CN104977277A

CN104977277A - Nanometer vesica capable of detecting wild type p53 protein and variant p53 protein in cells simultaneously

Info

Publication number: CN104977277A
Application number: CN201510423379.1A
Authority: CN
Inventors: 龙亿涛; 钱若灿; 曹玥
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2015-07-17
Filing date: 2015-07-17
Publication date: 2015-10-14
Anticipated expiration: 2035-07-17
Also published as: CN104977277B

Abstract

The invention discloses a nanometer vesica capable of detecting wild type p53 protein and variant p53 protein in cells simultaneously. Nanogold capable of specifically recognizing wild type p53 protein wraps inside the vesica, and an antibody resisting variant p53 protein marked by fluorescein isothiocyanate decorates the surface of the vesica. The nanometer vesica has the advantages that (1) the nanometer vesica recognizes different types of tumor suppressor proteins p53 with high specificity, and the detection specificity and the sensitivity are improved; (2) plasma resonance imaging and fluorescence imaging technologies are adopted, after the vesica enters the cell, the cell is subjected to in-situ imaging by adopting a microscope, and wild type p53 protein and variant p53 protein on the single-cell layers are detected simultaneously; (3) the nanometer vesica designed according to the technical scheme is capable of carrying out in-situ monitoring on variation of expression of p53 protein in the cell under the action of medicine, and a novel method is provided for researching physiological process in the cell participated by the vesica.

Description

A kind of nano vesicle that simultaneously can detect wild type and variation p53 albumen in cell

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, relate to a kind of nano vesicle and the application thereof that can detect wild type and variation p53 albumen in cell simultaneously.

Background technology

In recent years, plasma nano particle attracts wide attention, and these nano particles have special optics and physical property, effectively can absorb and scatter visible light, cause metal surface plasma to resonate.Have benefited from the development of dark field microscope and plasma resonance Rayleigh Scattering Spectra, plasma nano particle has been employed successfully in multiple research fields such as bio-sensing, single particle analysis, medicament transport.Research shows, plasma nano probe has become the powerful of research life entity especially cell, can be used for cell imaging, cell surface identification, wears film transport etc.

Tumor suppressor protein p53 carries out regulation and control trigger cell apoptosis by cell cycle, the generation of Tumor suppression and development.In most of tumour cell, the structure of p53 albumen is undergone mutation, and activity is suppressed.Therefore in-situ study and detection in cell are carried out to dissimilar p53, the diagnosis of tumour, treatment and Real-Time Monitoring are had great importance.

At present, the method for multiple detection p53 albumen is suggested.Wherein traditional detection method is the content being detected serum tumor Profilin by immunohistochemical method (IHC) or enzyme linked immunoassay (ELISA).Immunohistochemical method Application comparison is extensive, but its major defect is that between different batches, dyeing difference is comparatively large, poor repeatability.Enzyme linked immunoassay method utilizes the sandwich reaction detection target protein of antigen-antibody, has good stability, but the method complex operation step, sensitivity is not high.Electrochemical nano method for sensing is highly sensitive, reproducible, but the method adopts serum or cell extract as analytic target usually, therefore cannot realize in-situ study and the detection of p53 albumen, particular location and the distribution situation of p53 albumen in unicellular aspect cannot be obtained.

Summary of the invention

First object of the present invention is to provide a kind of nano vesicle that simultaneously can detect wild type and variation p53 albumen in cell.

Second object of the present invention is to provide a kind of application that simultaneously can detect the nano vesicle of wild type and variation p53 albumen in cell.

For realizing the present invention's first object, the present invention discloses following technical scheme: a kind of nano vesicle that simultaneously can detect wild type and variation p53 albumen in cell, it is characterized in that, described vesica internal package has the nm of gold of energy specific recognition wild-type p 53 protein, and vesicle surface is modified with the different p53 protein antibodies of resistance of marked by fluorescein isothiocyanate simultaneously.

