CN102680446A - Method for detecting location of resveratrol in target cells - Google Patents
Method for detecting location of resveratrol in target cells Download PDFInfo
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- CN102680446A CN102680446A CN2012101658438A CN201210165843A CN102680446A CN 102680446 A CN102680446 A CN 102680446A CN 2012101658438 A CN2012101658438 A CN 2012101658438A CN 201210165843 A CN201210165843 A CN 201210165843A CN 102680446 A CN102680446 A CN 102680446A
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Abstract
The invention discloses a method for detecting the location of resveratrol in target cells. Specifically, the method comprises the following steps of: firstly pretreating the target cells with resveratrol based on the autofluorescence property of resveratrol; then treating the target cells by using a specifically fluorescently-labeled subcellular organelle probe; and finally determining the distribution of resveratrol in the subcellular organelle of the target cells according to the overlap state of the autofluorescence of resveratrol and the fluorescence of the subcellular organelle probe in the cells. The method disclosed by the invention can be used for detecting the distribution of resveratrol in target cells, and has wide application prospect.
Description
Technical field
The present invention relates to detection range, particularly detect the method for resveratrol in the target cell location.
Background technology
Resveratrol is the phytochemical that extensively is present in multiple natural foods such as grape, peanut, giant knotweed, the fleece-flower root and the medicine, has significant reducing blood lipid, cardiovascular protection, antitumor, fat-reducing, anti-inflammatory, anti-ageing, hepatoprotective effect.It is the plurality kinds of health care food or the dietary supplements of principal ingredient, for example Longevity with the resveratrol that the U.S. has gone on the market
TMAmti-sanility health-care food.Be antitumor, anti-cardiovascular disease, the also successfully screening of anti senile dementia drug of lead compound exploitation with the resveratrol, the part medicine has got into clinical testing, has a good application prospect.Yet; The molecular mechanism of resveratrol various biological effect is not illustrated as yet fully; Action target spot is also indeterminate in the cell; The interaction of multiple big molecule (protein, nucleic acid, glycoprotein etc.) is closely related in existing research confirmation and resveratrol and the cell, and correlation molecule mechanism remains further research.Therefore, help to illustrate its action target spot and mechanism of action through the clear and definite resveratrol of certain technological means in biological utilisation and the Subcellular Localization of different target cells.People such as He Hui have reported about the absorbing state of resveratrol at enteron aisle, utilize concentration (He Hui, Chen Xijing, the Wang Guangji etc. of high effective liquid chromatography for measuring different time resveratrol in enteron aisle; The effect of drug transport body in the trans-resveratrol intestinal absorption. the journal .2008 of China Medicine University, 39 (4): 324 – 328).People such as Gao Xin have studied resveratrol pharmacokinetic parameter in vivo; Comprise (Gao Xin such as peak serum concentration, half life period, urine metabolism substrate concentration; Xie Wenli; Zhu Jiang etc. the comparative studies of nanoscale resveratrol and resveratrol Benexate Hydrochloride bioavilability. Chinese herbal medicine, 2011,42 (5): 966-968).People such as Gester adopt radioactive nuclide (
18F) or the method for fluorescence probe mark resveratrol detect its transhipment characteristics (Gester S in gut epithelium; Wuest F; Pawelke B; Bergmann R, Pietzsch J. Synthesis and biodistribution of an 18F-labelled resveratrol derivative for small animal positron emission tomography.
Amino Acids.2005 Dec; 29 (4): 415-28), these methods can be used to detect absorbing state and the transmembrane transport characteristics of resveratrol at enteron aisle, but sense cycle is long, the mark expense is high, have the human body radiologic hazard, and Routine Test Lab is difficult for carrying out.Therefore, be badly in need of a kind of method that detects location in the resveratrol cell, with action target spot and the mechanism of action of further research resveratrol different target cells.
Summary of the invention
In view of this, the object of the invention is to provide and detects the method for resveratrol in the target cell location, and its detection method is simple, and sense cycle is short, does not have radiologic hazard.
For realizing the foregoing invention purpose, technical scheme is:
Detect the method for resveratrol, may further comprise the steps in the target cell location:
A. target cell being used concentration is the resveratrol processing of 1～40 μ M, gets resveratrol and handles target cell;
B. step a gained resveratrol is handled target cell and add the organelle probe, get the target cell of organelle probe mark;
C. the target cell of step b gained organelle probe mark is observed the fluorescence position of resveratrol and organelle probe at laser confocal microscope, confirm the location of resveratrol target cell according to the overlapping situation of fluorescence.
