CN115656083A - Extracellular vesicle nano infrared spectrum detection device for tumor detection and malignancy and metastatic evaluation and application - Google Patents
Extracellular vesicle nano infrared spectrum detection device for tumor detection and malignancy and metastatic evaluation and application Download PDFInfo
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Abstract
The invention provides a detection device and application for tumor detection, malignancy evaluation and tumor metastasis evaluation. The device of the invention comprises: an extraction mechanism for extracellular vesicles; a characterization mechanism of extracellular vesicles; an extracellular vesicle nano infrared detection mechanism, an extracellular vesicle nano infrared spectrum and an extracellular vesicle protein component and secondary structure information analysis mechanism. The device has the advantages of simple and convenient operation and low cost, has high accuracy in the aspects of breast cancer detection, malignancy evaluation and metastatic evaluation, is expected to realize noninvasive detection of tumors and real-time tracking of diseases, can adjust a treatment scheme in time according to the development of the diseases, guides a patient to take medicine, and provides a new scheme for early screening, malignancy evaluation and metastatic evaluation of breast cancer.
Description
Technical Field
The invention belongs to the field of medical detection, and particularly relates to a nano infrared detection device for breast cancer detection, malignancy evaluation and metastatic evaluation and application thereof.
Background
The incidence and death rate of the tumor are high, and the public health is seriously harmed. The incidence of breast cancer is gradually increased year by year, and is a first killer threatening the health of women. The early accurate evaluation of the malignancy degree and the metastasis of tumors such as breast cancer is important for guiding a treatment scheme and prolonging the life of a tumor patient.
The protein dynamically changes in abundance and structure during tumorigenesis and development, and the tumors are relatedProtein heterogeneity provides information for elucidating tumor pathogenesis and is an important biomarker for tumor diagnosis and drug design. Extracellular vesicles are membrane vesicles released by cells, carry biomolecules such as proteins, nucleic acids, lipids, etc. from source cells, and transfer these biomolecules to recipient cells, affecting the phenotype and function of the recipient cells, thereby playing a key role in tumorigenesis and progression. The heterogeneity of the tumor-derived extracellular vesicle proteins reflects the degree of malignancy of the tumor and the ability of the tumor to progress and metastasize. The heterogeneity of extracellular vesicle tumor-associated protein expression has been demonstrated by extensive proteomic analysis, however, the heterogeneity of extracellular vesicle protein secondary structure features has been poorly studied. Meanwhile, bulk sample analysis may have an impact on detection sensitivity and specificity due to the heterogeneity of extracellular vesicles and difficulty in purification. The detection of the heterogeneity of the protein secondary structure characteristics of the single extracellular vesicles provides comprehensive information of the extracellular vesicle subpopulation, and directly identifies the heterogeneity of the tumor-associated proteins from a complex background, which is helpful for understanding the mechanism of malignant transformation and developing potential biomarkers of tumors. However, due to the nanoscale dimensions of extracellular vesicles, techniques for detecting the secondary structural features of proteins in individual extracellular vesicles are highly challenging. Nanometric IR is a combination of scanning probe microscopy and IR spectroscopy using a metallized tip as a single plasma amplifier, 100nm below the tip 2 In-situ optical absorption of the enhancing and trapping molecules within the range. The high optical spatial resolution enables the nano infrared spectrum to obtain the local absorption spectrum of a nano-scale biological sample, and can be used for detecting the structural heterogeneity of single extracellular vesicle proteins. The heterogeneity detection of the protein structure of the extracellular vesicles based on the nano infrared spectrum provides important information and a method for clarifying a tumor occurrence development mechanism and developing a tumor marker.
Disclosure of Invention
Therefore, the present invention aims to overcome the defects in the prior art and provide an extracellular vesicle protein secondary structure characteristic analysis device and application for breast cancer detection, malignancy evaluation and metastatic evaluation.
