CN111398595A - Application and detection method of protein TNFAIP8 in plasma small cell outer vesicle - Google Patents

Application and detection method of protein TNFAIP8 in plasma small cell outer vesicle Download PDF

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CN111398595A
CN111398595A CN202010095399.1A CN202010095399A CN111398595A CN 111398595 A CN111398595 A CN 111398595A CN 202010095399 A CN202010095399 A CN 202010095399A CN 111398595 A CN111398595 A CN 111398595A
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tnfaip8
protein
plasma
diabetic retinopathy
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李筱荣
张晓敏
肖静
张慧
杨付花
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TIANJIN MEDICAL UNIVERSITY EYE HOSPITAL
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Abstract

The invention relates to application of protein TNFAIP8 in plasma small extracellular vesicles as a marker for diagnosis of proliferative diabetic retinopathy. The invention discovers for the first time that the protein expression level of TNFAIP8 in plasma sEVs is closely related to diabetic retinopathy, and by detecting the TNFAIP8 expression in the plasma sEVs of diabetic patients, whether a subject has the risk of diabetic retinopathy can be judged more accurately and rapidly, so that a prevention or treatment scheme is provided for clinicians, and the TNFAIP8 expression is used as a target point for preparing a medicament for treating the diabetic retinopathy, so that a new treatment target point and a treatment way are provided for treating the diabetic retinopathy.

Description

Application and detection method of protein TNFAIP8 in plasma small cell outer vesicle
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to an application and a detection method of protein TNFAIP8 in plasma small extracellular vesicles.
Background
Diabetic Retinopathy (DR) is relatively hidden, no obvious symptom exists in the early stage, the disease usually progresses to the proliferative stage when a patient is in a visit, which is called Proliferative Diabetic Retinopathy (PDR), the existing treatment means such as laser photocoagulation, intravitreal injection of anti-Vascular Endothelial Growth Factor (VEGF) drugs, vitreoretinal surgery and other methods are mainly directed at the more advanced stage of the patient, only partial vision can be recovered and maintained, the structural and functional damage of the retina is difficult to reverse, and early diagnosis and timely treatment are very critical to the control and prognosis of the disease. However, there is currently no effective biomarker as a risk factor for developing DR and a clinical index for prognosis judgment. The development of novel efficient biomarkers is helpful for early diagnosis of DR and formulation of individual diagnosis and treatment schemes, reduces DR blindness rate, and has important clinical significance.
Extracellular Vesicles (EV) are a generic term for membrane-structure-carrying bodies released by cells such as exosomes, microvesicles, and apoptotic bodies. Wherein the exosome is a nano-scale microvesicle secreted by cells and having a diameter of between 30 and 200 nm. Exosomes originate in the endosome of cells, are double-layered lipid membranes on the surface, are rich in protein, lipid, mRNA and miRNA components from maternal cells, and are important mediators of intercellular signaling. The bilayer lipid membrane protects the components carried by the exosome from remaining biologically active for a long time. Small particle size EV (smallEV, sEV) with the diameter of less than 200nm can be separated by a traditional differential centrifugation method, and the main component of the small particle size EV is exosome. According to the consensus of experts published in 2018, sEV is recommended because it is difficult to directly prove that small-particle-size EV separated by the traditional differential centrifugation method is a purified exosome.
The occurrence of DR is associated with multiple factors, such as disease course, glycemic control, and inheritance. Currently, DR biomarkers are mainly classified into ocular and systemic sources. Ocular-derived indices include ocular multimodal imaging examination and electrophysiological examination, as well as intravitreal VEGF and Pigment epithelium-derived factor (PEDF), among others. Systemic biomarkers include: glycated Hemoglobin (Hemoglobin A1c, HbA1c), advanced glycation end products, expression levels of inflammatory factors and oxidative stress factors, GRB2 gene, epigenetics and the like. HbA1c levels are considered to be classical biomarkers. However, the association of HbA1c and the course of diabetes with DR was only 11% and 89% was due to other factors. Therefore HbA1c is not an ideal biomarker for determining DR development. Most of the new biomarkers studied at present still lack large sample validation, and the predicted effect does not exceed the traditional biomarkers such as HbA1c, and the biomarkers can only be used as research tools without changing clinical practice. In genomics, genetic biomarkers of DR have not been finalized, and detection of vitreal VEGF, PEDF requires invasive procedures with potential risks. Therefore, finding sensitive and noninvasive biomarkers which can carry out early diagnosis and prognosis judgment on DR so as to select appropriate means to effectively intervene on diseases in an early stage, improve the vision of patients and improve the quality of life is the research focus of DR.
