CN104977277B - It is a kind of to detect the nano vesicle of intracellular wild type and variation p53 albumen simultaneously - Google Patents
It is a kind of to detect the nano vesicle of intracellular wild type and variation p53 albumen simultaneously Download PDFInfo
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- CN104977277B CN104977277B CN201510423379.1A CN201510423379A CN104977277B CN 104977277 B CN104977277 B CN 104977277B CN 201510423379 A CN201510423379 A CN 201510423379A CN 104977277 B CN104977277 B CN 104977277B
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Abstract
The present invention disclose it is a kind of can and meanwhile detect intracellular wild type and make a variation p53 albumen nano vesicle, the vesica internal package has the nanogold of energy specific recognition wild-type p 53 protein, while vesicle surface is modified with the different p53 protein antibodies of resistance of marked by fluorescein isothiocyanate.The advantage of the invention is that：(1) nano vesicle high specific identification different type tumor suppressor protein p53, improves specificity and the sensitivity of detection.(2) using plasma resonance image-forming and Imaging-PAM, after vesica enters cell, in situ imaging is carried out using microscope to cell, is detected while realizing unicellular aspect wild type and variation p53 albumen.(3) change of intracellular p53 protein expressions carries out in-situ monitoring under the nano vesicle that designs of the present invention can be acted on medicine, has carried to study intracellular physiological process of its participation and has supplied new method.
Description
Technical field
The present invention relates to cytokines imaging analysis techniques, details in a play not acted out on stage, but told through dialogues microtechnic and field of nano material preparation, specifically
Say, be related to it is a kind of can and meanwhile detect intracellular wild type and variation p53 albumen nano vesicle and its application.
Background technology
In recent years, plasma nano particle is attracted wide attention, and these nano-particles have special optics and thing
Rationality matter, can effectively absorb and scatter visible light, cause metal surface plasma to resonate.Have benefited from dark field microscope and plasma
The development of resonance body Rayleigh Scattering Spectra, plasma nano particle has been employed successfully in bio-sensing, single particle analysis, medicine
Multiple research fields such as thing transport.Research shows that plasma nano probe has become research life entity especially cell
Powerful, available for cell imaging, cell surface identification, wears film transport etc..
Tumor suppressor protein p53 can by cell cycle carry out regulation and control trigger Apoptosis, suppress tumour generation and
Development.In most of tumour cell, the structure of p53 albumen is undergone mutation, and activity is suppressed.Therefore to different types of
P53 carries out intracellular in-situ study and detection, and the diagnosis, treatment and monitoring in real time to tumour have great importance.
At present, the method for a variety of detection p53 albumen has been suggested.Wherein traditional detection method is by immunohistochemistry
Method (IHC) or enzyme linked immunoassay (ELISA) detection serum tumor suppress the content of albumen.Immunohistochemical method application
Than wide, but its major defect is that dyeing difference is larger between different batches, poor repeatability.Enzyme linked immunoassay method is utilized
The sandwich reaction detection target protein of antigen-antibody, with preferable stability, but this method complex operation step, sensitivity is not
It is high.The sensitivity of electrochemical nano method for sensing is high, reproducible, but this method generally uses serum or cell extract conduct
Object is analyzed, therefore in-situ study and the detection of p53 albumen can not be realized, it is impossible to the tool of p53 albumen in unicellular aspect is obtained
Body position and distribution situation.
The content of the invention
First purpose of the present invention be to provide it is a kind of can be while detecting that intracellular wild type and variation p53 albumen are received
Rice vesica.
Second object of the present invention be to provide it is a kind of can be while detecting that intracellular wild type and variation p53 albumen are received
The application of rice vesica.
To realize first purpose of the invention, the present invention discloses following technical scheme：One kind can detect cell infield simultaneously
The nano vesicle of raw type and variation p53 albumen, it is characterised in that the vesica internal package has can specific recognition wild type
The nanogold of p53 albumen, while vesicle surface is modified with the different p53 protein antibodies of resistance of marked by fluorescein isothiocyanate.
As a preferred scheme, the nanogold of the energy specific recognition wild-type p 53 protein is by containing wild type
The double stranded DNA solutions of p53 protein recognition sites and 55-65nm nano-Au solution, which are mixed, to be obtained.The particle diameter of nanogold is excellent
Elect 60nm as.
To realize second purpose of the invention, the present invention discloses following technical scheme：Nano vesicle p53 eggs in living cells
Application in white image checking, and, nano vesicle answering in the image checking reagent of p53 albumen in living cells is prepared
With.