In order to achieve the above object, a first aspect of the present invention provides an apparatus for analyzing and detecting secondary structural features of a tumor extracellular vesicle protein, the apparatus comprising:
(1) Extracellular vesicle extraction mechanism: extracting extracellular vesicles from the cell culture supernatant by centrifugation;
(2) Extracellular vesicle characterization mechanism: and characterizing the extracellular vesicles extracted by the extracellular vesicle extraction mechanism to determine that the extracellular vesicles are actually extracted, wherein the characterization index is selected from one or more of the following indexes: morphology, size, concentration;
(3) Extracellular vesicle protein expression level information measuring means: measuring protein expression amount information of the extracellular vesicles extracted by the extracellular vesicle extraction mechanism; preferably, the protein of interest is selected from one or more of: CD63 protein, CD81 protein, calnexin protein, hsp70 protein;
(4) The extracellular vesicle nano infrared spectrum measuring mechanism comprises: and performing nano infrared spectrometry on the extracellular vesicles extracted by the extracellular vesicle extraction mechanism, wherein the characterization indexes are selected from one or more of the following indexes: morphology, near-field infrared amplitude images, near-field infrared absorption spectra;
(5) The mechanism for analyzing the secondary structure characteristic information of the extracellular vesicle protein comprises the following components: and (3) performing protein secondary structure characteristic analysis on the nano infrared absorption spectrum measured by the extracellular capsule nano infrared spectrum characterization mechanism, wherein the protein secondary structure characteristic is selected from one or more of the following characteristics: the ratio of the amide I band to the amide II band, the content ratio of alpha helix to curl, beta sheet, antiparallel beta sheet and beta turn;
the device according to the first aspect of the invention, wherein the cells are selected from one or more of: mammary epithelial cells MCF-10A, breast cancer cells MCF-7, MDA-MB-231.
The device according to the first aspect of the present invention, wherein, in the mechanism for extracting extracellular vesicles, the method for extracting extracellular vesicles is selected from one or more of the following: the ultracentrifugation method and the microporous membrane filtration method comprise the following steps:
(a) Centrifuging cell culture supernatant, discarding precipitate in the first centrifugation, centrifuging supernatant for the second time, and centrifuging supernatant again for the third time to discard precipitate;
preferably, the first centrifugation speed is 300g to 1000g, preferably 800g; the first centrifugation time was 10 minutes; the second centrifugal speed is 2000 g-5000 g, preferably 2000g; the second centrifugation time is 20 minutes; the third centrifugal speed is 8000 g-10000 g, preferably 10000g; the third centrifugation time is 1 hour; the centrifugation temperature is 4 ℃;
(b) Filtering the supernatant obtained in the step (a) by using a filter membrane, and centrifuging the filtered liquid again to obtain a precipitate, thus obtaining extracellular vesicles;
preferably, the pore size of the filter membrane is 100nm-500nm, preferably 100nm and 200nm; the centrifugal speed is 100000-150000g, preferably 100000g; the centrifugation time is 0.5 to 5 hours, preferably 2 hours; the centrifugation temperature was 4 ℃.
Preferably, the extraction of extracellular vesicles further comprises the steps of:
(c) The pellet from the centrifugation in step (b) was resuspended in PBS.
The device according to the first aspect of the present invention, wherein in the extracellular vesicle extraction mechanism, the medium of the cell culture supernatant contains 10% fetal bovine serum;
preferably, the extracellular vesicle extraction mechanism further comprises the following operating mechanisms:
replacing the culture medium of the cell culture supernatant with a culture medium prepared by fetal calf serum with the extracellular vesicles removed before the extracellular vesicles are extracted; preferably, the replacement time is 24 to 48 hours before extraction.
Preferably, the medium prepared from fetal bovine serum minus extracellular vesicles is prepared by:
(i) Adding the serum into a basic culture medium to ensure that the concentration of the serum in the cell culture medium is 30 percent;
(ii) (ii) subjecting the cell culture medium obtained in step (i) to ultracentrifugation at 4 ℃; the centrifugal speed is 100000-150000g, preferably 134000g, and the centrifugal time is 5-20 hours, preferably 10 hours;
(iii) After ultracentrifugation is finished, collecting supernatant, diluting a culture medium containing 30% serum by using a basic culture medium until the concentration of FBS is 10%, and adding 1% double antibody;
(iv) Filtering the culture medium with a filter membrane, and bottling for later use; preferably, the filter membrane pore size is 0.22 μm.