As a preferred version, the nm of gold of described energy specific recognition wild-type p 53 protein is by obtaining containing the double stranded DNA solutions of wild-type p 53 protein recognition site and the nano-Au solution mix and blend of 55-65nm.The particle diameter of nm of gold is preferably 60nm.

For realizing the present invention's second object, the present invention discloses following technical scheme: nano vesicle p53 albumen in living cells image checking in application, and, the application of nano vesicle in the image checking reagent preparing p53 albumen in living cells.

After this vesica enters cell, discharge inner nm of gold, in nm of gold and cell, the combination of wild-type p 53 protein can cause the gathering of nm of gold, the aggregation extent of the nm of gold that the wild-type p 53 protein of variable concentrations causes is different, the wavelength of its scattered light is different, and details in a play not acted out on stage, but told through dialogues scattering spectrum is also vicissitudinous.Therefore, observe the change of nm of gold scattered light color by dark field microscope and gather its scattering spectrum, thus the aggregation extent of nm of gold is characterized, and then obtaining the distributed intelligence of wild-type p 53 protein in cell.

Meanwhile, the FITC (fluorescein isothiocynate) that vesicle surface is modified marks resistance different p53 protein antibodies and can identify intracellular variation p53 albumen, and its fluorescence signal intensity is directly proportional to the concentration of the p53 albumen that makes a variation in cell.Therefore, obtain cell fluorescent images by fluorescent microscope, thus the distribution of the p53 albumen that makes a variation in cell is observed and characterized.

The invention has the advantages that: (1) is for operated in accordance with conventional methods complexity, specificity and repeated not high problem, process provides the nano vesicle of the dissimilar tumor suppressor protein p53 of a kind of high specific identification, improve specificity and the sensitivity of detection.(2) for the problem that p53 albumen monolayer surface analysis means lack, using plasma resonance image-forming of the present invention and Imaging-PAM, enter after cell until vesica, utilize microscope to carry out in situ imaging to cell, detect while achieving unicellular aspect wild type and variation p53 albumen.(3) nano vesicle of the present invention's design can carry out in-situ monitoring to the change of p53 protein expression in cell under drug effect, and the intracellular physiological process participated in for studying it has been carried for new method.

Accompanying drawing explanation

Fig. 1 is preparation and the fundamental diagram thereof of the nano vesicle simultaneously detecting wild type and variation p53 albumen in cell.

Fig. 2 is variable concentrations nutlin-3 (A1-5:0, B1-5:62.5 μ g mL-1, C1-5:125 μ gmL-1, D1-5:250 μ g mL-1) the HeLa cell that processed and nano vesicle and the micro-image of DAPI incubation after 2 hours (1: darkfield image, 2: bright field image, 3:DAPI transfect cell cores, 4:FITC marks the fluoroscopic image of the different p53 protein antibodies of resistance, 5: the details of darkfield image is amplified), i1-i12: the details in a play not acted out on stage, but told through dialogues scattering spectrum of nm of gold corresponding in cell.

Label in Fig. 1 is respectively:

1, nano vesicle; 2, nm of gold; 3, the hendecanal;

4, FITC marks the different p53 protein antibodies of resistance; 5, cell membrane.

Embodiment

Below in conjunction with specific embodiment, set forth the present invention further.The experimental technique used in following embodiment if no special instructions, is conventional method.Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.

Embodiment 1. synthesis can detect the nano vesicle of wild type and variation p53 albumen in cell simultaneously

(1) according to existing document, synthesis 13nm gold size is as seed.By 50mL HAuCl ₄(0.01%) solution add in round-bottomed flask be heated to boiling, then 5mL sodium citrate solution (38.8mM) is added fast and Keep agitation 30 minutes, until solution becomes red, the 13nm gold obtained is planted and is used to prepare the nm of gold of particle diameter at about 60nm.

(2) gold is utilized to plant the nm of gold of synthesis 60nm.1mL gold is planted solution and 100 μ LNH ₂25mL is diluted to pure water, afterwards by 3.0mLHAuCl after OHHCl (0.2M) mixing ₄(0.01%) slowly dropwise add in above-mentioned mixed solution, Keep agitation 30 minutes is until solution becomes kermesinus.By centrifugal for the nano-Au solution obtained, with pure water, preserve under 4 DEG C of conditions.

