| Summary: | 碩士 === 國立臺灣大學 === 生化科技學系 === 103 === Acute myocardial infarction (AMI) is the most common type of ischemic heart disease. Many clinical biomarkers have been used for detection AMI including cardiac troponin I (cTnI). cTnI is a cardiac-specific isoform of troponin I. Plasma cTnI increases rapidly after cardiomyocyte damage and remains detectable after days. Our goal is to identify high affinity peptides specific to cTnI and to apply in serum detection and molecular imaging for clinical use.
First, we used two different phage libraries to select cyclic 7-mer and linear 12-mer peptides by biopanning experiments. We found nine candidate peptides with high binding affinities to purified recombinant human cTnI after checking by ELISA binding assay. Since the N-terminal region is unique to the cardiac isoform of cTnI, we also performed biopanning against this region of cTnI and identified NC4-1. Next, we found all candidates bound to the central region, and three candidates bound to the N-terminal region. In addition, we found that the binding affinities of candidates to cTnI were not affected by culture media with fetal bovine serum.
For further analysis, the apparent dissociation constants of candidate phage clones and their corresponding synthesized peptides were measured. According to our results, we indicated that candidate NC4-1 had better binding affinity to cTnI. Moreover, candidate NC4-1 could target the N-terminal region of cTnI. We then used candidate NC4-1 to target cTnI in the rat heart myoblast cell line and ischemic-heart-failure rats. By using FITC-labeled NC4-1 peptide, we demonstrated that NC4-1 peptide could target to the cytoplasm of H9c2 (2-1) cells, and that was corresponding to the localization of cTnI studied by ICC using cTnI antibody. For in vivo study, PET imaging was performed using 68Ga-phage NC4-1 as the tracer to target cTnI in ischemic-heart-failure rats. Comparing the region identified as normal cardiac muscle by SPECT imaging using 99mTc sestamibi, we found that candidate NC4-1 could target to damage sites of cardiac muscles. In conclusion, NC4-1 peptide may have the potential to be developed as a diagnostic molecular imaging tracer in AMI detection.
|
|---|