The device according to the first aspect of the present invention, wherein in the extracellular vesicle characterization mechanism, the morphology characterization mechanism is a transmission electron microscope, preferably a biological transmission electron microscope; the mechanism for size characterization is a dynamic light scattering mechanism and/or a nanoparticle tracking analysis mechanism; the concentration characterization mechanism is a nanoparticle tracking analysis mechanism.
The device according to the first aspect of the present invention, wherein the means for characterizing the protein content is a BCA protein quantification means; the mechanism for measuring the protein expression quantity is an immunoblotting (western blot) mechanism.
The BCA protein quantification, specific steps and dosage vary according to different kits.
The immunoblotting mechanism for measuring the protein expression level comprises the following steps: according to the quantitative result of the BCA protein, taking the extracellular vesicles with equivalent weight of 6-20 mu g of protein, and separating and identifying the proteins with different molecular weights by running gel by using an electrophoresis method; the corresponding antibodies were incubated at the corresponding molecular weights and detected by chemiluminescence.
The apparatus according to the first aspect of the present invention, wherein the infrared spectrometry mechanism determines the protein secondary structure characteristics of the single extracellular vesicles, comprises the following steps:
(i) Dispersing and dripping the extracellular vesicles on a silicon wafer substrate;
(ii) The sample was scanned with a nano-infrared spectrometer and the infrared spectrum was collected on single extracellular vesicles. The spectrum collection range covers the characteristic wave bands of the amide I and the amide II;
(iii) Finding out characteristic peak positions in the infrared spectrum by utilizing second derivative spectral analysis, and calculating the area ratio of various protein structures in the spectrum by utilizing the characteristic peak positions;
(iv) And (3) obtaining correlation values of different protein structures of the sample and standard healthy and malignant samples by utilizing characteristic analysis of the subject, and evaluating the malignancy degree of the extracellular vesicles of the sample.
A second aspect of the invention provides the use of a device according to the first aspect in the manufacture of a medical product for the diagnosis and/or treatment of cancer; preferably, the cancer is breast cancer; more preferably, said use is the diagnosis and/or malignancy and/or metastatic assessment of breast cancer.
The invention relates to the aspect of breast cancer in-vitro detection, relates to the field of protein markers for breast cancer detection, and relates to the field of extracellular vesicle detection. The invention mainly aims to provide a method for detecting breast cancer in vitro by using the secondary structure characteristics of extracellular vesicle protein. By utilizing the nano infrared spectrum technology, the in-vitro diagnosis, the malignancy degree and the metastatic evaluation of the breast cancer patient can be realized. The method has the advantages of simple operation, low cost, rapid detection process, and reduced sample amount. In addition, the method is expected to track the state of an illness in real time, and a treatment scheme is adjusted in time according to the development of the state of the illness, so that a guidance thought is provided for realizing personalized medical treatment.
In order to achieve the purpose, the scheme of the invention is as follows:
the applications of the present invention include at least diagnosis of breast cancer, evaluation of malignancy, evaluation of metastasis, and the like.
The secondary structure characteristics of the target extracellular vesicle protein at least comprise an amide I band/amide II band ratio, alpha helix and curl, a beta sheet layer, an antiparallel beta sheet layer, a beta turn content ratio and the like.
Specifically, the step of extracting the extracellular vesicles from the tumor tissue comprises the following steps:
(i) Tumor tissue of breast cancer patients is extracted during operation, and the tumor tissue is stored in a refrigerator at the temperature of 80 ℃ below zero.
(ii) (ii) subjecting the tumor tissue obtained in step (i) to vortex digestion in a separation medium; the weight of the tumor tissue is 50-200 mg, preferably 150mg, and the separation solution is 12.5 muL of enzyme A, 50 muL of enzyme R, 100 muL of enzyme H and 2.2mL of serum-free cell cryopreservation culture medium; the number of vortexes is 2 to 10, preferably 3.