The vesica enters after cell, discharges the nanogold of inside, the combination of nanogold and intracellular wild-type p 53 protein
The aggregation of nanogold can be triggered, the aggregation extent of nanogold caused by the wild-type p 53 protein of various concentrations is different, its scattered light
Wavelength it is different, details in a play not acted out on stage, but told through dialogues scattering spectrum is also what is changed.Therefore, nanogold scattered light face can be observed by dark field microscope
The change of color simultaneously gathers its scattering spectrum, so as to be characterized to the aggregation extent of nanogold, and then obtains intracellular wild type
The distributed intelligence of p53 albumen.
Meanwhile, FITC (fluorescein isothiocynate) the mark different p53 protein antibodies of resistance of vesicle surface modification can be recognized
Intracellular variation p53 albumen, its fluorescence signal intensity is directly proportional to the concentration of intracellular variation p53 albumen.Therefore, it can pass through
Fluorescence microscope obtains cell fluorescent images, so that the distribution to intracellular variation p53 albumen is observed and characterized.
The advantage of the invention is that：(1) for operated in accordance with conventional methods complexity, specificity and not high repeatability the problem of, this
Method provides the nano vesicle that a kind of high specific recognizes different type tumor suppressor protein p53, improves the special of detection
Property and sensitivity.(2) the problem of lacking for p53 albumen monolayer surface analysis means, using plasma resonance of the present invention
Imaging and Imaging-PAM, after vesica enters cell, carry out in situ imaging to cell using microscope, realize slender
Detected while born of the same parents' aspect wild type and variation p53 albumen.(3) under the nano vesicle that the present invention is designed can be acted on medicine
The changes of intracellular p53 protein expressions carries out in-situ monitoring, has carried to study intracellular physiological process of its participation and has supplied new method.
Brief description of the drawings
Fig. 1 is while detects preparation and its fundamental diagram of the nano vesicle of intracellular wild type and variation p53 albumen.
Fig. 2 is various concentrations nutlin-3 (A1-5:0,B1-5:62.5μg mL-1,C1-5:125μg mL-1,D1-5:
250 μ g mL-1) treated HeLa cells incubated 2 hours with nano vesicle and DAPI after micro-image (1:Darkfield image,
2:Bright field image, 3:DAPI contaminates nucleus, 4:FITC marks the fluoroscopic image of the different p53 protein antibodies of resistance, 5:Darkfield image
Details is amplified), i1-i12:The details in a play not acted out on stage, but told through dialogues scattering spectrum of intracellular corresponding nanogold.
Label in Fig. 1 is respectively：
1st, nano vesicle；2nd, nanogold；3rd, the hendecanal；
4th, FITC marks the different protein antibodies of p 53 of resistance；5th, cell membrane.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.Experimental method used in following embodiments for example without
Specified otherwise, is conventional method.Material, reagent used etc. in following embodiments, unless otherwise specified, can be from business way
Footpath is obtained.It should be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.
Embodiment 1., which is synthesized, can detect the nano vesicle of intracellular wild type and variation p53 albumen simultaneously
(1) according to existing document, synthesis 13nm gold sizes are used as seed.By 50mL HAuCl4(0.01%) solution adds circle
Boiling is heated in the flask of bottom, 5mL sodium citrate solutions (38.8mM) is then quickly added into and continues stirring 30 minutes, until
Solution becomes red, and obtained 13nm gold, which is planted, is used to prepare nanogold of the particle diameter in 60nm or so.
(2) synthesis 60nm nanogold is planted using gold.1mL gold is planted into solution and 100 μ L NH2OHHCl (0.2M) is mixed
25mL is diluted to pure water afterwards, afterwards by 3.0mLHAuCl4(0.01%) it is slowly added dropwise in above-mentioned mixed solution, persistently stirs
30 minutes are mixed until solution becomes kermesinus.By the centrifugation of obtained nano-Au solution, with pure water, preserved under the conditions of 4 DEG C.
(3) nanogold of energy specific recognition wild-type p 53 protein is synthesized with 60nm nanogold.10 μ L are contained wild
The double stranded DNA solutions (100 μM) of type p53 protein recognition sites are mixed with 1mL nano-Au solutions, are stirred at room temperature overnight, so
0.1mL PBSs (NaCl containing 2M) are added dropwise to the nanogold for the DNA modification stablized in above-mentioned solution afterwards.Will
Above-mentioned solution centrifugal is simultaneously washed twice with PBS, and the energy specific recognition that DNA functionalization is obtained in 1mLPBS is finally dispersed in again
The nanogold of wild-type p 53 protein.
