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Development of Two-Photon Probes for Lipid Droplets Screening and NAD(P)H Quinone Oxidoreductase Activity
- Author(s)
- 조명기
- Advisor
- 김환명
- Department
- 일반대학원 에너지시스템학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2021-08
- Language
- eng
- Keyword
- NQO1; lipid droplets; two-photon fluorescent probe
- Alternative Abstract
- The fluorescent probes are useful in research observing various diseases and biological phenomena. Small molecule fluorescent probes have a low molecular weight, so they are easily loaded to cells and tissues. Thus, small molecule fluorescent probes can be easily applied to monitoring various biological events via confocal microscopy. There are several types of fluorescent probes, one of which is to use two-photon fluorescence. Two-photon fluorescent probes, with exceptional high transmittance originating from the use of a near-infrared excitation source, have been utilized in deep-tissue imaging which is out of scope for the applications of common one-photon fluorescent probes. In addition, two-photon microscopy utilizes relatively lower energy for excitation of photons damaging tissue samples minimally and thereby various tissue-imaging experiments with long-exposure time are feasible due to this high durability. Therefore, two-photon fluorescent probes can be applied to a variety of biomarkers and utilized in live tissues imaging. We employed two-photon probes for screening and detecting cellular organelles and enzymes. Lipid droplets (LDs) are one of the cellular organelles. LDs contain the neutral lipids such as triglycerides, cholesterol and acyl glycerol. The role of LDs is to regulate the amount of lipids in cells and supply stored lipids as a source of energy for cells. In particular, LDs rapidly increase in size and number when the endoplasmic reticulum (ER) is exposed to stress or when lipid metabolism is defective. Therefore, by tracking the size and number of LDs, it is possible to confirm the abnormal conditions or diseases. Thus, we designed a two-photon fluorescent probes for staining detecting LDs. We developed the probes that emit green and red fluorescence and observed changes in LDs under ER-stress and disease in which lipids were rapidly increased. Probes 3 and 4, which are selective for LDs, emit green and red fluorescence, respectively, and have a large stokes shift and narrow emission spectrum. Because of these characteristics, Probe 3 and 4 could perform multicolor imaging with other organelle trackers. In this study, the changes of LDs in drug-induced liver injuries were observed. Steatosis, one of the liver diseases, result in a large amount of lipids accumulated in cells due to liver toxicity, and steatosis can be caused by drugs which induce liver toxicity. Therefore, various drugs were treated in liver cells and tissues, and we monitored the liver toxicity. Based on the research results, Probe 3 and 4 can be applied to monitor of drug-induced liver toxicity. Through multicolor imaging with lysosome trackers, Probe 3 and 4 can distinguish between steatosis and phospholipidosis which is similar but different with steatosis. Thus, it is expected that Probes 3 and 4 can be useful in diverse liver disease studies and screening of various drugs. Human NAD(P)H quinone oxidoreductase type 1 (hNQO1) is an enzyme present in the cytoplasm that reduces intracellular toxic quinone derivatives, allowing cells to maintain homeostasis from oxidative stress. In particular, the expression of hNQO1 is significantly increased in cancer compared to normal. It is due to oxidative stress caused by the proliferation of cancer, especially liver and colon cancer show quite high activity of hNQO1. Therefore, it is possible to distinguish between normal and cancer by detecting the activity of hNQO1 enzyme. To detect the activity of hNQO1, we developed two ratiometric probes. The probes are reduced by hNQO1 enzyme, and fluorescence of probes were changed after response to hNQO1. The activity of the hNQO1 could be monitored by investigating the ratio of the changed fluorescence. SHC and SHC-E, developed for detecting hNQO1, are ratiometric probes, representing the difference in hNQO1 activity in normal and cancer. In addition, by applying to a biopsy sample of a patient with colorectal cancer, SHC and SHC-E could distinguish between normal and cancer. Furthermore, SHC could detect the progression stage of cancer. Therefore, SHC and SHC-E can be directly applied to the study of hNQO1 activity in normal and cancer, and can be used as a major tool for more detailed study of colorectal cancer and development of diagnostic methods.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Energy Systems > 4. Theses(Ph.D)
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