sEVs can also pass biological barriers of blood brain and blood eye due to a double-layer lipid membrane structure, and at present, sEVs are used for diagnosis and pathogenesis research of various diseases, for example, sEVs carrying glypican-1 in serum can be used as a biological marker for early pancreatic cancer diagnosis, and the expression level of α synuclein in plasma sEVs can be used as a biological marker for Parkinson patients to reflect the severity of intracerebral diseases.
Previous studies found that levels of sEVs carrying coated protein A in the circulatory system reflect the body's insulin resistance status. The c-megalin protein level in diabetic urine sEVs is positively correlated with the proteinuria level, and furthermore, aquaporins in sEVs are consistent with the tissue stage of diabetic nephropathy. When DR occurs, the amount of complement carried by sEVs increases, and the retinal microvasculature can be reached through blood circulation, promoting retinal microvascular leakage in patients with DR. The above studies suggest that sEVs may be involved in the development of DR, or that alterations in DR may result in corresponding changes in plasma sEVs. Therefore, changes in the composition of plasma sves may reflect changes in ocular diseases and are promising as biological markers of DR.
Through searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an application and a detection method of protein TNFAIP8 in plasma small extracellular vesicles, wherein the protein TNFAIP8 can provide a prevention or treatment scheme for clinicians, and can be used as a target for preparing a medicament for treating diabetic retinopathy, so that a novel treatment target and a novel treatment way are provided for treating the diabetic retinopathy.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the application of protein TNFAIP8 in plasma small extracellular vesicle as the marker of proliferative diabetic retinopathy diagnosis.
The application of protein TNFAIP8 in plasma small extracellular vesicle as target point for preparing medicine for treating diabetic retinopathy.
A method for detecting protein TNFAIP8 in plasma small cell outer vesicle comprises the following steps:
the reagents in the E L ISA kit and the desired 96-well plate were left at room temperature for 15 minutes;
preparing 1 × cleaning buffer solution, 1 × biotinylated alkaline phosphatase conjugate, 1 × biotin-HRP and standard substance, adding EVs into sample diluent, adding the diluted standard substance and a sample to be detected into a hole, incubating for two hours at 37 ℃, sucking the solution in the hole, adding 1 × cleaning buffer solution into each hole, washing for 3 times, adding 1 × biotinylated alkaline phosphatase conjugate, incubating for 1 hour at 37 ℃, washing a plate for 3 times, adding 1 × biotin-HRP, incubating for 1 hour at 37 ℃, washing the plate for 5 times, adding developing solution, incubating for 15-20min at 37 ℃, adding stop solution to terminate the reaction, preheating an enzyme labeling instrument in advance, measuring the 450nm light absorption value and subtracting the 540nm light absorption value to obtain the final result, establishing a standard curve, obtaining the value of each hole, and performing statistical analysis and mapping by using Prism;
wherein EVs is sample diluent, diluted standard or sample to be detected, 1 × washing buffer solution, 1 × biotinylated alkaline phosphatase conjugate, 1 × biotin-HRP, developing solution and stopping solution, and the proportion of the ratio is mu × 0, mu L, mu L, m L, mu L, mu L, mu L and mu L is 20: 80, 100: 100: 100: 50;
the sample diluent is 1% NaCl, 0.1% Tris base, 0.1% Proclin 300, H2And O, wherein the percentage is mass percent.
Furthermore, the E L ISA kit is Abebio-AE 14038H.
The invention has the advantages and positive effects that:
1. the invention discovers for the first time that the protein expression level of TNFAIP8 in plasma sEVs is closely related to diabetic retinopathy, and by detecting the TNFAIP8 expression in the plasma sEVs of diabetic patients, whether a subject has the risk of diabetic retinopathy can be judged more accurately and rapidly, so that a prevention or treatment scheme is provided for clinicians, and the TNFAIP8 expression is used as a target point for preparing a medicament for treating the diabetic retinopathy, so that a new treatment target point and a treatment way are provided for treating the diabetic retinopathy.
