Masters Theses

Date of Award

8-1995

Degree Type

Thesis

Degree Name

Master of Science

Major

Comparative and Experimental Medicine

Major Professor

Donita Frazier, Tuan Vo-Dinh

Committee Members

James Chen

Abstract

The purpose of this study was to utilize fluorescence spectroscopic techniques to differentiate between normal and neoplastic cells. Specific fluorescent biological compounds, i.e., tryptophan, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FADH2) and collagen, were examined in this study in order to determine if the cellular fluorescence could be attributed to them. Rat liver epithelial (RLE) cells, McA-RH7777 rat hepatoma (McA) cells, astrocytes and astrocytoma cells were investigated in this study. The autofluorescence of whole cells and cellular extracts of RLE and McA was studied using conventional and synchronous fluorescence, before and after dialysis. Synchronous fluorescence was used to study the astrocyte and astrocytoma cellular extract after dialysis. Polyacrylamide gel electrophoresis (PAGE) was performed with the polypeptide extracts. A charge-coupled device (CCD) was used to image fluorescent bands illuminated in the gels with a Helium-Cadmium laser. The use of synchronous fluorescence showed no differences with the astrocytes and astrocytomas. However, some differences in the spectra of RLE and MoA cells led to an attempt to identify the source of the fluorescence. Tryptophan, NADH, FADH2 and collagen did not appear to be the source of fluorescence differences in the normal or neoplastic cells because the peak positions for the synchronous spectra for the cells did not correspond to peak positions for synchronous measurements of tryptophan, NADH, FADH2 and collagen. McA cells have a synchronous fluorescence peak that is shifted to shorter wavelengths with respect to the peak position of RLE cells by 4-5nm. Differences in the peak positions are even greater in the cellular extracts. The McA cellular extract exhibits a synchronous fluorescence peak blue-shifted by 10nm to that of the RLE cellular extract. When imaging gels to detect fluorescent bands, a 70-75 kD polypeptide was found to fluoresce in the McA cell line, whereas no fluorescent band was observed in the RLE cell line. No spectral differences were detected in the astrocytes or astrocytomas when performing synchronous fluorescence. No fluorescent bands were detected in astrocytes or astrocytomas when imaging gels to detect fluorescent bands. In conclusion, conventional, synchronous fluorescence and CCD imaging have been employed in order to detect differences in the emission of RLE and McA cells. Spectral differences were detected in RLE and McA cells using synchronous fluorescence, and a fluorescent polypeptide detected in the McA cells using CCD imaging. Although these techniques did not detect differences in the other cells studied, it may be possible to utilize synchronous fluorescence excited at other wavelengths to detect differences in other cell lines. Synchronous fluorescence may be an effective way to determine differences in normal and neoplastic cells.

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