Preferably, said step a is that target cell is added concentration is in 1～40 μ M resveratrol, is 37 ℃, 100% saturated humidity in temperature, and volume fraction is 5% CO
2Incubator was cultivated 1 hour at least.
Method among the present invention can be used to detect mammiferous any cell, and preferred target cell is vascular endothelial cell, liver cell or tumour cell.
The organelle probe that uses among the present invention can be according to acceptable probe on the organelle different choice cell biology that detects, and preferred organelle probe is cell membrane fluorescence probe, mitochondria fluorescence probe or nucleus fluorescence probe.Preferred, said cell membrane fluorescence probe be cell membrane red fluorescence probe (1,1'-dioctadecyl-3,3; 3', 3'-tetramethylindocarbocyanine perchlorate, Dil probe), said mitochondria fluorescence probe is a mitochondrial membrane potential fluorescence probe (5; 5 ', 6,6 '-Tetrachloro-1,1 '; 3,3 '-tetraethyl-imidacarbocyanine iodide 5,5 ', 6; 6 '-Tetrachloro-1,1 ', 3,3 '-tetraethylbenzimidazolocarbocyanine iodide CBIC2 (3); JC-1), and the fluorescence probe that said nucleus fluorescence probe is labeled cell DNA (Propidium Iodide, PI).
Preferably, the resveratrol excitation wavelength is 322nm among the said step c, and receiving wavelength is to detect under 350～500nm condition.
Preferred, said reception wavelength is 405nm.
The beneficial effect of this method is: the invention discloses the method for resveratrol in the target cell location that detect; Utilized the autofluorescence characteristic of resveratrol; And the fluorescently-labeled organelle probe of binding specificity; The utilization laser confocal microscope detects the subcellular fraction distribution of resveratrol and organelle probe in the different target cells; Confirm that according to the overlapping situation of fluorescence the subcellular fraction of resveratrol in target cell distributes, for action target spot and mechanism of action in the cell of further studying resveratrol are laid a good foundation.
Description of drawings
Fig. 1 is a resveratrol molecule structural drawing of the present invention.
Fig. 2 excites and absorption spectrum full wavelength scanner figure for resveratrol autofluorescence of the present invention.
Fig. 3 is the detection and localization figure of resveratrol of the present invention in Human umbilical vein endothelial cells.
Fig. 4 is the cell membrane detection and localization figure of resveratrol of the present invention in Human umbilical vein endothelial cells.
Fig. 5 is resveratrol Human umbilical vein endothelial cells mitochondria detection and localization figure of the present invention.
Fig. 6 is that resveratrol of the present invention is at Human umbilical vein endothelial cells nucleus detection and localization figure.
Fig. 7 is that resveratrol of the present invention is at hepatocellular detection and localization figure.
Fig. 8 is that resveratrol of the present invention is at hepatocellular cell membrane detection and localization figure.
Fig. 9 is that resveratrol of the present invention is at liver cell mitochondria detection and localization figure.
Figure 10 detects figure for resveratrol of the present invention at the liver cell nucleus.
Figure 11 is the detection and localization figure of resveratrol of the present invention at breast cancer cell.
Figure 12 is the cell membrane detection and localization figure of resveratrol of the present invention at breast cancer cell.
Figure 13 is that resveratrol of the present invention is at breast cancer cell mitochondria detection and localization figure.
Figure 14 is that resveratrol of the present invention is at breast cancer cell nucleus detection and localization figure.
Embodiment
Below will carry out detailed description to the preferred embodiments of the present invention with reference to accompanying drawing.The experimental technique of unreceipted actual conditions in the preferred embodiment, usually according to normal condition, or the condition of advising according to manufacturer.
This method is mainly utilized the autofluorescence characteristic of resveratrol, and utilization laser confocal microscope inspection resveratrol is characteristic distributions in the location of different target tissues.The molecular structure of resveratrol is as shown in Figure 1, and the result is as shown in Figure 2 to the resveratrol full wavelength scanner.
