مقالات پذیرفته شده در نهمین کنگره بین المللی زیست پزشکی
Studies on activity of executioner caspase enzymes in formalin-fixed tissue samples
Studies on activity of executioner caspase enzymes in formalin-fixed tissue samples
Soheila Danesh,1Kamaloddin Hamidi Nokhostin,2,*Esmat Abdi,3
1. Cellular and Molecular Section, Department of Biology, Faculty of Sciences, University of Mohaghegh Ardabili , Iran 2. Cellular and Molecular Section, Department of Biology, Faculty of Sciences, University of Mohaghegh Ardabili , Iran
Introduction: Apoptosis, or programmed cell death, is a crucial physiological process that plays an essential role in tissue development, growth, and homeostasis. A series of signaling cascades, operating under specific conditions, precisely regulate this process. Among these, the caspase cascade system has a vital role in initiating, transmitting, and amplifying intracellular apoptotic signals.
Caspases are cysteine-dependent proteolytic enzymes within the protease family which play a key role in maintaining cellular homeostasis and executing programmed cell death. Based on their functions, caspases are broadly classified into apoptosis-related caspases (such as caspase-3, -6, -7, -8, and -9 in mammals) and inflammation-related caspases (such as caspase-1, -4, -5, and -12 in humans, Apoptosis-related caspases are further categorized, according to their mechanism of action, into initiator caspases (caspase-8 and -9) and executioner caspases (caspase-3, -6, and -7). In addition to apoptosis, caspases are also involved in other forms of non-apoptotic cell death, such as necroptosis, pyroptosis, and autophagy.
10% formalin, whose active compound is formaldehyde, is one of the most important and widely used tissue fixatives in pathology laboratories. The mechanism of action of formalin is primarily through cross linking between amino groups in the side chains of lysine residues, which, over time, leads to the formation of methylene bridges. Formaldehyde also reacts with the amino groups of other amino acids, including arginine, tyrosine, asparagine, histidine, glutamine, and serine, thereby stabilizing protein structures.
Methods: In the study, a fresh sheep liver sample was obtained, and two pieces weighing 40 g each were separated. One piece was lysed in caspase lysis buffer for 5 minutes using a probe homogenizer, and then centrifuged at 12,000 rpm for 15 minutes at 4 °C. According to the protocol provided with the caspase-3 /7 activity assay kit (Kiazist, Life Sciences, Iran), The samples were analyzed in duplicates, and absorbance was measured at a wavelength of 405 nm using a BioTek microplate reader (USA) to obtain the results.
The second piece was placed in a container of 10% formalin for two weeks . After fixation, caspase enzymatic activity was measured following the same procedure described above.
Results: The average activity of caspase-3/7 in the tissue sample before fixation was 1854 nmol/min/l, whereas in the tissue sample after two weeks fixation, it was 253 nmol/min/l.
Conclusion: The enzyme activity results indicate that the activity of executioner caspases in sheep liver sample fixed in 10% formalin for two weeks, compared to the freshly sample, is decreased by more than 80%, although the activity is not completely eliminated.