(iii) Taking the tissue suspension obtained in the step (2), centrifuging for the first time to remove precipitates, taking the supernatant to centrifuge for the second time to remove the precipitates, taking the supernatant again to centrifuge for the third time to remove the precipitates;
preferably, the first centrifugation speed is 300g to 1000g, preferably 300g; the first centrifugation time was 10 minutes; the second centrifugal speed is 2000 g-5000 g, preferably 2000g; the second centrifugation time is 10 minutes; the third centrifugal speed is 8000 g-10000 g, preferably 10000g; the third centrifugation time is 20 minutes; the centrifugation temperature is 4 ℃;
(iv) (iv) filtering the supernatant obtained in step (iii) with a filter membrane, and centrifuging the filtered liquid again to obtain a precipitate, thereby obtaining extracellular vesicles;
preferably, the pore size of the filter membrane is 0.22 μm; the centrifugal speed is 100000-150000g, preferably 150000g; the centrifugation time is 0.5 to 10 hours, preferably 7 hours; the centrifugation temperature was 4 ℃.
Preferably, the extraction of extracellular vesicles further comprises the steps of:
(v) (iv) centrifuging the pellet and resuspending in PBS.
The invention provides a method for analyzing a secondary structure of a single extracellular vesicle nano infrared spectroscopic protein, which comprises the following steps:
(1) Use of detection of the ratio of the vesicular amide I band/the amide II band outside the tumor tissue-derived cell of a subject, the alpha helix and coil, the beta sheet, the antiparallel beta sheet and the beta turn content in the diagnosis or in an auxiliary diagnosis of whether a subject is a breast cancer patient.
(2) The application of detecting the ratio of the vesicular amide I band/the vesicular amide II band outside the tumor tissue of the subject, alpha helix and curl, beta sheet, antiparallel beta sheet and beta turn content in evaluating the malignancy degree and the metastasis of the breast cancer subject.
The extracellular vesicle detection device for breast cancer detection, malignancy evaluation and metastatic evaluation of the present invention may have the following advantageous effects, but not limited thereto:
the invention discloses application of detecting the secondary structure characteristics of extracellular vesicle protein by means of a nano infrared spectrum technology to diagnosis of breast cancer and malignancy and metastatic evaluation thereof.
The secondary structure characteristics of the protein comprise amide I band/amide II band ratio, alpha helix and curl, beta sheet, antiparallel beta sheet and beta turn content.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
figure 1 shows the results of particle size measurements of extracellular vesicles extracted from the three cell lines.
Fig. 2 shows transmission electron microscopy characterization of extracellular vesicles extracted from the three cell lines.
Fig. 3 shows the results of the immunoblot assay for the expression of CD63, CD81, calnexin, hsp70, and GAPDH proteins from the three cell lines.
Fig. 4 shows the values at v =1430-1800cm -1 And (3) near-field infrared intensity characterization results of the extracellular vesicles extracted from the cell line under the wavelength.
Fig. 5 shows the nano-infrared absorption spectra of extracellular vesicles extracted from the cell lines.
Figure 6 shows the extracellular vesicular amide I band/amide II band ratio extracted from the three cell lines.
Figure 7 shows the assignment of protein secondary structure features to the second derivative of the nano-infrared absorption spectra of extracellular vesicles extracted from the cell line.
Figure 8 shows the results of the extracellular vesicle alpha helix and coil, antiparallel beta sheet and beta turn, parallel beta sheet content ratios extracted from the three cell lines.
Figure 9 shows the results of the analysis of the characteristics of the subjects (ROC) for sensitivity and specificity of the sensitivity and specificity of assessing tumor malignancy for the ratios of extracellular vesicular amide I band/amide II band ratios, helices and curls, antiparallel beta folds and beta turns, and content ratios of parallel beta folds extracted from the three cell lines.