Embodiment 1, laser confocal microscope detect the location of resveratrol in Human umbilical vein endothelial cells
Get in Human umbilical vein endothelial cells to the living cells double dish, the final concentration that in nutrient culture media, adds resveratrol to resveratrol is 20 μ M, is 37 ℃, 100% saturated humidity in temperature then, and volume fraction is 5% CO
2Handled 1 hour in the incubator, get the Human umbilical vein endothelial cells that resveratrol is handled; With the pretreated endothelial cell of gained resveratrol add cell membrane red fluorescence probe (1,1'-dioctadecyl-3,3; 3'; 3'-tetramethylindocarbocyanine perchlorate, Dil) (available from green skies Bioisystech Co., Ltd) dissolves with DMSO; To final concentration be 10 μ M, be to handle under 25 ℃ of conditions 5 minutes in temperature; In excitation wavelength is 322nm, and receiving wavelength is to detect resveratrol fluorescence site under the 397nm condition, and the result is as shown in Figure 3.Be 549nm in excitation wavelength simultaneously, emission wavelength is the luminous position that detects Dil under the 565nm condition, and the result is as shown in Figure 4.Can know that by Fig. 3 20 μ M resveratrols are handled endothelial cell can detect resveratrol after 1 hour blue-fluorescence.Can know that by Fig. 4 red fluorescence and blue-fluorescence overlap, show that there be a small amount of the distribution in resveratrol at the cell membrane of Human umbilical vein endothelial cells.
The pretreated Human umbilical vein endothelial cells of resveratrol being added mitochondria fluorescence probe JC-1 (available from green skies Bioisystech Co., Ltd), uses DMSO to be dissolved to final concentration and be 5mg/mL, is processing 30 minutes under 25 ℃ of conditions in temperature; In excitation wavelength is 322nm, and the maximum wavelength that receives is to detect the resveratrol luminescence sites under the 405nm condition, is 514nm in excitation wavelength simultaneously, and emission wavelength is the luminous position that detects JC-1 under the 585nm condition, and the result is as shown in Figure 5.Can know that by Fig. 5 red punctate fluorescence is depicted as the position of JC-1, overlapping on a small quantity with the blue-fluorescence of resveratrol, show that resveratrol can be distributed in the mitochondria of Human umbilical vein endothelial cells.
The pretreated Human umbilical vein endothelial cells of resveratrol being added the nucleus fluorescence probe, be specially the fluorescence probe PI (available from Sigma company) of cell DNA, use DMSO to be dissolved to final concentration and be 1mg/mL, is to handle 10 minutes under 4 ℃ of conditions in temperature; In excitation wavelength is 322nm, and the maximum wavelength that receives is to detect the resveratrol luminous position under the 500nm condition, is 540nm in excitation wavelength simultaneously, and emission wavelength is the luminous position that detects PI under the 600nm condition, and the result is as shown in Figure 6.Can be known that by Fig. 6 red fluorescence is depicted as the position of nucleus fluorescence probe, no blue-fluorescence exists, and shows that resveratrol can not be positioned in the nucleus of Human umbilical vein endothelial cells.
In sum, resveratrol mainly is positioned the cytoplasm of endothelial cell in Human umbilical vein endothelial cells, and a small amount of distribution is arranged in cell membrane and mitochondria, in nucleus, does not see distribution.
Embodiment 2, laser confocal microscope detect resveratrol and locate liver cell
Get liver cell HepG2 and to the living cells double dish, cultivate, the final concentration that in nutrient culture media, adds resveratrol to resveratrol is 1 μ M, is 37 ℃, 100% saturated humidity in temperature then, and volume fraction is 5% CO
2Handled 1.5 hours in the incubator, get the liver cell that resveratrol is handled; The pretreated liver cell of gained resveratrol is added cell membrane red fluorescence probe Dil (available from green skies Bioisystech Co., Ltd), and using DMSO to be dissolved to final concentration is 5 μ M, is to handle 20 minutes under 25 ℃ of conditions in temperature; In excitation wavelength is 322nm, and receiving wavelength is to detect the resveratrol luminescence sites under the 350nm condition, and the result is as shown in Figure 7; Be 549nm in excitation wavelength simultaneously, emission wavelength is the luminous position that detects Dil under the 565nm condition, and the result is as shown in Figure 8.Can know that by Fig. 71 μ M resveratrol is handled liver cell can detect resveratrol after 1.5 hours blue-fluorescence.Can know that by Fig. 8 the red fluorescence and the blue-fluorescence of cell membrane red fluorescence probe overlap, show that resveratrol has distribution at cell membrane.
