CN113105521B - Application of rhizoma paridis saponin I in detection of lysosome - Google Patents
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The invention discloses a method for detecting paris polyphylla saponin I in lysosomeApplication is carried out. Experiments show that the paris polyphylla saponin I has the function of specifically targeting lysosomes, and in order to facilitate observation and detection, the paris polyphylla saponin I and a fluorescent dye are further synthesized into a red fluorescent probe named as Polyphyllin I-Cy3, and the structural formula of the red fluorescent probe is shown as (II). The Polyphenyllin I-Cy3 red fluorescent probe synthesized by the invention can permeate cell membranes, can be used for specific fluorescent staining of live cell lysosomes, and has the advantages of good targeting property, high detection speed, good repeatability, convenience in use, no cytotoxicity and the like.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to application of paris polyphylla saponin I in lysosome detection.
Background
Lysosomes (lysosomes) are single-layer membrane-coated saccular structures, have the diameter of about 0.025-0.8 microns, contain a plurality of hydrolytic enzymes, are organelles with certain morphological structures and important physiological functions in biological cells and are special for decomposing various exogenous and endogenous macromolecular substances. Since Cristian de Duve et al discovered lysosomes in rat hepatocytes in 1955, the lysosome research involved various aspects and was found to be closely related to the development of many diseases such as metabolic diseases, inflammation, tumors, and immunity. Therefore, it is of great interest to develop probes that can accurately label and visualize lysosomes.
The Cy3 dye is an amino reactive dye with a molecular weight of 668.86 useful for immunocytochemistry applications, labeling nucleic acids, and the like. The Cy3 fluorescent dye, excited in the 532nm band, emitted a bright orange-red light, generally described as red, as indistinguishable to the naked eye.
Paris polyphylla saponin I (Polyphyllin I, PD) is a small molecular monomer with molecular weight of 855.02, is extracted from rhizome of Paris yunnanensis Franch belonging to Liliaceae, and has a structural formula shown in formula (I):
although the potential anti-tumor activity of the paris polyphylla saponin I is reported earlier, no report that the paris polyphylla saponin I can target lysosomes exists so far.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide the application of the paris polyphylla saponin I in lysosome detection.
The invention also aims to provide the application of the paris polyphylla saponin I in preparing a fluorescent probe for detecting lysosomes.
It is still another object of the present invention to provide a red fluorescent probe targeted to lysosomes.
The invention further aims to provide the application of the red fluorescent probe targeting the lysosome in lysosome detection.
The purpose of the invention is realized by the following technical scheme:
application of rhizoma paridis saponin I in detection of lysosome is provided.
The structural formula of the paris polyphylla saponin I is shown as the formula (I):
the lysosome is lysosome in living cells; preferably lysosomes in living cells in vitro.
The cells are preferably liver cancer cells; more preferably HepG2 cells.
The application of the paris polyphylla saponin I in preparing a fluorescence probe for detecting lysosomes.
The paris polyphylla saponin I has the function of specifically targeting lysosomes, and can be further synthesized into a probe with fluorescence characteristics with fluorescent dyes for convenient observation (such as equipment such as a flow cytometer, a confocal microscope or a fluorescence microplate reader).
The fluorescent dye is red fluorescent dye; preferably a Cy3 fluorescent dye; more preferably Cy 3-COOH.
A red fluorescent probe targeting lysosomes is named Polyphylllin I-Cy3, and the structural formula of the red fluorescent probe is shown as the formula (II):
the method for synthesizing the red fluorescent probe targeting the lysosome comprises the following steps:
dissolving the paris polyphylla saponin I in Dimethylformamide (DMF), adding Cy3-COOH, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 4-Dimethylaminopyridine (DMAP), stirring for reaction at 50 +/-5 ℃, decompressing and carrying out rotary evaporation to remove the solvent after the reaction is finished, washing, recrystallizing and drying to obtain the red fluorescent probe (Polyphyllin I-Cy3) for targeting lysosomes.
The mole ratio of the paris polyphylla saponin I, Cy3-COOH, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 4-Dimethylaminopyridine (DMAP) is 1:1.2:1.2: 0.1.
The dosage of the dimethylformamide is calculated according to the proportion of 0.1-0.2 mL of dimethylformamide per milligram (mg) of paris polyphylla saponin I.
The stirring reaction time is 5-8 h; preferably 6 h.
The conditions for removing the solvent by reduced pressure rotary evaporation are as follows: the temperature is 40 ℃ and the rotating speed is 120 r/min.
The washing is acetone washing; preferably, the washing is carried out 3 or more times with acetone.
The recrystallization is carried out by adopting ethanol.
The drying is vacuum drying.