FIG. 10 shows the ratio of vesicamide I band/amide II band outside the tumor tissue-derived cells of patients with non-metastatic breast cancer and those with lymph node metastatic breast cancer.
Figure 11 shows the results of the content ratio of the tumor tissue-derived extracellular vesicle helices and curls, antiparallel beta-sheet and beta-turn, parallel beta-sheet.
Figure 12 shows the results of analysis of the extracellular vesicular amide I band/amide II band ratios, helices and curls, antiparallel beta-folds and beta turns, content ratios of parallel beta-folds extracted from the tumor tissue on subject characteristics to assess the sensitivity and specificity of tumor metastasis.
Detailed Description
The invention is further illustrated by the following specific examples, but it should be understood that these examples are included merely for purposes of illustration and description in more detail, and are not intended to limit the invention in any way.
This section generally describes the materials used in the testing of the present invention, as well as the testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. It will be apparent to those skilled in the art that the materials and methods of operation used in the present invention are well within the skill of the art in this context, if not specifically mentioned.
The cell lines MCF-10A, MCF-7 and MDA-MB-231 used in the following examples were purchased from the national academy of medical sciences, unless otherwise specified.
Unless otherwise indicated, the tumor tissues of breast cancer patients used in the examples below were obtained from the first hospital affiliated with the university of science and technology of china.
The solvents of the aqueous solutions used in the examples below were sterile ultrapure aqueous solutions unless otherwise specified.
Unless otherwise indicated, all reagents used in the following examples were analytical reagents.
The reagents and instrumentation used in the following examples are as follows:
reagent:
PBS was from Thermo fisher.
Filter membranes, from Millex-GP.
Carbon film supported copper mesh, purchased from mesoscope.
PVDF membrane, available from Millipore, bedford, MA.
BCA protein quantification kit, purchased from Solebao.
All antibodies were purchased from abcam.
The instrument comprises the following steps:
ultracentrifuge, from beckman, model Optima XPN.
Transmission electron microscopy, purchased from hitachi. Model number HITACHI 7700.
Nanoparticle tracer available from malvern, model NanoSight LM14.
An ultra-high sensitivity chemiluminescent imaging system, available from Bio-Rad.
Nano infrared spectroscopy, (available as neaspc).
Example 1
This example illustrates the extraction of extracellular vesicles derived from cell lines according to the method of the invention.
(1) When the cells grew to 50% density, the cells were replaced with a medium prepared by subtracting the serum of the extracellular vesicles, and the cells were placed in a cell incubator for 48 hours.
(2) The cell culture supernatant of 12 dishes of cells was put into a 50mL centrifuge tube and centrifuged at 4 ℃ for 800g for 10 minutes.
(3) Taking the supernatant obtained after centrifugation in the step (2), centrifuging the supernatant at 2000g for 20 minutes at 4 ℃.
(4) Centrifuging the supernatant obtained in the step (3) to 10000g for 1 hour at 4 ℃.
(5) And (5) taking the supernatant obtained after the centrifugation in the step (4), filtering the supernatant by using 100nm and 200nm filter membranes, and collecting the filtrate into a new centrifuge tube.
(6) The filtered sample was loaded into an ultracentrifuge tube and centrifuged at 100000g,2 hours, 4 ℃.
(7) After ultracentrifugation, the supernatant medium was discarded, and the bottom pellet was resuspended in PBS buffer, centrifuged 100000-150000g for 2 hours at 4 ℃.
(8) The supernatant of PBS buffer was discarded, and the extracellular vesicles obtained by extraction were resuspended in a volume of 100-500. Mu.L of PBS buffer previously filtered through a 0.22 μm filter.
Example 2
This example illustrates the characterization of the morphology and size of the extracted extracellular vesicles by the method of the present invention.