The pretreated liver cell of resveratrol being added mitochondria fluorescence probe JC-1 (available from green skies Bioisystech Co., Ltd), uses DMSO to be dissolved to final concentration and be 5mg/mL, is processing 60 minutes under 25 ℃ of conditions in temperature; In excitation wavelength is 322nm, and receiving wavelength is to detect the resveratrol luminescence sites under the 400nm condition, is 514nm in excitation wavelength simultaneously, and emission wavelength is the luminous position that detects JC-1 under the 585nm condition, and the result is as shown in Figure 9.By shown in Figure 9, red punctate fluorescence is depicted as mitochondria, and blue-fluorescence is a resveratrol, and visible two kinds of fluorescence exist overlapping on a small quantity, and being illustrated in hepatocellular mitochondria has resveratrol to distribute.
The pretreated liver cell of resveratrol being added the nucleus fluorescence probe, be specially the fluorescence probe PI (available from Sigma company) of cell DNA, use DMSO to be dissolved to final concentration and be 1mg/mL, is to handle 5 minutes under 4 ℃ of conditions in temperature; In excitation wavelength is 322nm, and receiving wavelength is to detect the resveratrol luminescence sites under the 405nm condition, is 540nm in excitation wavelength simultaneously, and emission wavelength is the luminous position that detects PI under the 600nm condition, and the result is shown in figure 10.Can know that by Figure 10 red fluorescence shows nucleus,, show that resveratrol is in hepatocellular nucleus distribution-free with the blue-fluorescence zero lap of resveratrol.
In sum, resveratrol mainly is positioned cytoplasm in liver cell HepG2, has a small amount of distribution, distribution-free in nucleus at cell membrane and mitochondria.
Embodiment 3, laser confocal microscope detect the location of resveratrol at breast cancer cell
Get breast cancer cell MDA-MB-231 and to the living cells double dish, cultivate, the final concentration that in nutrient culture media, adds resveratrol to resveratrol is 40 μ M, is 37 ℃, 100% saturated humidity in temperature then, and volume fraction is 5% CO
2Handled 2 hours in the incubator, get the tumour cell that resveratrol is handled; The breast cancer cell that the gained resveratrol is handled adds cell membrane red fluorescence probe Dil (available from green skies Bioisystech Co., Ltd), and using DMSO to be dissolved to final concentration is 8 μ M, is to handle 10 minutes under 25 ℃ of conditions in temperature; In excitation wavelength is 322nm, and receiving wavelength is to detect the resveratrol luminescence sites under the 500nm condition, and the result is shown in figure 11; Be 549nm in excitation wavelength simultaneously, emission wavelength is that the luminous position result of detection cell membrane fluorescence probe Dil under the 565nm condition is shown in figure 12.Can know that by Figure 11 40 μ M resveratrols are handled breast cancer cell can detect resveratrol after 2 hours blue-fluorescence.Can know by Figure 12, shown in the cell membrane of red fluorescence mark, have less with the blue-fluorescence of resveratrol and to overlap, show that resveratrol has distribution at the breast cancer cell film.
The tumour cell that resveratrol is handled adds mitochondria fluorescence probe JC-1 (available from green skies Bioisystech Co., Ltd), and uses DMSO to be dissolved to concentration and be 5mg/mL, be processing 45 minutes under 25 ℃ of conditions in temperature; In excitation wavelength is 322nm, and receiving wavelength is to detect the resveratrol luminescence sites under the 500nm condition, is 514nm in excitation wavelength simultaneously; Emission wavelength is the luminous position that detects JC-1 under the 585nm condition; The result is shown in figure 13, and red punctate fluorescence is depicted as mitochondria, and blue-fluorescence is a resveratrol; It is thus clear that it is more overlapping that two kinds of fluorescence exists, and shows that there is distribution in resveratrol in breast cancer cell.
