神经调节蛋白-1(NRG-1)组,取正常培养的为ADSCs组。诱导3周后,通过RT-PCR检测各组NKx2.5、HCN4、TBX3、TBX2基因,Westernblot检测TBX3蛋白,膜片钳检测各组动作电位的表达差异。结果 ADSCs加5-Aza诱导3周后表达心脏早期转录因子Nkx2.5及肌钙蛋白,证明5-Aza可以将ADSCs诱导成心肌样细胞。而AG1478组起搏相关基因(HCN4、TBX3、TBX2)的表达明显强于对照组及NRG-1组(P<0.05),TBX3蛋白也强于对照组(P<0.05),并能产生窦房结样动作电位。而NRG-1组Nkx2.5的表达高于对照组及AG1478组(P<0.05),亦能检测到心室肌样动作电位。结论在大鼠AD-SCs向心肌样细胞分化的一定时间内通过干预NRG-1/ErbB通路可使其定向分化为起搏样细胞或工作肌样细胞,这为今后干细胞生物起搏研究做了有益探讨。
Aim To investigate whether mouse adipose tissue - derived adult stem cells ( ADSCs ) could differentiate into sinus node-like cells or working-type cells through intervening the pathway of Neuregulin-1/ErbB in a certain period of time when ADSCs differentiate in- to cardiomyocytes. Methods ADSCs were separated and cultured. Then, culture medium containing 10 μmol L-1 5-Aza and 10 μmol L-1 bFGF was joined into three generations of ADSCs,24 hours after treat- ment, it was divided into three groups to differentiate into cardiomyocytes, including control group, and AG1478 group, NRG-1 group and normal cultivation of ADSCs group. 3 weeks later, the expression of NKx2.5, HCN4, TBX3, TBX2 was detected by RT- PCR. Western blot was used to detect the difference in the expression of protein of TBX3. Patch clamp was a- dopted to detect the direct action potential phenotyping in the induced cells. Results 3 weeks later, ADSCs expressed heart early transcription factor Nkx2.5 and troponin I after treated with 10μmol L-1 5-azacyti- dine, which proved ADSCs could differentiate into cardiomyocyte--like cells in vitro. The inhibition of neu- regulin-1/ErbB signaling (used AG1478 )greatly en- hanced the gene expression of HCN4, TBX3, TBX2 compared with control group and NRG-1 group (P 〈 O. 05 ). The protein of TBX3 was higher than control group too(P 〈0.05),and even produced distinct nod- al-type action potentials. The expression of Nkx2.5 in NRG-I group was higher than the other two groups (P 〈 0.05),and the working-type action potentials could also be detected. Conclusions Intervening the path- way of Neuregulin/ErbB in a certain period of time when mouse ADSCs differentiate into cardiomyocytes could obtain more sinus pacemaker-like cells or work- ing-type cells, which contributes to the future research of biological pacemaker.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2013年第7期937-942,共6页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 81041097)
江西省自然科学基金资助项目(No 2008GZY0045)