2. The invention provides a protein biomarker and a detection method for diabetic plasma sEVs to reliably diagnose diabetic retinopathy. The protein TNFAIP8 is differentially expressed in plasma shevs of proliferative diabetic retinopathy patients compared to plasma shevs of healthy control patients, and the differentially expressed protein represents the severity of the disease, the protein expression biomarker being indicative of a diagnosis of diabetic retinopathy.
3. Previous studies by the applicant have found for the first time that TNFAIP8 in PDR patient plasma sves is significantly elevated in plasma sves and intraocular vitreous of PDR patients compared to normal, DR-free diabetic patients, but has no significant change in plasma and medium-sized extracellular vesicles (mEVs) with diameters greater than 200 nm. Further in vitro experiments show that in an in vitro oxidative stress model, the expression of TNFAIP8 of human retinal microvascular endothelial cells treated by 4-hydroxynonenal (4-hydroxynenal, 4-HNE) is remarkably up-regulated, and TNFAIP8 can promote the proliferation and migration of cells. The results of the above studies indicate that TNFAIP8 is involved in the pathogenesis of DR, especially PDR, and is positively correlated with disease changes. TNFAIP8 in plasma shevs is consistent with intraocular changes and can serve as a biomarker for PDR in peripheral blood circulation.
Drawings
FIG. 1 is a graph showing the results of identification of extracellular vesicles according to the present invention; wherein, the left graph is sEVs, and the right graph is mEVs;
FIG. 2 is a transmission electron micrograph of extracellular vesicles according to the present invention; wherein, the upper graph is sEVs, and the lower graph is mEVs;
FIG. 3 is a graph showing the results of protein mass spectrometry analysis of TNFAIP8 protein in plasma small extracellular vesicles according to the present invention; wherein, the upper graph is up-regulated protein, and the lower graph is down-regulated protein;
FIG. 4 is a graph showing the results of the detection of the expression level of TNFAIP8 protein in plasma extracellular vesicles in plasma according to the present invention by E L ISA;
FIG. 5 is a graph showing the results of the detection of the expression level of TNFAIP8 protein in plasma small extracellular vesicles in extracellular vesicles sEVs according to the present invention by E L ISA;
FIG. 6 is a graph showing the results of the detection of the expression level of TNFAIP8 protein in plasma small extracellular vesicles in extracellular vesicles mEVs according to the present invention by E L ISA;
FIG. 7 is a graph showing the results of detection of the expression level of TNFAIP8 protein in the plasma small extracellular vesicles in the vitreous body according to the invention by E L ISA;
FIG. 8 is a graph showing the results of 4-HNE-induced proliferation of HRMECs in the present invention;
FIG. 9 is a graph showing the results of Western Blot detection of TNFAIP8 expressed by 4-HNE-stimulated cells in the present invention;
FIG. 10 is a graph showing the results of the TNFAIP8 protein in plasma small extracellular vesicles promoting the proliferation of HRMECs in the present invention;
FIG. 11 is a graph showing the results of the TNFAIP8 protein in plasma small extracellular vesicles promoting tube formation of HRMECs in the present invention;
FIG. 12 is a graph showing the results of the TNFAIP8 protein in plasma small extracellular vesicles promoting migration of HRMECs in the present invention;
FIG. 13 is a WB result of low expression of HRMECs by TNFAIP8 protein in plasma small extracellular vesicles according to the present invention;
FIG. 14 is a graph showing the results of inhibiting the proliferation of HRMECs by the low expression of TNFAIP8 protein in plasma small extracellular vesicles according to the present invention;
FIG. 15 is a graph showing the results of inhibiting HRMECs tube formation by the low expression of TNFAIP8 protein in plasma small extracellular vesicles according to the present invention;
FIG. 16 is a graph showing the results of inhibiting migration of HRMECs by the low expression of TNFAIP8 protein in plasma small extracellular vesicles of the present invention.
Detailed Description
The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.
The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The application of protein TNFAIP8 in plasma small extracellular vesicle as the marker of proliferative diabetic retinopathy diagnosis.
