Objective To investigate the feasibility of recombinant lentivirus (LVs) mediated hyperpolarization- activated cyclic nucleotide-gated cation channel 4 (HCN4) gene transfecting rat bone mesenchymal stem cells (BMSCs) so as to construct the biological pacemaker cells. Methods Sprague Dawley rats at the age of 3-5 weeks were selected to isolate and culture BMSCs using modified whole bone marrow adherent culture method. LVs was used as carrier, and enhanced green fluorescent protein (EGFP) as marker to build LVs-HCN4-EGFP virus liquid. The BMSCs at passage 3 were transfected with LVs-HCN4-EGFP virus liquid (experimental group) and LVs-EGFP null virus liquid (control group). Fluorescence microscope was used to observe the green fluorescent protein expression after 24, 48, and 72 hours of transfection; Western blot method was used to detect the HCN4 protein expression. The electrophysiology was used to detect the pacemaker current in the experimental group. Results After transfection, BMSCs in the experimental group showed normal morphology and good growth; scattered green fluorescence could be seen at 48 hours under fluorescence microscope, with a transfection efficiency of about 10%; the fluorescence expression increased slightly, with the transfection efficiency of 20% to 25% at 72 hours. While no expression of green fluorescence was seen in the control group. Western blot results showed that the same band expression as a relative molecular mass of HCN4 protein were found at 72 hours after transfection in the experimental group, only weak expression of protein band was seen in the control group; the gray value of the experimental group (33.75 ± 0.41) was significantly higher than that of the control group (23.39 ± 0.33) (t=17.524, P=0.013). In the experimental group, the pacemaker current was recorded, and it could be blocked by CsCl, in accordance with the characteristics of pacemaker current. Conclusion The recombinant LVs mediated HCN4 gene is successfully transfected into rat BMSCs, and the expression of HCN4 protein and the pacemaker current can be detected.
Coronary angiography (CAG) as a typical imaging modality for the diagnosis of coronary diseases hasbeen widely employed in clinical practices. For CAG-based computer-aided diagnosis systems, accurate vessel segmentation plays a fundamental role. However, patients with bradycardia usually have a pacemaker which frequently interferes the vessel segmentation. In this case, the segmentation of vessels will be hard. To mitigate interferences of pacemakers and then extract main vessels more effectively in CAG images, we propose an approach. At first, a pseudo CAG (pCAG) image is generated through a part of a CAG sequence, in which the pacemaker exists. Then, a local feature descriptor is employed to register the relative location of pacemaker between the pCAG image and the target CAG image. Finally, combining the registration result and segmentation results of main vessels and pacemaker, interferences of pacemaker are removed and the segmentation of main vessels is improved. The proposed method is evaluated based on 11 CAG images with pacemakers acquired in clinical practices. An optimization ratio of the Dice coefficient is 12.04%, which demonstrates that our method can remove overlapping pacemakers and achieve the improvement of main vessel segmentation in CAG images.Our method can further become a helpful component in a CAG-based computer-aided diagnosis system, improving its diagnosis accuracy and efficiency.