Differentiating The Cd117+ Cardiac Stem Cells Via Contact With HCN4+ Sinus Node Cells

Objectives: To explore whether CD117+ cardiac stem cells in contact with the HCN4+ sinus node cells can enhance the possibility of the former to differentiate into sinus node-like cells, preliminarily analyze the differentiation mechanism and provide theoretical foundation for using biological pacemaker to treat such diseases as slow rhythm of heartbeat.Methods:(1) Adopting digestion method to digest sinus node tissue and form cell suspension, regarding HCN4 as markers and using magnetic cell separation system to separate HCN4+ sinus node cells;(2) Using digestion method to digest heart tissue, obtaining cell suspension and separating CD117+ cardiac stem cells with magnetic cell separation instrument;(3) Using transwell Petri dish to contact and coculture HCN4+ sinus node cells and CD117+ cardiac stem cells, choosing cardiac stem cells which have been induced for 10 days, 20 days and 30 days, using immunofluorescence technique to observe the differentiation of cardiac stem cells and RT-PCR technique to detect the m RNA expression of HCN2 and HCN4.Results:(1) Using HCN4 as markers, magnetic cell separation instrument can successfully separate HCN4+ sinus node cells and improve the purity of sinus node cells;(2) After induction for 10 days, the positive rates of expression of HCN2 and HCN4 in cardiac stem cells are respectively 5.2±3.4% and 5.8±3.7%; the above positive rates of protein are respectively 7.5±3.6% and 7.1±3.5% after induction for 20 days, 10.2±3.8% and 11.9±4.4% after induction for 30 days. Among them, the positive rate difference of markers between the 30-days-induction group and 10-days-induction group has obvious statistical significance(P<0.05); Using RT-PCR technique to detect m RNA of HCN2 and HCN4 in the cells, we find that both HCN2 and HCN4 have m RNA expressions after induction for 10 days, 20 days and 30 days. The expression has significantly increased with the time and the difference has statistical significance(P<0.05).Conclusion:(1) By using HCN4 as marker and magnetic cell separation instrument, we can obtain purified HCN4+ sinus node cells.(2) Cardiac stem cells can differentiate into endothelial-like, cardiac-like and smooth muscle-like cells and also have the potential of differentiating into sinus node-like cells.(3) Cardiac stem cells in contact with the sinus node cells can improve the efficiency of the former to differentiate into sinus node-like cells.