The application of protein TNFAIP8 in plasma small extracellular vesicle as target point for preparing medicine for treating diabetic retinopathy.
The extraction process of TNFAIP8 protein in the plasma small cell outer vesicle includes the following steps:
centrifuging diabetic plasma for 2000g × 15min, extracting supernatant, transferring the supernatant into an ultracentrifuge tube, centrifuging for 10000 + 80000g × 30min to obtain supernatant, transferring the supernatant into a new ultracentrifuge tube, performing 110,000 g × 120min to obtain precipitate, fully resuspending the precipitate with PBS, performing 110,000 g × 120min centrifugation to obtain precipitate, and cracking the precipitate with 0.1% TritonX-100 to obtain the TNFAIP8 in the plasma small cell outer vesicle.
The method for detecting the protein TNFAIP8 in the plasma small extracellular vesicles comprises the following steps:
the reagents in the E L ISA kit and the desired 96-well plate were left at room temperature for 15 minutes;
preparing 1 × cleaning buffer solution, 1 × biotinylated alkaline phosphatase conjugate, 1 × biotin-HRP and standard substance, adding EVs into sample diluent, adding the diluted standard substance and a sample to be detected into a hole, incubating for two hours at 37 ℃, sucking the solution in the hole, adding 1 × cleaning buffer solution into each hole, washing for 3 times, adding 1 × biotinylated alkaline phosphatase conjugate, incubating for 1 hour at 37 ℃, washing a plate for 3 times, adding 1 × biotin-HRP, incubating for 1 hour at 37 ℃, washing the plate for 5 times, adding developing solution, incubating for 15-20min at 37 ℃, adding stop solution to terminate the reaction, preheating an enzyme labeling instrument in advance, measuring the 450nm light absorption value and subtracting the 540nm light absorption value to obtain the final result, establishing a standard curve, obtaining the value of each hole, and performing statistical analysis and mapping by using Prism;
wherein EVs is sample diluent, diluted standard or sample to be detected, 1 × washing buffer solution, 1 × biotinylated alkaline phosphatase conjugate, 1 × biotin-HRP, developing solution and stopping solution, and the proportion of the ratio is mu × 0, mu L, mu L, m L, mu L, mu L, mu L and mu L is 20: 80, 100: 100: 100: 50;
the sample diluent is 1% NaCl, 0.1% Tris base, 0.1% Proclin 300, H2And O, wherein the percentage is mass percent.
Preferably, the E L ISA kit is Abebio-AE 14038H.
More specifically, the relevant preparations are as follows:
1. materials and methods
1.1 test subjects
The study was approved by the ethical committee of the affiliated ophthalmic hospital of Tianjin medical university. All patients of the subsidiary ophthalmic hospital of Tianjin medical university signed an informed consent
For proteomics analysis, plasma of healthy subjects was used as a control group (N ═ 5), diabetic retinopathy without complications (diabetes without clinical DR) (N ═ 5), non-proliferative diabetic retinopathy (N ═ 5) and diabetic retinopathy (N ═ 5), obtained as an experimental group, another group of individuals was recruited, including normal control (N ═ 7), diabetes (N ═ 10), non-proliferative diabetic retinopathy (N ═ 13) and proliferative diabetic retinopathy (N ═ 14), for E L ISA validation of specific protein candidates from proteomics analysis, inclusion criteria for DM and diabetic retinopathy were (1)40 to 80 years, (2) type 2 diabetes mellitus, exclusion criteria were (1) uveitis (2) other metabolic syndrome with complications of the eye (3) immune system disease with complications of the eye (4) retinal vessel occlusion.
1.2 diabetic retinopathy Classification
Judging the fundus of the examinee after mydriasis according to the super-wide-angle photographing result of the fundus of the examinee or by a fundus doctor:
a. no apparent retinopathy (NDR): only diabetes exists, and the eyeground has no abnormal change;
b. non-proliferative stage diabetic retinopathy (NPDR): microaneurysms, intraretinal hemorrhage; venous beading, microvascular abnormalities
c. Proliferative Diabetic Retinopathy (PDR): 1 or more alterations occur, including neovascularization, vitreal blood, or pre-retinal hemorrhage.