The tumour cell that resveratrol is handled adds the nucleus fluorescence probe, is specially the fluorescence probe PI (available from Sigma company) of cell DNA, and use DMSO to be dissolved to final concentration and be 1mg/mL, be to handle 8 minutes under 4 ℃ of conditions in temperature; In excitation wavelength is 322nm, and receiving wavelength is to detect the resveratrol luminescence sites under the 500nm condition, is 540nm in excitation wavelength simultaneously, and emission wavelength is the luminous position that detects PI under the 600nm condition, and the result is shown in figure 14.Can know that by Figure 14 the nucleus by red fluorescence shows with the obvious zero lap of the blue-fluorescence of resveratrol, shows resveratrol distribution-free in breast cancer cell nuclear.
In sum, resveratrol mainly is positioned on cell membrane and the mitochondria a small amount of distribution is arranged on cell membrane in breast cancer cell, and in nucleus distribution-free.
Explanation is at last; Above embodiment is only unrestricted in order to technical scheme of the present invention to be described; Although through invention has been described with reference to the preferred embodiments of the present invention; But those of ordinary skill in the art should be appreciated that and can make various changes to it in form with on the details, and the spirit and scope of the present invention that do not depart from appended claims and limited.
Claims (7)
1. detect the method for resveratrol, it is characterized in that, may further comprise the steps in the target cell location:
A. target cell being used concentration is the resveratrol processing of 1～40 μ M, gets resveratrol and handles target cell;
B. step a gained resveratrol is handled target cell and add the organelle probe, get the target cell of organelle probe mark;
C. the target cell of step b gained organelle probe mark is observed the fluorescence position of resveratrol and organelle probe at laser confocal microscope, confirm the location of resveratrol target cell according to the overlapping situation of fluorescence.
2. according to the method for the said detection resveratrol of claim 1 in target cell location, it is characterized in that: said step a is that target cell is added concentration is in 1～40 μ M resveratrol, is 37 ℃, 100% saturated humidity in temperature, and volume fraction is 5% CO
2Incubator was cultivated 1 hour at least.
3. according to the method for the said detection resveratrol of claim 1 in the target cell location, it is characterized in that: said target cell is vascular endothelial cell, liver cell or tumour cell.
4. according to the method for the said detection resveratrol of claim 1 in the target cell location, it is characterized in that: among the said step b, said organelle probe is cell membrane fluorescence probe, mitochondria fluorescence probe or nucleus fluorescence probe.
5. according to the method for the said detection resveratrol of right claim 4 in the target cell location; It is characterized in that: said cell membrane fluorescence probe is a cell membrane red fluorescence probe; Said mitochondria fluorescence probe is the mitochondrial membrane potential fluorescence probe, and said nucleus fluorescence probe is the fluorescence probe of labeled cell DNA.
6. according to the method for the said detection resveratrol of claim 5 in the target cell location, it is characterized in that: the resveratrol excitation wavelength is 322nm among the said step c, and receiving wavelength is to detect under 350～500nm condition.
7. according to the method for the said detection resveratrol of claim 6 in the target cell location, it is characterized in that: said reception wavelength is 405nm.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103558164A (en) * | 2013-08-30 | 2014-02-05 | 黄宏南 | Method for simultaneously determining proanthocyanidins and resveratrol in wine |
| CN104529893A (en) * | 2014-12-30 | 2015-04-22 | 中国科学技术大学 | Novel quinoline dye capable of being used as Golgi apparatus organelle probe |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1624456A (en) * | 2003-12-04 | 2005-06-08 | 中国农业大学 | Method of fast detecting cis-trans verlkaol and its glucoside |
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| 周曦 等: "SIRT1在白藜芦醇抑制血管内皮细胞氧化应激损伤中的作用及机制研究", 《中国西部第六届营养与健康学术会议》, 17 August 2011 (2011-08-17) * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103558164A (en) * | 2013-08-30 | 2014-02-05 | 黄宏南 | Method for simultaneously determining proanthocyanidins and resveratrol in wine |
| CN103558164B (en) * | 2013-08-30 | 2016-04-13 | 黄宏南 | A kind of method of Simultaneously test grape wine procyanidins and resveratrol |
| CN104529893A (en) * | 2014-12-30 | 2015-04-22 | 中国科学技术大学 | Novel quinoline dye capable of being used as Golgi apparatus organelle probe |
| CN104529893B (en) * | 2014-12-30 | 2016-08-24 | 中国科学技术大学 | One class can be as the quinoline dye of golgi cell device probe |
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