(3) by the nm of gold of the nm of gold synthesis energy specific recognition wild-type p 53 protein of 60nm.The double stranded DNA solutions (100 μMs) 10 μ L being contained wild-type p 53 protein recognition site mixes with 1mL nano-Au solution, at room temperature stir and spend the night, then 0.1mL PBS damping fluid (containing 2M NaCl) is dropwise added the nm of gold obtaining stable DNA modification in above-mentioned solution.Above-mentioned solution centrifugal is washed twice with PBS, is finally again dispersed in 1mLPBS the nm of gold of the energy specific recognition wild-type p 53 protein obtaining DNA functionalization.

(4) preparation can detect the nano vesicle of wild type and variation p53 albumen in cell simultaneously.The nano-Au solution (20 μ L) of the DNA modification of preparation in (3) is mixed with 20 μ L serum free mediums; In addition 1.0 μ L Lipo-2000 liposomes are mixed with 20 μ L serum free mediums.At room temperature leave standstill after 10 minutes, above-mentioned two kinds of solution are mixed then incubated at room 20 minutes, obtain the liposome being enclosed with nm of gold.Then the 1.0 μ L hendecanals are added above-mentioned solution, react 10 minutes under room temperature, then add 1mg FITC and mark the different p53 protein antibodies of resistance, hold over night at 4 DEG C.By above-mentioned solution centrifugal and with PBS washing, be then dispersed in 60 μ L serum free mediums, obtain nano vesicle.

Embodiment 2. utilizes nano vesicle, carries out in situ imaging and detection to the distribution of p53 albumen in cell

Utilize HeLa cervical cancer tumer line as model, first by HeLa cell (1.0mL, 1 × 10 ⁶mL ^-1) plant in the burnt double dish of copolymerization, cultivate 24 hours.Then 60 μ L nano vesicles are added double dish, incubation 2 hours at 37 DEG C.With PBS washing double dish and by cell submergence, then at dark field microscope and fluorescence microscopy Microscopic observation.Cell imaging is see Fig. 2.Find in tumour cell HeLa, in darkfield image, the color of nm of gold is green, does not obviously assemble, illustrates that the content of wild type p53 is lower; And the fluorescence signal of FITC is comparatively strong, illustrate that the content of variation p53 is higher.With nutlin-3 drug solution (250 μ gmL ^-1) to cell (1.0mL, 1 × 10 ⁶mL ^-1) carry out pre-service, by cell and medicine Dual culture 48h, then 60 μ L nano vesicles are added, at 37 DEG C, incubation used microscopic examination after 2 hours, find in the cell that drug treating is crossed, the color of nm of gold is obviously partially orange red, and FITC fluorescence signal obviously reduces, and to illustrate in cell that the activity of wild type p53 increases and the activity of the p53 that makes a variation is suppressed.

The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, 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 considered as protection scope of the present invention.

Claims

1. one kind can be detected the nano vesicle of wild type and variation p53 albumen in cell simultaneously, it is characterized in that, described vesica internal package has the nm of gold of energy specific recognition wild-type p 53 protein, and vesicle surface is modified with the different p53 protein antibodies of resistance of marked by fluorescein isothiocyanate simultaneously.

2. a kind of nano vesicle that simultaneously can detect wild type and variation p53 albumen in cell according to claim 1, it is characterized in that, the nm of gold of described energy specific recognition wild-type p 53 protein is by obtaining containing the double stranded DNA solutions of wild-type p 53 protein recognition site and the nano-Au solution mix and blend of 55-65nm.

3. a kind of nano vesicle that simultaneously can detect wild type and variation p53 albumen in cell according to claim 2, it is characterized in that, the particle diameter of described nm of gold is 60nm.

4. the application in the image checking of nano vesicle according to claim 1 p53 albumen in living cells.

5. the application of nano vesicle according to claim 1 in the image checking reagent preparing p53 albumen in living cells.