(1) 20. Mu.L of the extracellular vesicle suspension isolated in example 1 was diluted 50-fold to 1mL with PBS. Detection was carried out with a nanoparticle tracer, laser 405nm. Injecting the diluted extracellular vesicles into a sample cell by using a syringe, detecting by using a high-sensitivity metal oxide semiconductor (CMSO) camera, collecting for 60s, setting the detection limit to be 7, performing three parallels on each sample, and carefully washing the sample cell for three times by using PBS (phosphate buffer solution) between every two samples. After the detection is finished, the testing result is analyzed by using data analysis software NTA software, version 2.3 of the nanoparticle tracer. FIG. 1 shows the size distribution and number of extracellular vesicles of example 1 measured by a nanoparticle tracer.
(2) 10 mu L of the extracellular vesicle suspension separated in example 1 was dropped on an ultrathin carbon film-supported copper mesh, washed twice, negatively stained with 1% uranyl acetate, photographed by a 80Kv transmission electron microscope, and the morphology is characterized as shown in FIG. 2.
Example 3
This example illustrates the protein expression assay of extracted extracellular vesicles according to the method of the invention.
(1) 100. Mu.L of the extracellular vesicle suspension isolated in example 1 was taken, 100. Mu.L of a protein lysate (containing a protease inhibitor) was added thereto, mixed well, and lysed on ice for 1 hour. After lysis was complete, extracellular vesicle protein content was quantitated according to the BCA protein quantitation kit instructions.
(2) The extracellular vesicular protein, which was loaded in an amount of 10. Mu.g, was added to polyacrylamide gel and subjected to electrophoresis at 110V for 100 minutes under a constant pressure. The gel was transferred to a PVDF membrane having a pore size of 0.45. Mu.m. 5% skimmed milk powder was sealed at room temperature for one hour with shaking. The blocked PVDF membrane was soaked in primary antibody and incubated at 4 ℃ for 12 hours. Tween 20-Tris buffered saline was 0.1-vol%, and washed 3 times with shaking at room temperature for 5 minutes each. The washed PVDF membrane is soaked in a secondary antibody solution containing horseradish peroxidase label, incubated for 1 hour at room temperature with shaking, then washed with 0.1 percent Tween 20-Tris solution, and washed 3 times with shaking at room temperature for 5 minutes each time. Finally, the membrane was immersed in a fluorescent developer and imaged in an ultra-high sensitivity chemiluminescent imaging system, with the results shown in FIG. 3.
Example 4
This example illustrates the nano-infrared spectral characterization and the secondary structural characterization of extracellular vesicle proteins by the method of the present invention.
(1) Dispersing and dripping the extracellular vesicles on a silicon wafer substrate.
(2) The sample was scanned by a nano-infrared spectrometer and the results are shown in figure 4. The results of infrared spectra collected on single extracellular vesicles are shown in figure 5. The spectrum collection range covers the characteristic bands of amide I and amide II, and the ratio of the amide I band/the amide II band is calculated, and the result is shown in FIG. 6.
(3) The second derivative spectral analysis was used to find the characteristic peak position in the infrared spectrum, the results are shown in fig. 7. The area ratio of the secondary structure of each protein in the spectrum was calculated using the characteristic peak position, and the result is shown in fig. 8.
(4) The correlation values of different protein structures of the sample and the samples without metastasis and lymph node metastasis tumor are obtained by using the characteristic analysis of the subject, and are used for evaluating the malignancy and the metastasis of extracellular vesicles of the sample, and the result is shown in fig. 9.
Example 5
This example serves to illustrate the extraction and morphological characterization of the tumor tissue-derived extracellular vesicles by the method of the invention.
(1) Taking 150mg of tumor tissue of the breast cancer patient, adding 2.5 mu L of enzyme A, 50 mu L of enzyme R, 100 mu L of enzyme H and 2.2mL of serum-free cell cryopreservation medium, and vortexing for 3 times.
(2) The tissue suspension obtained in step (1) was centrifuged at 300g for 10 min at 4 ℃.
(3) Taking the supernatant obtained after centrifugation in the step (2), centrifuging the supernatant at 2000g for 10 minutes at 4 ℃.
(4) Taking the supernatant obtained after centrifugation in the step (3), centrifuging at 10000g for 20 minutes at 4 ℃.
