CN113252895A

CN113252895A - Application of serum cathepsin D in lymphedema diseases

Info

Publication number: CN113252895A
Application number: CN202010084796.9A
Authority: CN
Inventors: 张曼; 孟倩
Original assignee: Beijing Shijitan Hospital
Current assignee: Beijing Shijitan Hospital
Priority date: 2020-02-10
Filing date: 2020-02-10
Publication date: 2021-08-13

Abstract

The invention provides a clinical application specification of a protein named cathepsin D (CTSD), in particular to preparation of serum cathepsin D, which is used for auxiliary diagnosis of a patient with lymphedema. The invention proves that compared with normal population (normal control), the expression of CTSD in the serum of a patient with lymphedema is increased, and the CTSD can be used for monitoring and auxiliary diagnosis of the lymphedema diseases.

Description

Application of serum cathepsin D in lymphedema diseases

Technical Field

The invention relates to a new application of serum cathepsin D, in particular to an application of the serum cathepsin D in lymphedema diseases.

Background

Lymphedema is a chronic disease caused by dysplasia or severe damage of lymphatic system, and is characterized by unbalanced generation and drainage of lymphatic fluid, retention of a large amount of interstitial fluid in interstitial space and wide-spread edema of tissues. Lymphedema is a highly disabling disease, ranked 2 in the array of disabling diseases by the world health organization. The main reason is that the disease is usually not regarded at the early stage of the disease, and the disease gradually progresses from the accumulation of body fluid in the superficial soft tissues of limbs, and undergoes the irreversible disease course of chronic inflammation, fibrous connective tissue hyperplasia, fat sclerosis, fascia thickening and the thickening of the whole limb. The thickened skin, hyperkeratosis of epidermis and hyperplasia of subcutaneous tissue comprise a large amount of fiber components, which make the lesion tissue of the limbs in the late stage hard like elephant skin, also called elephantiasis. In addition to the thickening of the limbs, patients often suffer from erysipelas, excrescence-like skin hyperplasia, ulcer and the like, and part of patients can cause disability and lose labor capacity. Lymphedema is classified into primary and secondary 2 major categories based on etiology. About 10% of patients have primary lymphedema caused by congenital lymphatic system defect or dysplasia, mostly develop diseases before the age of 35 years, have latent diseases, or are caused by infection and trauma, and are difficult to diagnose. Although less common, some primary lymphedema is familial, such as Milroy and Meige syndromes. The remaining 90% are secondary lesions resulting from filariasis, local infection, trauma, post-tumor resection surgery. The secondary lesions often have definite medical history of operations, infection and the like.

At present, the inspection means of lymphedema is mainly an imaging method, and the treatment is mainly surgical treatment mainly for controlling symptoms, but various conventional inspection and treatment methods have certain limitations: 1) the invasive lymphography is an imaging method which is firstly used for lymphatic vessel examination, and has the defects of being invasive, limited in examination range, incapable of displaying the surrounding conditions of lymphatic vessels, easy to generate incision infection, lymphatic leakage, lymphangitis, pulmonary embolism and other complications. 2) The difference nuclide lymphography is considered as a main examination method for limb lymphedema, but the method has the defects of poor spatial and time resolution, radiation and the like, and limits the evaluation of the degree of lymphatic circulation disorder. 3) The imaging of the precious MRI lymphatic system, the three-dimensional MRI examination and the dynamic MRI lymphatic system imaging are considered as an alternative method and an effective supplement of the current diagnostic means, but the examination cost is high, so the method cannot be used as a conventional screening means. 4) The imaging examination equipment and the treatment operation of the scanty lymphatic system are generally developed in few special hospitals for lymphatic diseases, and cannot be realized in general primary hospitals. For the reasons mentioned above, often delaying diagnostic treatment brings serious consequences: 5) repeated contact imaging and surgery can be a psychological and economic burden and nuisance on patients and their families.

For the diagnosis and identification of lymphedema diseases, the current auxiliary examination means is limited, and no effective conventional blood and body fluid biomarkers are applied to clinic. In terms of treatment, targeted therapy of the disease becomes a focus of attention in recent years, but currently, there are very few therapeutic targets that can be applied clinically, and the effect of many genes and proteins identified in primary diseases for targeted therapy of secondary diseases is unclear and related studies are few. Therefore, compared with the normal population, the protein cathepsin D related to the damage and reconstruction of the lymphatic tissues is screened and identified in the serum of the patient with lymphedema.