The red fluorescent probe targeting lysosomes is applied to lysosome detection.
The application is for non-disease diagnosis and treatment purposes.
The lysosome is lysosome in living cells; preferably lysosomes in living cells in vitro.
The cells are preferably liver cancer cells; more preferably HepG2 cells.
The safe concentration range of the red fluorescent probe is below 1000 nmol/L; preferably 10-1000 nmol/L; further preferably 10 to 100 nmol/L; more preferably 50 nmol/L.
The red fluorescent probe targeting lysosomes is applied to the preparation of the lysosome specific fluorescent probe.
Compared with the prior art, the invention has the following advantages and effects:
(1) the synthesized Polyphenyllin I-Cy3 red fluorescent probe is a novel lysosome specific fluorescent labeling probe, can permeate cell membranes, and can be used for living cell lysosome specific fluorescent staining.
(2) The Polyphenyllin I-Cy3 probe synthesized by the invention can highly select target lysosome and effectively mark living cells at nanomolar concentration.
(3) The Polyphenyllin I-Cy3 probe synthesized by the invention has the advantages of good targeting property, high detection speed, good repeatability, convenient use, no cytotoxicity and the like, realizes lysosome fluorescence labeling under low concentration, and can detect through flow type, confocal and the like.
Drawings
FIG. 1 is a synthetic scheme of Polyphyllin I-Cy 3.
FIG. 2 is a HPLC peak plot of Polyphenyllin I reacted with Cy3 for 0 h.
FIG. 3 shows HPLC peaks of Polyphenyllin I reacted with Cy3 for 6 hours (peaks appearing around 12 minutes and 35 seconds in the figure are the peaks of Polyphenyllin I-Cy3 products).
FIG. 4 is an excitation spectrum and an emission spectrum of Polyphyllin I-Cy 3.
FIG. 5 is a graph showing the effect of Polyphenyllin I-Cy3 co-localization with lysosomes in cells (lysosome staining with the commercial lysosome probe Lyso-Tracker Green, lysosomes appear Green fluorescence; Polyphenyllin I-Cy3 stains lysosomes in HepG2 cells, and appears red fluorescence).
FIG. 6 is a graph showing the effect of no co-localization of Polyphenyllin I-Cy3 to mitochondria and autophagosomes in cells (commercial Mito-Tracker Green stained lysosomes from HepG2 cells, showing Green fluorescence; autophagosomes labeled with LC3-GFP Green fluorescent protein, showing Green fluorescence; Polyphenyllin I-Cy3 stained lysosomes from cells, showing red fluorescence).
FIG. 7 is a graph showing the results of activity measurements of cells treated with Polyphyllin I-Cy 3.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.
Example 1 Synthesis of Polyphyllin I-Cy3
1.1 Experimental procedures
Synthesis of Polyphylllin I-Cy 3: weighing 5mg of paridis saponin I (Polyphenyllin I) and dissolving in 0.5mL of dimethylformamide (DMF, Shanghai Tantake technology Co., Ltd.), adding 3.45mg of Cy3-COOH (Rexiliny organism), 1.34mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC, Bizhi medicine) and 71.47 μ g of 4-dimethylaminopyridine (DMAP, Adama reagent Co., Ltd.) and dissolving completely (paridis saponin I: Cy 3-COOH: EDC: DMAP ═ 1:1.2:1.2:0.1 (molar ratio)) and stirring and reacting at 50 ℃ for 6h, removing the solvent by rotary evaporator (model YRE2000E, consolidation to Wajke instruments Co., Ltd.) and rotary evaporating at 40 ℃ and 120r/min under reduced pressure, then washing with acetone 3 times, recrystallizing with ethanol, vacuum drying to obtain the product, hangzhou Xinqiao Biotechnology Co., Ltd.) verified the synthesized product. The HPLC detection method comprises the following steps:
the apparatus used was an Agilent 1100HPLC &1946D MS, the column was Waters XTerra MS C18 (3.0X 100mm, 3.5 μm);
liquid phase conditions: the mobile phase is acetonitrile-water (containing 10mM ammonium acetate), and is eluted at 70:30 (volume ratio) isocratic with the flow rate of 0.5mL/min, the column temperature of 25 ℃ and the injection volume of 10 mu L;
mass spectrum conditions: ESI ion source, positive ion mode, drying gas temperature 350 ℃, drying gas flow 10L/min, atomization gas pressure 40 psi.