(5) The supernatant obtained in step (4) was filtered through a 0.22 μm filter, and the filtered liquid was centrifuged at 150000g for 7 hours, and the resulting precipitate was resuspended in PBS buffer at 4 ℃.
(6) And (3) dripping 10 mu L of the extracellular vesicle suspension separated in the step (5) on an ultrathin carbon film-supported copper net, washing with water twice, carrying out negative staining by 1% uranyl acetate, carrying out shooting by using an 80Kv transmission electron microscope, and carrying out morphology characterization as shown in FIG. 10.
Example 6
This example illustrates the nano-infrared spectroscopic characterization and protein secondary structure analysis of extracellular vesicles extracted from the tumor tissue by the method of the present invention.
(1) Dispersing and dripping the extracellular vesicles on a silicon wafer substrate.
(2) The sample was scanned with a nano-infrared spectrometer and the infrared spectrum was collected on single extracellular vesicles. The spectrum collection range covers the characteristic bands of amide I and amide II, and the ratio of the amide I band/the amide II band is calculated, and the result is shown in FIG. 10.
(3) The second derivative spectrum analysis was used to find the characteristic peak position in the infrared spectrum, and the area ratio of the secondary structure of each protein in the spectrum was calculated using the characteristic peak position, the result is shown in fig. 11.
(4) The correlation values of different protein structures of the sample and the sample without metastasis and lymph node metastasis tumor are obtained by using the characteristic analysis of the subject, and are used for evaluating the malignancy and the metastasis of the extracellular vesicles of the sample, and the result is shown in fig. 12.
Claims (10)
1. An extracellular vesicle nano infrared spectrum detection device for tumor detection, malignancy and metastatic evaluation, characterized in that the device comprises:
(1) Extracellular vesicle extraction mechanism: extracting extracellular vesicles by centrifugation;
(2) Extracellular vesicle characterization mechanism: and characterizing the extracellular vesicles extracted by the extracellular vesicle extraction mechanism, wherein the characterization indexes are selected from one or more of the following indexes: morphology, size, concentration;
(3) Extracellular vesicle protein expression level information measuring means: measuring protein expression amount information of the extracellular vesicles extracted by the extracellular vesicle extraction mechanism;
(4) The extracellular vesicle nano infrared spectrum measuring mechanism comprises: and (3) carrying out nano infrared spectrum measurement on the extracellular vesicles extracted by the extracellular vesicle extraction mechanism, wherein the characterization indexes are selected from one or more of the following indexes: morphology, near-field infrared amplitude images, near-field infrared absorption spectra;
(5) The mechanism for analyzing the secondary structure characteristic information of the extracellular vesicle protein comprises the following components: and (3) carrying out protein secondary structure characteristic analysis on the nano infrared absorption spectrum measured by the extracellular capsule nano infrared spectrum characterization mechanism, wherein the protein secondary structure characteristics are selected from one or more of the following characteristics: and (3) carrying out breast cancer detection, malignancy evaluation and metastatic evaluation on the ratio of the amide I band to the amide II band, alpha helix and curl, beta sheet, antiparallel beta sheet and beta turn content ratio.
2. The apparatus according to claim 1, wherein the extraction mechanism of extracellular vesicles comprises:
(a) Centrifuging cell culture supernatant, discarding precipitate in the first centrifugation, centrifuging supernatant for the second time, and centrifuging supernatant again for the third time to discard precipitate;
the first centrifugation speed is 300 g-1000 g, and the first centrifugation time is 10 minutes; the second centrifugation speed is 2000 g-5000 g, and the second centrifugation time is 20 minutes; the third centrifugation speed is 8000 g-10000 g, and the third centrifugation time is 1 hour; the three centrifugation temperatures are all 4 ℃;
(b) Filtering the supernatant obtained in the step (a) by using a filter membrane, and centrifuging the filtered liquid again to obtain a precipitate, thus obtaining extracellular vesicles; the aperture of the filter membrane is 100nm-500nm; the centrifugal speed is 100000-150000 g; the centrifugal time is 0.5 to 5 hours; the centrifugation temperature is 4 ℃;
(c) The pellet from the centrifugation in step (b) was resuspended in PBS.