Disclosure of Invention

The invention aims to provide a preparation method of serum cathepsin D, which is used for the auxiliary diagnosis of a patient with lymphedema.

Preferably, the amino acid sequence of the serum cathepsin D is shown as SEQ ID NO.1 (MQPSSLLPLALCLLAAPASALVRIPLHKFTSIRRTMSEVGGSVEDLIAKGPVSKYSQAVPAVTEGPIPEVLKNYMDAQYYGEIGIGTPPQCFTVVFDTGSSNLWVPSIHCKLLDIACWIHHKYNSDKSSTYVKNGTSFDIHYGSGSLSGYLSQDTVSVPCQSASSASALGGVKVERQVFGEATKQPGITFIAAKFDGILGMAYPRISVNNVLPVFDNLMQQKLVDQNIFSFYLSRDPDAQPGGELMLGGTDSKYYKGSLSYLNVTRKAYWQVHLDQVEVASGLTLCKEGCEAIVDTGTSLMVGPVDEVRELQKAIGAVPLIQGEYMIPCEKVSTLPAITLKLGGKGYKLSPEDYTLKVSQAGKTLCLSGFMGMDIPPPSGPLWILGDVFIGRYYTVFDRDNNRVGFAEAARL); or an amino acid sequence which is derived from the amino acid sequence shown in SEQ ID NO.1 and has the same function with the amino acid sequence shown in SEQ ID NO. 1.

Preferably, the cathepsin D is derived from blood.

Preferably, the serum cathepsin D is highly expressed in lymphedema patients.

Preferably, the preparation is a serum cathepsin D detection kit for a patient with lymphedema.

Preferably, the kit comprises one or more of an aptamer antibody or antibody fragment capable of specifically binding to cathepsin D protein.

Preferably, the kit further comprises a component selected from the group consisting of: the kit comprises a solid phase carrier, a diluent, a reference substance, a standard substance, a quality control substance, a detection antibody, a second antibody diluent, a luminescent reagent, a washing solution, a color development solution and a stop solution, wherein the solid phase carrier is any one or a combination of a plurality of the solid phase carrier, the diluent, the reference substance, the standard substance, the quality control substance, the detection antibody, the second antibody and the second antibody diluent.

Preferably, the standard comprises a cathepsin D protein standard, a humanized tag antibody standard; preferably, the quality control product comprises: a cathepsin D protein quality control product and a humanized label antibody quality control product; preferably, the solid support comprises: microparticles, microspheres, slides, test strips, plastic beads, liquid phase chips, microwell plates, or affinity membranes.

Preferably, the solid phase carrier is made of any one of polyvinyl chloride, polystyrene, polyacrylamide and cellulose.

The inventor firstly collects primary lymphedema, secondary lymphedema and normal control serum samples and extracts protein. Uniformly mixing 1 mu l of sample and 10 mu l of matrix, taking 1 mu l of point on an Anchorchip target plate, ionizing the sample, carrying out mass spectrometry, and collecting data within the range of 1000-10000Da to obtain a mass spectrometry polypeptide diagram consisting of protein peaks with different mass-to-charge ratios. And analyzing all mass spectrograms of the normal control group, the secondary lymphedema group and the primary lymphedema group by using BioExplorer analysis software, and screening the differential polypeptides. Then the inventor carries out matrix-assisted laser desorption ionization time-of-flight mass spectrometry identification on the differential polypeptide with statistical significance, obtains the cathepsin D protein with differential expression by database retrieval, and compared with a normal control, the cathepsin D protein is high expressed in the serum of a patient with lymphedema.

The invention proves that compared with a normal control group, the cathepsin D is highly expressed in the serum of a patient with lymphedema through research. Thus, it is proposed that the detection of serum cathepsin D can be used for monitoring or aided diagnosis of lymphedema diseases.

In order to make the aforementioned and other objects, features and advantages of the present invention comprehensible, preferred embodiments accompanied with figures are described in detail below.

