摘要
目的观察环磷酸核苷对重组mHCN4通道动力学特性的影响。方法用免疫磁珠法分选、纯化2月大昆明小鼠的骨髓间充质干细胞。以pUC18、pcDNA3-mHCN4、pIRES2-EGFP、pMSCV-puro等质粒载体为架构,构建pMSCV-mHCN4-EGFP逆转录病毒载体并转染纯化的mMSCs,采用常规膜片钳技术,观察环磷酸核苷对阳性转染细胞mHCN4通道的调控作用。结果(1)mHCN4基因转染组细胞有超极化激活的内向电流,该电流对细胞外Cs+高度敏感。mHCN4通道电流的半数最大激活电压为(-99.0±5.8)mV,反转电位是(-22.5±5.2)mV,在-140mV电压时的激活时间常数为(451±61)ms。(2)当细胞内液加入cAMP后,阳性转染细胞超极化激活的内向电流明显变大,通道的激活曲线向右移位,半数最大激活电压绝对值变小,反转电位负值加大,在不同指令电压下的激活时间常数变小。cGMP的调控作用与cAMP相似,但作用略弱于后者。结论环磷酸核苷对重建的HCN4通道有类生理性的调控作用,mHCN4基因修饰后的mMSCs有可能成为一种有效的生物起搏种子细胞。
Objective To study the effects of eyelie nueleotide on I(f) channels reconstructed in mMSCs with mHCN4 gene. Methods mMSCs were collected from bone marrow of Kunming mice and purified by immuno-magnetie mierobeads, pMSCV-mH- CN4-EGFP and pMSCV-EGFP retrovirus vectors were constructed by enzymatic reeonneetion of target segments from vectors pUC18, mHCN4/peDNA3, pIRES2-EGFP and pMSCV-puro. To eolleet the mMSCs transfeeted with retrovirus and examine the kinetie eharaeteristies of mHCN4 ehannel, and its ehanges to different drugs with whole-eell pateh elamp teehnique. Results (1) The reeorded I(f) eurrent from mMSCs transfeeted with mHCN4 had signifieant time and voltage dependeneies. The half-maximal activation was (-99.0 ± 5.8) mV, the reversal potential was (-22.5±5.2)mV, time constants of activation was (451±61) ms under-140mV. (2) Compared with the experimental group, I(f) current in mMSCs transfeeted with mHCN4 increased obviously and activation curve moved to right in the present of cAMP or eGMP. The absolute values of half-maximal activation and slop decreased, the active potential moved to more negetive values; time constant of activation diminished under the same prepotential. The modulation of cAMP was stronger than that of eGMP. Conclusion The reconstructed mHCN4 channels have the similar physiologieal reaetion to eyelie nueleotide. The mMSCs modified with mHCN4 may be a souree of effeetive biologieal paeemaker eells.
出处
《重庆医学》
CAS
CSCD
2007年第9期815-817,共3页
Chongqing medicine
基金
国家自然科学基金资助项目(30370585)