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바이오와 나노기술을 이용한 세포사멸 기전과 구조에 대한 연구
- Alternative Title
- Kwon Hyuk-Kwon
- Author(s)
- KWON HYUK-KWON
- Alternative Author(s)
- Kwon Hyuk-Kwon
- Advisor
- 최상돈
- Department
- 일반대학원 분자과학기술학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2015-02
- Language
- eng
- Keyword
- Cell death; Biotechnology; Nanotechnology
- Alternative Abstract
- Even though various chemotherapeutic drugs are being used for clinical trials, until now, the field of scientific research keeps on carrying several studies aiming the development of relatively safe and stable anti-cancer drugs. Unfortunately, the continued use of the chemotherapeutic drug has the major limitation of severe side-effects in various organs. In particular, in case of p53, which is a key transcription factor known for its important role in the modulation of multiple cellular signaling pathways. The mutation occurring in p53 gene has been reported to be the cause of 75% of cancer’s cases suggesting that the loss of p53 function might lead to tumorigenesis. However, the exact mechanism by which the p53 causes cell death by DNA damage through apoptosis or necrosis, is not yet fully understood. Moreover, a clear understanding of the various modifications occurring in both cell adhesion and nuclear envelope features related to cell death remains largely unknown. To address this subject, our present study investigated the observed modifications in cell adhesion ability and nuclear envelope changes as well as DNA damage caused by p53 regulated apoptosis and necrosis, by using the Topoisomerase II targeting chemotherapeutic drugs such as etoposide and/or doxorubicin. For this, a variety of bio/nano-technological methods were used during. The first results of this study showed that the treatment of the human renal proximal tubule derived cells (HK-2) by etoposide induced DNA damage, p53 activation, cell cycle arrest and triggered the generation of reactive oxygen species (ROS) as well the mitochondrial biogenesis. Moreover, cell morphological changes such as cell swelling and plasma membrane rupture were also confirmed by carbon nanotube atomic force microscopy (CNT/AFM) analysis. In the other hand, the treatment of the p53-defective cells by etoposide limited cell cycle arrest and the mitochondrial biogenesis while it induced the increase of DNA damage, ROS mitochondrial generation and nitric oxide (NO) synthesis. Moreover, mitochondria outer membrane protein degradation as well as caspase 3 activation also increased. The morphological changes such cell shrinkage and the formation of apoptotic body were confirmed by the use of the CNT/AFM analysis. These findings show that the p53 activation, through the DNA damage signals, is responsible for the inhibition of cell death by apoptosis and the activation of necrosis. The second result of this study showed that though the treatment of the HK-2 cell by etoposide and doxorubicin induced the change of morphological features into those noticed in necrosis, in the case of cells treated by etoposide both apoptosis and necrosis features were observed while in the case of doxorubicin only necrosis morphological characteristic were reported. Based on the above experimental conditions, the morphological changes of cell during the mechanism of cell death by apoptosis were measured in real time by the xCELLigene: cells size, cell adhesion area and cell adhesion speed were formulated based on that adhesive properties of the cells were confirmed. The results showed that in apoptosis the cells presented a decrease in cell adhesion area as well as a decrease in their speed of adhesion. In the other hand in case of necrosis the speed of cell adhesion didn't present any significant change while an increase in the cell adhesion area was reported. In addition, in both cases of apoptosis and necrosis the changes of the nuclear envelope were investigated using CNT/AFM analysis and the result of the investigation showed that in necrotic cells the nuclear envelope did not present any damage. The nuclear pore complex as well as the general form of the nuclear envelope was confirmed. However, in case of apoptosis the rupture of the nuclear envelope as well as the nuclear pore destruction due to the activated caspase were also showed. Moreover, in the present work, DNA fragmentation protein translocation in nucleus through the rupture of the nuclear envelope caused by caspase, were also detected. In the present study, even though apoptosis and necrosis features such as changes in cell adhesion properties and changes in the nuclear envelope features were characterized, new methods of measurement using the nano-technology will be recommended in order to overcome the limitations of conventional measurement methods. In conclusion, through the fusion of various bio- and nano-chemical technologies we studied cell death, used a variety of methods that helped to understand cell death mechanisms.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Molecular Science and Technology > 4. Theses(Ph.D)
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