1.2 results and conclusions
Since polypylin I does not have fluorescence per se, and detection is inconvenient by using equipment such as a flow cytometer, a confocal microscope or a fluorescence microplate reader, the method firstly introduces Cy3 red fluorescence to synthesize a polypylin I-Cy3 red fluorescence probe (figure 1), and the specific synthetic steps are shown in the experimental steps. After the synthetic product is obtained, the synthetic effect is detected by adopting an HPLC technology, and the result proves that the synthesis of the Polyphenyllin I-Cy3 is successful by comparing the HPLC peak (figure 2) of 0h at the beginning of the reaction and the peak of the Polyphenyllin I-Cy3 (figure 3) which appears about 12 minutes and 35 seconds after the 6h reaction HPLC peak. Polyphyllin I-Cy3, molecular weight 1505.86, excitation spectrum and emission spectrum see FIG. 4. laser lines at 532nm with emission wavelength of 577nm can be used.
Example 2 Polyphenyllin I-Cy3 targeting lysosomes
2.1 Experimental procedure:
2.1.1 cell culture
Liver cancer HepG2 cells were purchased from American Type Culture Collection (ATCC). At 37 deg.C, 5% CO2The culture was carried out in a DMEM medium (Invitrogen) containing 10% (v/v) fetal bovine serum (Gibco) and 1% (w/v) penicillin/streptomycin (Invitrogen). When the cells are subcultured, the original culture medium is removed, washed twice with PBS buffer, cells are digested with trypsin (Gibco Co.), and left to stand in a cell culture box for about 1min until the cells are detached, and the medium is added to stop the digestion. The cells were gently pipetted down, centrifuged at 216g for 3min, the supernatant removed, and the cells were resuspended in fresh DMEM medium containing 10% (v/v) fetal bovine serum, 1% (w/v) penicillin/streptomycin at a 1:4 passaging ratio. The cells for confocal observation were all cultured in a confocal dish (NEST Co.).
2.1.2 plasmid construction and transfection
LC3-GFP green fluorescent protein indicating autophagosome is obtained by transfecting liver cancer HepG2 cells with pEGFP-LC3b plasmid (purchased from vast Lingzi). The specific transfection steps are as follows: HepG2 cells were seeded at 50% density into a petri dish (middle dish) and cultured for 12 h; the pEGFP-LC3b plasmid (0.5. mu.g) was mixed with a cationic transfection reagent Lipo3000 (2. mu.L, Invitrogen corporation) and P3000 (2. mu.L, Invitrogen corporation) in 200. mu.L of Opti-MEM medium (Invitrogen corporation), and the mixture was allowed to stand for 20 min; changing the culture medium of HepG2 cells into an Opti-MEM culture medium, adding the mixed Opti-MEM culture medium, and placing the mixed Opti-MEM culture medium in an incubator for culturing for 6 hours; the transfected cells were digested by changing fresh DMEM medium containing 10% (v/v) fetal bovine serum and 1% (w/v) penicillin/streptomycin for 48h, and seeded at 50% density in confocal culture dishes for subsequent experimental observation.
2.1.3 Probe staining
Polyphyilin I-Cy3 staining: polyphyllin I-Cy3 was added to DMEM medium containing 10% (v/v) fetal bovine serum, 1% (w/v) penicillin/streptomycin to prepare 50nM working solution, which was preincubated at 37 ℃. The cells obtained by 2.1.2 transfection (used for observing the co-localization condition of the Polyphenyllin I-Cy3 and mitochondria) and HepG2 cells (used for observing the co-localization condition of the Polyphenyllin I-Cy3, lysosomes and autophagosomes) are removed from the cell culture solution, and the prepared and pre-incubated Polyphenyllin I-Cy3 staining working solution at 37 ℃ is added to be co-incubated with the cells at 37 ℃ for 5-20 minutes (the experiment is co-incubated for 15 min).
② Lyso-Tracker Green (from Biyunyan day) staining: Lyso-Tracker Green was added to a DMEM medium containing 10% (v/v) fetal bovine serum and 1% (w/v) penicillin/streptomycin at a ratio of 1:13333 to 1:20000 to a final concentration of 50 to 75 nM. After mixing, pre-incubation was carried out at 37 ℃. Removing the cell culture solution from the HepG2 cells, adding the prepared Lyso-Tracker Green staining working solution pre-incubated at 37 ℃, and incubating the cells for 5-60 minutes at 37 ℃ (the incubation time is 15min in the experiment).
③ Mito-Tracker Green (from Biyun sky) staining: adding a small amount of 1mM Mito-Tracker Green stock solution into a DMEM medium containing 10% (v/v) fetal bovine serum and 1% (w/v) penicillin/streptomycin according to the proportion of 1: 5000-1: 50000 to enable the final concentration to be 20-200 nM, uniformly mixing to obtain the Mito-Tracker Green working solution, and pre-incubating at 37 ℃ before use. The cells obtained by 2.1.2 transfection were removed from the cell culture medium, added with the Mito-Tracker Green staining working solution pre-incubated at 37 ℃ and incubated with the cells at 37 ℃ for 15-45 minutes (15 min in the experiment).