1.3 extraction and preservation of plasma and its extracellular vesicles
1.3.1 plasma, namely taking 10m L of blood from a vein, preserving the blood by using an EDTA anticoagulant tube, taking supernatant plasma after 1800g is centrifuged for 15 minutes, preserving the plasma at minus 80 ℃, centrifuging the plasma for 2000g × 15min, then extracting the supernatant and transferring the supernatant into an ultracentrifuge tube, centrifuging 10000g × 30min to obtain the supernatant, transferring the supernatant into a new ultracentrifuge tube to carry out 110,000 g × 120min to obtain a precipitate, fully resuspending the precipitate by using PBS, centrifuging the precipitate for 110,000 g × 120min to obtain the precipitate, and cracking the precipitate by using 0.1 percent TritonX-100 to obtain the protein TNFAIP8 in the plasma small cell outer vesicle.
1.3.2mEVs 1.5ml plasma was centrifuged for 2000g × 15min, the supernatant was removed and transferred to a 15m L centrifuge tube, the volume was adjusted to 12m L, and 10000g × 30min was centrifuged to obtain a precipitate mEVs.
1.3.3sEVs, 1.5ml of plasma is taken, centrifuged for 2000g × 15min, the supernatant is extracted and transferred into an ultracentrifuge tube, centrifuged for 80000g × 30min to obtain the supernatant, the supernatant is transferred into a new ultracentrifuge tube and centrifuged for 110,000 g × 120min to obtain a precipitate, the precipitate is resuspended by 1m L PBS, the volume is supplemented to 11.5m L, and centrifuged for 110,000 g × 120min to obtain the precipitate sEVs.
1.4 identification of extracellular vesicles
1.4.1 Nanoparticle Tracking Analysis (NTA)
The resuspended extracellular vesicles were diluted to 1ml volume with PBS and then transferred to clear tubes using Nano sight ns 300 according to the protocol to measure the size and effect of the extracellular vesicles.
1.4.2 Transmission Electron microscopy of extracellular vesicles
Thawing the subpackaged mEVs and sEVs, then suspending in 200 mu L PBS liquid for uniformly mixing, mixing 10 mu L exosome solution and 4% PFA according to the volume ratio of 1: 1, dripping on a clean plastic film to form liquid drops, then buckling the front surface of an electron microscope carbon net on the liquid drops, standing for 20min, carrying out negative staining on 10 mu L phosphotungstic acid for 90s, baking the carbon net, observing by using a HitacW-7500 transmission electron microscope, and taking pictures.
1.5 protein Mass Spectrometry method
Processing sEVs by using a special lysate for protein mass spectrum sample preparation, removing precipitates through ultrasound and centrifugation, extracting upper-layer protein based on an ultrafiltration-assisted sample preparation enzyme digestion method to extract cell protein, and performing liquid phase tandem mass spectrum analysis. The detection is carried out by adopting a data independent acquisition mode (SWATH-MS) method. The obtained raw mass spectral data were subjected to database search using the ProteinPilot (5.0) software. And importing the ProteinPilot file generated after the library is searched into a SWATH plug-in unit built in Peakview software, and selecting corresponding SWATH original data. The translation number of each peptide segment and the number of peptide segments of each protein in the process setting are both 5, the post-translation modified peptide segments and homologous peptide segments are excluded, the credibility of the peptide segments is more than or equal to 90 percent, and the false positive rate is less than or equal to 1 percent.
1.6 ELISA
(1) The reagents in the E L ISA kit (abebaio-AE 14038HU) and the required 96-well plate were left at room temperature for 15 minutes.
(2) Configuration 1 × wash buffer, 1 × biotinylated alkaline phosphatase conjugate, 1 × biotin-HRP, and standard.
(3) Adding 20 mu L lysed mEVs/sEVs into 80 mu L sample diluent, or adding 50ul of plasma into 50ul of normal saline, or adding 50ul of plasma into 50ul of TritonX-100 for membrane rupture treatment, or adding 100ul of vitreous body sample into the well, incubating at 37 ℃ for two hours, sucking out the solution in the well, and adding 250m L1 × washing buffer into each well for 3 times.
