| Apoptosis is controled by cell genes and is the process of programmed cell death genetically.It is a survival ability that biological cells adapt to the changes of the internal and external environment,and involves a series of activation of gene expression and regulation role.The investigations of apoptosis,including the molecular mechanisms of the apoptosis,the apoptosis initiation pathway,biochemical process of the apoptosis,are the focus of current research in life sciences.Mitochondria is an important organelle for the energy metabolism in eukaryotic cells and is the control center of cell life activities,which are not only the center of energy metabolism,but also cell the participant of the apoptosis.Many research results shown that the mitochondrial Cyt c release into the cytoplasm is a common manifestation of the cell apoptosis.Laser tweezers Raman spectroscopy(LTRS)is a biological photon technology that combines laser tweezers with Raman spectroscopy technology,which applies to research of the suspension cells,organelles,macromolecular particles without destructive or labeling.Yeast is a kind of low unicellular eukaryotes and a simple biological model species.In yeast fermentation process,the byproducts,such as acetic acid,ethanol,formate,glycerin,are toxic to the physiological activity of yeast and prohibitive to the yeast fermentation.If the metabolites of the yeasts further accumulate,massive yeast cells would be death.But so far.It is still not entirely clear how the yeast metabolites induce the cell death process.Preliminary research suggested that the release of mitochondrion inclusions induced by metabolites could start cell irreversible apoptosis.Aluminum(Al)toxicoty severely limits crop production on acidic soils.Gradually,mitochondria and Cyt c play important roles in caspase activation and cell death after plant cells stressed by aluminum.But intriguing questions,such as if cell apoptosis can be induced by Al in plants,how Aluminum induces the cell apoptosis,how the signal transduction in vivo after cells apoptosis,are not very clear.In this paper,the LTRS was used to real-time monitor the physiological and biochemical processes of cells apoptosis,and the release of the mitochondria inclusions as well,while a single yeast cell and a single mitochondria stressed by acetic acid,or a single plant cell stressed by the aluminum.The main results are as follows:(1)We used the laser Raman spectroscopy(LTRS)to monitor the process of a single yeast cell’s apoptosis while induced by acetic acid in real-time,and analysed the spectral information of biological macromolecules combined with repeated measurement variance method.The results showed that,under the treatment of 300 mM acetic acid,the intensity of raman peak(721,1083,1301 cm-1),(721,858,1001,1301,1445,1608,1657 cm-1),(1301,1445 cm-1)that attribute to nucleic acid,protein and lipid respectively had a decreasing tendency significantly with the extension of treatment time,indicated that the content of nucleic acid,protein and lipid were reduced during the apoptosis process of yeast cells.According to the changed of the intensity of the raman peak of 1,608 cm-1,we could acquire the information that the energy metabolism of cells was blocked due to the damage of mitochondrial function.In the process of apoptosis of yeast cells induced by acetic acid,LTRS could record the process of the changing of the physiological based at a single cell level in real time,and we could acquire the real-time information of the changing of intracellular biological macromolecular in the apoptosis process of a cell,in which all information can reflect the physiological and biochemical process of the apoptosis process truly.LTRS can find a aensitive information of the spectral change of the biological macromolecule in a single cell but the information always is concealed while monitoring group cells.and LTRS is a effective tool to monitor the process of apoptosis at the level of a single cell in real-time.(2)The mitochondriaes were isolated after the yeast cells were stressed by acetic acid,and LTRS was used to real-time monitor the mitochondrial and recorded the Raman spectroscopy.Combined with the conventional method to measure mitochondrial physiological and biochemical changes,the change of mitochondria biological macromolecules was tested and valided.The results showed that Raman peaks(1301,1445 and 1657 cm-1)associated with mitochondrial lipid were significantly decreased as a function of the increase in the concentration of acetic acid and treatment time.accordingly,yeast mitochondrial malonyl aldehyde(MDA)content measured by a conventional method also was observed to increase with the increase in acetic acid concentration and treatment time,indicating that the unsaturated fatty acids was continuous to be degraded in oxidbillity,and leading to a decrease in mitochondrial lipid content.The intensity of characteristic peak(750 and 1125 cm-1)related with Cyt c in the mitochondria significantly decreased with the increase of acetic acid concentration and treatment time,and the reduced content of Cyt c was similar with the the content of mitochondrial Cyt c/a and the Cyt c measured with conventional method,suggesting that the yeast mitochondrial membrane swelled by the stress of acetic acid,leading to the membrane rupture and Cyt c release into the mitochondrial outer.1604 cm-1 is the characterized peak of mitochondrial respiration,with the increase of the acetic acid concentration and treatment time increases,the respiratory peak was observed to decrease gradually,indicating that the mitochondrial activity become weaker,energy metabolism had been hampered;The mitochondrial Cyt c oxidase activity,mitochondrial respiratory rate and the phosphorus oxygen ratio are consistent with the result measured by conventional method.The intensity of the nucleic acid peaks(1081 and 1301 cm-1)and the protein peaks(872,1445,1604 and 1657 cm-1)were significantly reduced with the increase of the acetic acid concentration and treatment time.These results illustrated that nucleic acid,protein and lipid content in mitochondria were a downward trend in the process of apoptosis.(3)LTRS was used to real-time monitor the mitochondrial Raman spectroscopy in vitro after root tip cells of Vicia faba were stressed by aluminum.Combined with the conventional method to measure mitochondrial physiological and biochemical changes,the information changes of mitochondria biological macromolecules was analyzed.DAPI staining experiments showed that there was a characteristics of apoptosis after the Vicia faba root tip was induced by aluminum,the concentration of mitochondrial malondialdehyde(MDA)increased with the increase of the aluminum concentration,and mitochondrial Cyt c/a ratio,the content of mitochondrial Cyt c,mitochondrial Cyt c oxidase activity were on the decline,indicating that the increases of the mitochondrial reactive oxygen species(ROS),the contents of Cyt c/a,Cyt c and mitochondrial Cyt c oxidase activity were associated with the apoptosis;The increase of mitochondrial MPTP and the reduction of the membrane potentialcould lead to the decline of the mitochondrial respiratory rate(RCR)and P/O,suggested that mitochondrial membrane structure and function were damaged after apoptosis induced by aluminum,.The Raman spectral data from the mitochondria showed that the peak intensity of nucleic acid peaks(825,1262 cm-1),protein peaks(930,1330,1360,1457 and 1602cm-1)and mitochondrial lipid peaks(1065,1457 cm-1)were significantly reduced with the increase of aluminum concentration,indicatinf that the nucleic acid,protein and lipid content within the mitochondria were all reduced during apoptosis.The intensity reduction of mitochondrial lipid peaks may be due to the oxidation degradation of unsaturated fatty acids in the mitochondrial membrane.It was consistent with the results of the MDA.1602 cm-1 is mitochondrial respiration characterized peak,the respiratory peak was observed to decreases with the increase of aluminum concentration.It was consistent with the mitochondrial respiration rate measured by conventional method.750 and 1127 cm-1 is the characteristic peak of the cell dye C,and the spectrum peak intensity was significantly reduced with the increase of aluminum concentration.It is consistent with Cyt c results trend measured by the conventional method.The results of these experiments showed that LTRS is an effective tool to real-time monitor the changing process of the mitochondria. |
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