2.1.4 confocal laser observation
The staining solution was removed, and fresh cell culture solution was added, followed by observation by a confocal laser microscope (zeiss).
2.1.5 WST-1 assays for cellular Activity
As an analog of MTT, WST-1, when mixed with an electron coupling reagent, can be reduced by certain dehydrogenases in the mitochondria to produce the yellow-orange formazan compound. Generally, the more rapidly the cells proliferate, the darker the color of the detection reagent. The specific operation is as follows: digesting HepG2 cells in a good state by pancreatin, collecting and counting, paving 3000 cells per well in a 96-well plate, and culturing overnight in an incubator; adding Polyphyllin I-Cy3 to the adherent cells to make the final concentration of 0, 10, 100, 1000nM respectively, and culturing for 24 and 48h respectively; firstly, preparing a WST-1 solution (Biyunyan) according to an instruction, and preparing a WST-1 mixed solution from the WST-1 solution and a culture medium in a ratio of 1:10, namely 10 mu L of the WST-1 solution and 100 mu L of the culture medium; after the cells were treated with Polyphyllin I-Cy3, the original medium was removed and 100. mu.L of the prepared mixed solution of WST-1 was added to each well. A blank control with a corresponding amount of the mixed solution of WST-1 added but no cells is added to the blank wells of the same plate; placing the incubator at 37 ℃ for 2h, detecting the absorbance value under 450nm by using an enzyme-labeling instrument, and finally analyzing the data.
2.2 Experimental results and conclusions:
polyphyllin I-Cy3 can specifically target lysosome, has excellent selectivity, and can be used for labeling and tracing lysosome in living cells. Before imaging, cells are incubated by a Polyphyllin I-Cy3 probe, and the specific concentration and incubation time of the probe can be searched according to the self experiment condition and the specific cell type to achieve a satisfactory staining effect. The staining effect of Polyphylllin I-Cy3 in live cells HepG2 is shown in FIGS. 5 and 6. Good co-localization of lysosomes labeled with Polyphyllin I-Cy3 and a commercial Lyso-Tracker Green probe (Byunnan) was observed by confocal experiments at a final concentration of 50nM and a duration of action of 15min (FIG. 5). Furthermore, we can observe that polypylin I-Cy3 does not co-localize with other organelles, such as mitochondria and autophagosomes, by confocal focusing (fig. 6). The above results indicate that the Polyphenyllin I-Cy3 probe synthesized by the present invention is indeed capable of specifically targeting lysosomes.
To evaluate the safety of the Polyphenyllin I-Cy3 probe, we evaluated the effect of different concentrations of Polyphenyllin I-Cy3 on HepG2 cell activity by WST-1 (Biyunyan) assay. As shown in FIG. 7, 1000nM of Polyphyllin I-Cy3 treated cells for 48h had no significant effect on cell viability. The results prove that the recommended concentration of the probe is 50nM which is safe and has no obvious influence on cell growth.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (6)
1. A red fluorescent probe targeted to lysosomes, characterized in that: under the name of Polyphyllin I-Cy3, the structural formula is shown in formula (II):
2. the method of synthesizing a red lysosomal-targeting fluorescent probe of claim 1, comprising the steps of:
dissolving the paris polyphylla saponin I in dimethylformamide, adding Cy3-COOH, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, stirring and reacting at 50 +/-5 ℃, decompressing and performing rotary evaporation to remove the solvent after the reaction is finished, washing, recrystallizing and drying to obtain the red fluorescent probe for targeting lysosomes.
3. The method of synthesizing a red lysosomal-targeting fluorescent probe according to claim 2, wherein:
the mole ratio of the paris polyphylla saponin I, the Cy3-COOH, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the 4-dimethylaminopyridine is 1:1.2:1.2: 0.1;
the stirring reaction time is 5-8 h;
the conditions for removing the solvent by reduced pressure rotary evaporation are as follows: the temperature is 40 ℃, and the rotating speed is 120 r/min;
the washing is acetone washing;
the recrystallization is to adopt ethanol for recrystallization;
the drying is vacuum drying.
4. The use of a red lysosomal-targeting fluorescent probe of claim 1 for detection of lysosomes, characterized in that: the lysosome is the lysosome in vitro living cells.
5. Use according to claim 4, characterized in that: the safe concentration range of the red fluorescent probe is below 1000 nmol/L.
6. Use of the red lysosomal-targeting fluorescent probe of claim 1 for the preparation of a lysosomal-specific fluorescent probe.
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