(4) 100 μ L1 × biotin-conjugated biotin was added and incubated for 1 hour at 37 ℃.
(5) After washing the plate 3 times, 100 μ L1 × streptavidin was added and incubated for 1 hour at 37 ℃.
(6) After washing the plate for 5 times, adding 100 mu L color development solution, and incubating for 15-20min at 37 ℃.
(7) Adding 50 mu L stop solution to stop the reaction, preheating the microplate reader in advance, and measuring the absorbance value of 450nm minus the absorbance value of 540nm to obtain the final result.
(8) A standard curve is established giving the value for each well. Statistical analysis and mapping were performed using Prism.
1.7 cell culture
Human Retinal Microvascular Endothelial Cells (HRMECs) were purchased from angiopromie and cultured in endothelial cell minimal medium (ECM) with 5% fetal bovine serum, 1% penicillin-streptomycin and Endothelial Cell Growth Supplement (ECGS). Cells were incubated at 37 ℃ and 5% carbon dioxide. The medium was changed every 2-3 days. The third to sixth generation cells were taken for experiments.
1.8 measurement of cell proliferation potency of CCK-8
Cell proliferation of HRMECs was determined by cell counting kit-8 (CCK-8; Dojindo Molecular Technologies, USA) in media with different concentrations of 4-HNE and TNFAIP8, and HRMECS was seeded in 96-well plates at a density of 2,000 cells/well. Cell growth was analyzed 24 or 48 hours after treatment. The Optical Density (OD) was measured at 450nm using a microplate reader.
1.9 vascular endothelial cell lumen formation
Matrigel was allowed to sit overnight at 4 ℃ and then placed in 48-well plates and allowed to solidify at 37 ℃ for 30 minutes HRMECs were treated with 500ng/ml TNFAIP8 for 48 hours and then cells were trypsinized, 5 × 10^4HRMECs were seeded into Matrigel and then incubated at 37 ℃ under 5% CO2, 4 high resolution images were collected in each group using microscopy and the tubes and networks formed therein were counted using ImageJ software, 3 times for this assay.
1.10 cell migration Capacity test
Cell confluence was 80-90%, HRMEC was trypsinized, resuspended in serum-free medium, and seeded at 1 × 10^5 cells per well on Transwell upper chamber (8 μm, Corning), then medium containing 20% FBS and 500ng/ml TNFAIP8 was added to the bottom well 600 μ L cells were incubated for 48 hours under 5% CO2 conditions, then cells migrating from the upper chamber to the lower chamber were fixed with 4% PFA and stained with crystal violet buffer, the number of cell migrations in five fields was calculated using a microscope at × 200, the average number of cells in the five fields was taken as the migrating cells in the set, the assay was performed 3 times.
1.11Western Blot
Proteins were extracted from extracellular vesicles and cells using RIPA buffer (R0010, Solarbio, beijing), the homogenate was centrifuged at 12000 × g for 15 minutes at 4 ℃, the supernatant was transferred to another clear tube, then 10 μ g of protein from the sample and cell lysate added with 5 fold protein loading buffer was heated at 95 ℃ for 5 minutes, all samples were loaded onto SDS-PAGE gels for electrophoresis, then the proteins that entered the gel were transferred to polyvinylidene fluoride (PVDF) membranes, after incubation with 5% milk for 1 hour at room temperature, the membranes were incubated with primary antibodies including CD9, CD63, CD81 or TNFAIP8 (1: 2000) overnight at 4 ℃ and washed 3 times with TBST (10 minutes each), the membranes were placed in HRP-conjugated secondary antibodies (abcam), finally, images were taken using enhanced chemiluminescence (EC L).
2. Results
2.1 results of extracellular vesicle identification
The NTA result shows that the features of sEVs and mEVs separated by the conventional method are obviously distinguished (figure 1), and the transmission electron microscope picture shows that the sEVs and the mEVs are different in size and shape (figure 2, the scale is 200 nm).
2.2 protein Mass Spectrometry results
The data were subjected to bioinformatic analysis using the R language (version 3.5.3) psych package, including differential protein screening between samples (FIG. 3: a up-regulated protein, b down-regulated protein).