Drawings

FIG. 1 is the mass spectrometric identification protein cluster analysis of the normal control group, the secondary lymphedema group and the primary lymphedema group.

FIG. 2 is the differential expression of serum cathepsin D in the normal control group, the secondary lymphedema group, and the primary lymphedema group.

FIG. 3 is a Western blot to confirm differential expression of serum cathepsin D in normal control, secondary lymphedema, primary lymphedema.

Detailed Description

Example 1Collection and processing of serum samples

Sample source: collecting the primary and secondary lymphedema patients who are treated in 12 months from 2019 to 2021, wherein the age is more than 4 and less than 60, and the patients are unlimited. Collecting random urine and blood samples; the experiment was performed following the declaration of helsinki, approved by the ethics committee of hospitals.

Primary, secondary lymphedema is included as a criterion:

primary and secondary lymphedema are definitely diagnosed by lymphography, and the clinical stages of the diseases are in stages I to III;

no other lower limb vascular diseases, no chronic diseases such as skin diseases, blood diseases and diabetes and the like, and the secondary lymphedema patient reaches the clinical cure standard;

12 cases of lymphedema group, disease control group and normal control group respectively; the inclusion standard of healthy people is as follows:

the physical edema expression or medical history, chronic disease and tumor medical history are not clear, indexes such as blood sugar, blood fat, blood pressure, liver and kidney functions and the like are normal, obvious discomfort is not caused in nearly 1 week, and long-term medication history is not caused; exclusion criteria:

except for those that do not meet inclusion criteria;

except for those with unclear diagnosis;

except for pregnant women and patients with mental diseases.

Example 2Lable free mass spectrometric detection

The extracted protein concentration was determined by the Bradford method. Firstly, diluting a sample by a certain multiple with a protein lysate to enable the final concentration to fall within a standard curve range, taking 10 mu l of each diluted sample and a standard substance (bovine serum albumin is dissolved into standard proteins with series concentrations with the protein lysate), respectively reacting with 300 mu l of protein quantitative dye in a dark place for 20min, simultaneously measuring the light absorption values of the standard substance and the sample at 595nm by using an enzyme labeling instrument, drawing a standard curve according to the relation between the light absorption value and the concentration of each tube of the standard substance, and then calculating the concentration of the sample. Mu.g of each sample was subjected to polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Brilliant blue for 30min, and destained until the background was clear. Cutting each piece of glue into 3 pieces with a diameter of 1mm by a scalpel, and placing the pieces in a 1.5ml test tube; washing with 200 μ l double distilled water for 2 times, each for 10 min; adding combo-destaining solution (50 mM ammonium bicarbonate (NH 4HCO 3) and acetonitrile (ACN, 1: 1)) or silver stain destaining solution 200 μ l (K3 (Fe (CN)6 and Na2S2O3(1: 1)), destaining for 15 min, double distilled water washing, repeating for 3 times until destaining is complete, adding ACN 100 μ l and dehydrating until colloidal particles become white, vacuum-drying for 10 min, adding 10mM Dithiothreitol (DTT) 200 μ l, dissolving with 25mM NH4HCO3, water-bathing for 1h at 37 deg.C, adding ACN 100 μ l and dehydrating until colloidal particles become white, adding 55mM indole-3-acetic acid (IAA) 200 μ l, dissolving with 25mM NH4HCO3, placing in dark room for 30min, adding ACN 100 μ l and dehydrating until colloidal particles become white, sequentially adding double distilled water (1), ACN (1), double distilled water (1) and ACN (1 min), diluting with trypsin (82.01 μ l) and diluting with trypsin (NH 4O) 2 min), adding 100 μ l of the enzyme solution into each tube, centrifuging slightly, allowing the enzyme solution to contact with the colloidal particles, and standing at 4 deg.C for 30 min. When the enzyme solution is completely absorbed by the colloidal particles, 3100 mu l of 25mM NH4HCO is added, and the mixture is kept at 37 ℃ overnight; the next day, the enzymolysis supernatant was collected by centrifugation and placed in another Eppendorf tube; extracting the residual micelle with extraction buffer (5% TFA, 95% ddH 2O) for 1h, collecting the enzymolysis supernatant, and placing the enzymolysis supernatant in an Eppendorf tube for combination; the remaining micelles were then extracted with extraction buffer (2.5% TFA, 50% ACN, 47.5% ddH 2O) for 1h, and the enzymatic supernatants were collected and combined in the previous Eppendorf tube; the fractions obtained by high pH reverse phase separation were redissolved with 20ul of 2% methanol, 0.1% formic acid. Centrifuging at 12000 rpm for 10 min, and sucking the supernatant. The sample loading volume is 10ul, and the sample loading is carried out by adopting a sandwich method. LoadingPump flow rate 350nl/min, 15 minutes. The separation flow rate was 350 nl/min. An EASY-nLC1000 HPLC system was used for the separation. Liquid phase a was 0.1% formic acid in acetonitrile (2% acetonitrile) and liquid phase B was 0.1% formic acid in acetonitrile (98% acetonitrile). The 100% A solution was equilibrated on a 100X 100mm BEHnanoACquisity column at a flow rate of 400 nl/min. The chromatographic spray needle is PN: FS 360-20-10-N-20-C12. Samples were separated by capillary HPLC and analyzed by Q-exact mass spectrometer (Thermo). The ion source voltage is 2.5kv, the analysis time is 120min, and the scanning range of the parent ion is 350-. Data analysis software PD (Proteome scanner 1.4, thermo) was used for the search. The search database is Mascot. The parent ion error was set to 20ppm, the fragment ion error was set to 1Da, the digestion mode was non-digestion, and the modification was oxidation (M) of methionine. Data card value, Percolator algorithm, FDR < 1%.