(4) nano vesicle of intracellular wild type and variation p53 albumen can be detected simultaneously by preparing.By what is prepared in (3)
The nano-Au solution (20 μ L) of DNA modification is mixed with 20 μ L serum free mediums；In addition by 1.0 μ L Lipo-2000 liposomes with
20 μ L serum free mediums are mixed.After standing 10 minutes at room temperature, above two solution is mixed and then is incubated at room temperature 20 points
Clock, obtains being enclosed with the liposome of nanogold.Then the 1.0 μ L hendecanals are added into above-mentioned solution, reacts 10 minutes, connect at room temperature
The addition 1mg FITC mark different p53 protein antibodies of resistance, are stood overnight at 4 DEG C.Washed above-mentioned solution centrifugal and with PBS
Wash, be then dispersed in 60 μ L serum free mediums, obtain nano vesicle.
Embodiment 2. utilizes nano vesicle, and the distribution to intracellular p53 albumen carries out in situ imaging and detection
By the use of HeLa cervical cancer tumer lines as model, first by HeLa cells (1.0mL, 1 × 106mL-1) plant in copolymerization
In burnt culture dish, cultivate 24 hours.Then 60 μ L nano vesicles are added into culture dish, incubated 2 hours at 37 DEG C.Washed with PBS
Wash culture dish and submerge cell, then in dark field microscope and fluorescence microscopy Microscopic observation.Cell imaging is referring to Fig. 2.
It was found that in tumour cell HeLa, the color of nanogold is green in darkfield image, does not significantly build up, illustrates wild type p53
Content it is relatively low；And FITC fluorescence signal is stronger, illustrate that variation p53 content is higher.With nutlin-3 drug solutions (250 μ
g mL-1) to cell (1.0mL, 1 × 106mL-1) pre-processed, cell and medicine are co-cultured into 48h, 60 μ L is then added and receives
Rice vesica, use in micro- sem observation, the cell that discovery drug-treated is crossed, the color of nanogold is obvious after incubating 2 hours at 37 DEG C
It is partially orange red, and FITC fluorescence signals are substantially reduced, and illustrate that the activity of intracellular wild type p53 increases and the activity for the p53 that makes a variation
It is suppressed.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (5)
1. a kind of can detect the nano vesicle of intracellular wild type and variation p53 albumen simultaneously, it is characterised in that in the vesica
Portion is enclosed with the nanogold of energy specific recognition wild-type p 53 protein, while vesicle surface is modified with fluorescein isothiocynate mark
The different p53 protein antibodies of resistance of note.
2. it is according to claim 1 it is a kind of can detect simultaneously intracellular wild type and variation p53 albumen nano vesicle, its
It is characterised by, the nanogold of the energy specific recognition wild-type p 53 protein is by containing wild-type p 53 protein recognition site
Double stranded DNA solutions and 55-65nm nano-Au solution, which are mixed, to be obtained.
3. it is according to claim 2 it is a kind of can detect simultaneously intracellular wild type and variation p53 albumen nano vesicle, its
It is characterised by, the particle diameter of the nanogold is 60nm.
4. application of the nano vesicle described in claim 1 in living cells in the image checking of p53 albumen.
5. application of the nano vesicle in the image checking reagent of p53 albumen in living cells is prepared described in claim 1.
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| DE102017005543A1 (en) * | 2017-06-13 | 2018-12-13 | Forschungszentrum Jülich GmbH | Method for detecting extracellular vesicles in a sample |
| CN107329249B (en) * | 2017-08-28 | 2023-03-24 | 重庆三峡医药高等专科学校 | Unicellular drug delivery and SPR detection experimental apparatus |
| CN107656059B (en) * | 2017-09-26 | 2019-06-18 | 中南大学 | A kind of Fluorescent detector and its preparation method and application for p53 albumen |
| CN110387416B (en) * | 2018-04-16 | 2022-06-17 | 华东理工大学 | Nano-gold dual-probe system with controllable polymerization regulation function and application thereof |
| CN110346334B (en) * | 2018-12-27 | 2022-02-11 | 华东理工大学 | Nano-gold dual-probe system based on DNA coding circulation program |
| CN114796521B (en) * | 2021-01-18 | 2023-05-30 | 华东理工大学 | Molecularly imprinted nanostructure capable of specifically recognizing PD-L1 and application thereof |
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