3. The apparatus according to claim 2, wherein in the extracellular vesicle extraction mechanism, the culture medium of the cell culture supernatant contains 10% fetal bovine serum; the extracellular vesicle extraction mechanism further comprises the following operations:
replacing the culture medium of the cell culture supernatant with a culture medium prepared by fetal calf serum with the extracellular vesicles removed before the extracellular vesicles are extracted; the replacement time is 24 to 48 hours before extraction;
the medium prepared from fetal bovine serum minus extracellular vesicles was prepared by the following method:
(a) Adding the serum into a basic culture medium to ensure that the concentration of the serum in the cell culture medium is 30 percent;
(b) Ultracentrifuging the cell culture medium obtained in the step (a) at 4 ℃, wherein the centrifugation speed is 100000-150000g, and the centrifugation time is 5-20 hours;
(c) After ultracentrifugation is finished, collecting supernatant, diluting a culture medium containing 30% serum by using a basic culture medium until the concentration of FBS is 10%, and adding 1% double antibody;
(d) Filtering the culture medium with a filter membrane, and bottling for later use; the filter pore size was 0.22 μm.
4. The device according to claim 3, wherein the extracellular vesicle characterization mechanism comprises a protein content characterization mechanism, a morphology characterization mechanism, a size characterization mechanism and a concentration characterization mechanism, and the protein content characterization mechanism is a BCA protein quantification mechanism; the appearance characterization mechanism is a transmission electron microscope; the mechanism for size characterization is a dynamic light scattering mechanism and/or a nanoparticle tracking analysis mechanism; the concentration characterization mechanism is a nanoparticle tracking analysis mechanism.
5. The device according to claim 4, wherein the extracellular vesicular protein expression level information measuring means is a receptor selected from the group consisting of CD63, CD81, calnexin and Hsp70.
6. The device according to claim 5, wherein the means for measuring the expression level of the protein is an immunoblotting western blot device.
7. The device of claim 6, wherein the immunoblotting mechanism determining the amount of protein expression comprises the steps of: according to the BCA protein quantitative result, taking the extracellular vesicles with equivalent weight of 6-20 mug protein, and separating and identifying the proteins with different molecular weights by running gel by using an electrophoresis method; the corresponding antibodies were incubated at the corresponding molecular weights and detected by chemiluminescence.
8. The apparatus according to claim 7, wherein the nano infrared spectrometry mechanism for extracellular vesicles comprises the following steps:
(a) Dispersing and dripping the extracellular vesicles on a silicon wafer substrate;
(b) Scanning a sample by using a nano infrared spectrometer, and collecting a near-field infrared amplitude image and a near-field infrared absorption spectrum on the single extracellular vesicle; the spectral collection range covers the characteristic bands of amide I and amide II.
9. The apparatus according to claim 8, wherein the means for analyzing the secondary structural feature information of the extracellular vesicle protein comprises:
(a) Finding out characteristic peak positions in an infrared spectrum by utilizing second derivative spectral analysis, and calculating the area ratio of various protein structures in the spectrum by utilizing the characteristic peak positions, wherein the secondary structure characteristics of the protein comprise amide I band/amide II band ratio, alpha helix and curl, beta sheet layer, anti-parallel beta sheet layer and beta corner content;
(b) And (4) obtaining correlation values of different protein structures of the sample and standard healthy and malignant samples by utilizing characteristic analysis of the subject, and evaluating the malignancy degree of the extracellular vesicles of the sample.
10. Use of a device according to any one of claims 1 to 9 for the manufacture of a medical product for the treatment of tumours.
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| CN116559107A (en) * | 2023-04-27 | 2023-08-08 | 中国人民解放军海军军医大学 | System for diagnosing early liver cancer based on exosome infrared spectrum characteristics |
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| CN116559107A (en) * | 2023-04-27 | 2023-08-08 | 中国人民解放军海军军医大学 | System for diagnosing early liver cancer based on exosome infrared spectrum characteristics |
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