2.3 Using E L ISA method to detect TNFAIP8 expression level in plasma, extracellular vesicles, vitreous
TNFAIP8 was expressed differently in plasma and in plasma that was not lysed, and after membrane disruption treatment with lysate, it was possible to detect a significantly higher level of TNFAIP8 protein expression than in plasma that was not lysed (FIG. 4). No statistical difference was detected for each group of levels in the mEVs (figure 5). Significant differences were detected in the levels of TNFAIP8 in each group in the svs, with the NPDR and PDR groups being significantly higher than the control group (fig. 6). TNFAIP8 was significantly different in the NPDR and PDR groups as detected in the vitreous of each group of patients (fig. 7).
2.44-HNE induces proliferation of HRMECs and high expression of TNFAIP8
The 4-HNE was added to the culture medium of HRMECs, and the concentration gradient was found to cause the cell to proliferate significantly (FIG. 8). The cell expression of TNFAIP8 in the 4-HNE stimulated group was significantly increased at this concentration as measured by Western Blot (FIG. 9).
2.5 TNFAIP8 promotes the proliferation, tube formation and migration of HRMECs.
TNFAIP8 was added to HRMECs medium and found to cause significant cell proliferation at 500ng/ml concentration by concentration gradient (FIG. 10), and functional experiments were performed at this concentration: enhanced luminal formation of HRMECs (fig. 11) and enhanced migration of HRMECs (fig. 12).
2.6 Low expression of TNFAIP8 inhibits proliferation, tube formation and migration of HRMECs
HRMECs were transfected with lentiviruses, and their expression in cells was reduced by ShRNA knock-down of TNFAIP8 in HRMECs (FIG. 13), and functional assays were performed using the knocked-down cells: decreased cell viability of HRMECs (fig. 14) decreased luminal formation of HRMECs (fig. 15) and decreased migration of HRMECs (fig. 16).
3. Discussion of the related Art
According to the results of protein mass spectrometry experimental analysis, the E L ISA verifies that the expression of TNFAIP8 in plasma small cell outer vesicles and vitreous bodies of DR patients is increased, and the expression of the TNFAIP8 in plasma and mEVs is not different.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.

Claims (4)

1. The application of protein TNFAIP8 in plasma small extracellular vesicle as the marker of proliferative diabetic retinopathy diagnosis.
2. The application of protein TNFAIP8 in plasma small extracellular vesicle as target point for preparing medicine for treating diabetic retinopathy.
3. A method for detecting protein TNFAIP8 in plasma small cell outer vesicle, which is characterized in that: the method comprises the following steps:
the reagents in the E L ISA kit and the desired 96-well plate were left at room temperature for 15 minutes;
preparing 1 × cleaning buffer solution, 1 × biotinylated alkaline phosphatase conjugate, 1 × biotin-HRP and standard substance, adding EVs into sample diluent, adding the diluted standard substance and a sample to be detected into a hole, incubating for two hours at 37 ℃, sucking the solution in the hole, adding 1 × cleaning buffer solution into each hole, washing for 3 times, adding 1 × biotinylated alkaline phosphatase conjugate, incubating for 1 hour at 37 ℃, washing a plate for 3 times, adding 1 × biotin-HRP, incubating for 1 hour at 37 ℃, washing the plate for 5 times, adding developing solution, incubating for 15-20min at 37 ℃, adding stop solution to terminate the reaction, preheating an enzyme labeling instrument in advance, measuring the 450nm light absorption value and subtracting the 540nm light absorption value to obtain the final result, establishing a standard curve, obtaining the value of each hole, and performing statistical analysis and mapping by using Prism;
wherein EVs is sample diluent, diluted standard or sample to be detected, 1 × washing buffer solution, 1 × biotinylated alkaline phosphatase conjugate, 1 × biotin-HRP, developing solution and stopping solution, and the proportion of the ratio is mu × 0, mu L, mu L, m L, mu L, mu L, mu L and mu L is 20: 80, 100: 100: 100: 50;
the sample diluent is 1% NaCl, 0.1% Tris base, 0.1% Proclin 300, H2And O, wherein the percentage is mass percent.
4. The method for detecting the protein TNFAIP8 in the plasma small extracellular vesicles according to claim 3, wherein the E L ISA kit is Abebio-AE 14038H.
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