Example 2Western blot detection

Extracting serum protein for detection. 20ug of protein was subjected to 12% SDS-PAGE, voltage 12V was applied for 60 min, 5% skimmed milk powder was sealed at room temperature for 2 h, and cathepsin D monoclonal antibody was added thereto and incubated overnight at 4 ℃. PBST rinsing 3 times, each time 20min, adding secondary antibody, incubating at room temperature for 90 min, PBST rinsing, DAB developing.

Compared with the normal control group, the cathepsin D protein is highly expressed in the serum of the patient with lymphedema, and as shown in figure 3, the expression of the cathepsin D protein in the serum of the normal control group, the primary lymphedema and the secondary lymphedema is obviously different.

Although the present invention has been described with respect to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> Beijing century bed Hospital affiliated to capital medical university

<120> use of serum cathepsin D in lymphedema diseases

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Claims

1. Blood cathepsin D is used for the aid and differential diagnosis of lymphedema patients.

2. The use according to claim 1, wherein the amino acid sequence of the cathepsin D protein is as set forth in SEQ ID No. 1; or an amino acid sequence which is derived from the amino acid sequence shown in SEQ ID NO.1 and has the same function with the amino acid sequence shown in SEQ ID NO. 1.

3. The use according to claim 1, wherein cathepsin D is derived from blood.

4. The use according to claim 1, wherein cathepsin D is highly expressed in the serum of patients with lymphedema.

5. The use of claim 1, wherein the preparation is a serum cathepsin D test kit for normal persons or lymphedema patients.

6. The use of claim 5, wherein the kit comprises one or more of an antibody or antibody fragment capable of specifically binding cathepsin D aptamer.

7. The use according to claim 5, wherein the kit further comprises a component selected from the group consisting of: the kit comprises a solid phase carrier, a diluent, a reference substance, a standard substance, a quality control substance, a detection antibody, a second antibody diluent, a luminescent reagent, a washing solution, a color development solution and a stop solution, wherein the solid phase carrier is any one or a combination of a plurality of the solid phase carrier, the diluent, the reference substance, the standard substance, the quality control substance, the detection antibody, the second antibody and the second antibody diluent.

8. The use of claim 7, wherein the standard comprises a cathepsin D protein standard, a humanized tag antibody standard; preferably, the quality control product comprises: a cathepsin D protein control substance and a humanized label antibody quality control substance; preferably, the solid support comprises: microparticles, microspheres, slides, test strips, plastic beads, liquid phase chips, microwell plates, or affinity membranes.

9. The use of claim 8, wherein the solid phase carrier is made of any one of polyvinyl chloride, polystyrene, polyacrylamide and cellulose.