| Laser tweezers Raman spectroscopy(LTRS)is able to capture a single living cell or organelles by forming a three-dimensional trap and detect a single living cell in real time.The information of molecular structure in acquisition is output as a spectrum,and the advantages of this technique are high sensitivity,high specificity and rapid detection.LTRS was applied in a real time monitor of single yeast cell that was incubated in high concentration of ethanol.Physiological and biochemical changes of their mitochondrion during apoptosis were studied by LTRS combined with the technologies of spectrophotometer and oxygen electrode.Moreover,the physiological and biochemical changes of apoptotic plasma cells of Vicia faba tip induced by AICl3 were also monitored by the same methods.The main results were listed as follows:(1)Laser tweezers Raman spectroscopy was employed in this study to aim at monitoring the dynamic of the intracellular biological macromolecular in real-time during the apoptosis process of yeast cells stressed with high concentrations of ethanol at group cells level and a single yeast level respectively.The results showed that the intensities of Raman peaks of nucleic acids(721,1083,1301 cm-1),proteins(721,858,1001,1301,1445,1608,1657 cm-1)and lipids(1083,1301,1445 cm-1)decreased significantly along with the extension of treatment time.These phenomena indicated that the contents of nucleic acids,proteins and lipids decreased gradually while the yeast cells were undergoing apoptosis induced by high concentrations of ethanol.However,the results of single factor analysis and repeated measures analysis showed that the intensitie changes of Raman peaks of the group cells and the single cells were significantly different,and it implied that the heterogeneities of the single cells were covered by the average spectrum of the population cells.The results display that LTRS can be used to detect the kinetic apoptosis process directly and truthfully under high concentrations of ethanol stress at the single cell level and probe cellular heterogeneity.High concentration of ethanol will induce yeast apoptosis.LRST and Oxygen electrode were employed to detect the changes of the structure and function of mitochondrion during yeast apoptosis.The results showed as follow:the activation of Cytochrome c oxidase,NDAH oxidase and succinate oxidase was restricted after the yeast stressed by ethanol,and their activation receded either with the ethanol concentration increasing or the stress time extending.Meanwhile,the changes of MDA contents had similar results.These turned out that the important oxidase in mitochondrion were restricted,even if inactivation,and the mitochondrial membrane is damaged,as well as the membrane permeability increased after cells incubated with high concentration ethanol;The reducing of RCR and P/O further illustrated that the structure and function of mitochondrion were damaged;The reducing of the content of cytochrome C in this process indicated that Cyt c was released because of the increasing permeability of mitochondrion membrane during apoptosis induced by high concentration ethanol;The intensities of Raman peaks of nucleic acids(1083,1301 cm-1),proteins(1001,1301,1445,1608,1657 cm-1),lipids(1083,1301,1448 cm-1)and Cyt c(750?1125 cm-1)decreased significantly along with the increasing concentration of ethanol and the extension of treatment time,which indicated the losing of these structural material in mitochondrion;Similar to the results of a normal inspection results,the decreasing of peaks 750 and1125 cm-1 showed a cutting down of Cyt c,and the decreasing of 1603 cm-1 showed a damage of its energy metabolism further in LTRS.(2)The Vicia faba tips were used as research materials,and the cells apoptosis induced by AlCl3 in the different concentration and a certain concentration(200 ?M).The DAPI staining was used to test the apoptosis cells,and LRST was used to monitor the ripe and uniform size plasma cells wich were released from the tips by enzymolysis approach and separated by saccharose gradient.The results were listed as follow:AICl3 could induce the tip cells apoptosis,the color of DAPI staining in nucleus became heavier after 4d culture with 50?M AICl3;After the tip cells were incubated for 4d in 300 ?M AICl3,some special changes,such as the nucleus were out of shape,the irregular nucleus became more seriously,and the blue-fluorescence was lighter,were observed.These indicate that the cells were undergoing apoptosis.The result of the tip cells,which were incubated by 200 ?M AICl3 for different time,showed that the color of DAPI staining became heavier after longer time treated,for example,the cells were inspired a light blue fluorescence after 4 d stress.These results indicated that apoptosis was happed during stress process,and the result of Raman spectra supported the views that the intensities of the peaks assigned to nucleic acids,proteins,lipids and carbohydrates showed a regular decrease.For the intensities of Raman peaks of nucleic acids(716?1050?1083?1379 cm-1)?proteins(863?941?1211?1340?1460 cm-1)?carbohydrates(766?1050?1460 cm-1)and lipids(612?716?1127?1266 cm-1)decrease with the treat-time prolonging,it indicates that the content of nucleic acids,proteins,carbohydrates and lipids in the cells lost gradually during apoptosis induced by AICl3. |
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