Application of secreted protein CTSB in prediction of onset risk of peripheral vasculopathy of DFU patient

Abstract

The invention provides an application of secreted protein CTSB (cathepsin B) in vascular disease risk prediction of diabetic foot ulcer (DFU) patients, and belongs to the technical field of biological medicines. Single cell sequencing research finds that secreted protein CTSB is significantly enriched in vascular endothelial cells in blood vessels of DFU patients, PPI connectivity is high, and expression is low; an overexpression / knockout experiment of the CTSB shows that the CTSB is related to proliferation and tubule generation of vascular endothelial cells in human blood vessels; the ELISA experiment also finds that the level of secretory protein CTSB in serum can be used as a marker for predicting the vasculopathy risk of a DFU patient, and the expression level of CTSB is negatively correlated with Wanger grading of diabetic foot; the method can predict the onset risk of peripheral vasculopathy of DFU patients, has the advantages of simple operation, high sensitivity and the like, can reduce false detection and missing detection, has stable and reliable detection results, and is suitable for large-scale detection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the application of secretory protein CTSB in serum in predicting the risk of developing vascular lesions in DFU patients. Background Art

[0002] Diabetic foot ulcer (DFU) is a common chronic complication of diabetes mellitus (DM) and one of the main causes of disability and death in DM patients. Among DM patients worldwide, 4%-10% suffer from DFU, with a lifetime morbidity of 25%, an annual mortality rate of 11%, and amputation patients as high as 22%, with a 5-year mortality rate of more than 70%. At the same time, the median healing time of DFU is 32 weeks, which is a major public health problem that places a heavy burden on society. Peripheral vascular disease, peripheral neuropathy and foot deformity are the main causes of DFU, of which 50% of DFU patients suffer from peripheral vascular disease. The destruction of peripheral blood vessels, the reduction of new blood vessels, the difficulty of vascular regeneration and vascular dysfunction in peripheral vascular disease are important factors that make diabetic ulcer wounds difficult to heal. Early detection, early diagnosis and early treatment can effectively improve the outcome of DFU, but about 50% of DFU patients often miss the best treatment period due to the lack of typical manifestations. In addition, the blood vessels of diabetic patients are often accompanied by arterial calcification and difficulty in contraction, which makes the diagnosis of DFU challenging, resulting in some DFU ulcers developing into chronic ulcers and unable to heal.

[0003] At present, the diagnostic methods for peripheral vascular disease of DFU include ankle-brachial index, toe-brachial index, transcutaneous oxygen partial pressure, Doppler ultrasound, magnetic resonance angiography, digital subtraction angiography, etc. However, these diagnostic methods for peripheral vascular disease of DFU have some limitations: first, they are all non-invasive screening tests, with insufficient sensitivity and specificity, and cannot completely and reliably exclude peripheral vascular disease of DFU. In addition, the revascularization time of DFU is short, and the diagnosis of these diagnostic methods is lagging. These limitations mean that the clinic urgently needs to find sensitive, specific, comprehensive and rapid diagnostic markers for the risk of peripheral vascular disease in patients with DFU. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide the application of secretory protein CTSB in the risk prediction of vascular lesions in patients with DFU. The present invention found through single-cell sequencing that the secretory protein CTSB is significantly enriched in the vascular endothelial cells in the blood vessels of DFU patients, with high PPI connectivity and low expression; the overexpression / knockout experiment of CTSB found that CTSB is associated with the proliferation and tubule formation of vascular endothelial cells in human blood vessels under a high-sugar environment, indicating that CTSB is associated with the vascular lesions of DFU; the ELISA experiment also found that the level of secretory protein CTSB in serum can be used as a marker for predicting the risk of vascular lesions in patients with DFU, and the expression level of CTSB is negatively correlated with the Wanger grade of diabetic foot. Therefore, the secretory protein CTSB in serum can be used as a marker for predicting the risk of vascular lesions in patients with DFU.

[0005] The present invention provides the use of a reagent for detecting the content of secretory protein CTSB (i.e., cathepsin B) in the preparation of a reagent or kit for predicting the risk of vascular lesions in DFU patients. When the content of secretory protein CTSB is significantly lower than that of a healthy individual, it is determined that the individual to be tested has a risk of vascular lesions.

[0006] Preferably, the secretory protein CTSB is the secretory protein CTSB in peripheral blood; the sample used to detect the secretory protein CTSB is preferably peripheral blood, serum or plasma.

[0007] Preferably, the reagent for detecting the content of secretory protein CTSB includes an ELISA detection reagent.

[0008] Preferably, overexpression of the secretory protein CTSB in a high-glucose environment promotes the proliferation and tubulogenesis of vascular endothelial cells in the blood vessels of the DFU wound.

[0009] Preferably, knocking down the secretory protein CTSB in a high-glucose environment inhibits the proliferation and tubule formation of vascular endothelial cells in the blood vessels of the DFU wound.

[0010] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides the use of a reagent for detecting the content of secretory protein CTSB (cathepsin B) in the preparation of a reagent or kit for predicting the risk of vascular lesions in DFU patients. When the content of secretory protein CTSB is significantly lower than that of a healthy individual, it is determined that the individual to be tested has a risk of vascular lesions.

[0011] The present invention found through single cell sequencing that the secretory protein CTSB is significantly enriched in the vascular endothelial cells in the blood vessels of DFU patients, with high PPI connectivity and low expression; the overexpression / knockout experiment of CTSB found that CTSB is related to the proliferation and tubulogenesis of vascular endothelial cells in human blood vessels; the ELISA experiment also found that the secretory protein CTSB level in serum can be used as a marker for predicting the risk of vascular lesions in DFU patients, and the expression level of CTSB is negatively correlated with the Wanger grade of diabetic foot, so the secretory protein CTSB in serum is used as a marker for predicting the risk of peripheral vascular lesions in DFU patients. The present invention provides a new method for predicting the risk of peripheral vascular lesions in DFU patients, which has important clinical value.

[0012] The method provided by the present invention is helpful to predict the risk of peripheral vascular disease in DFU patients at an early stage. The present invention has the advantages of simple operation and high sensitivity, and is easy to use and can reduce false detection and missed detection.

[0013] The present invention adopts the ELISA method to detect the content of secretory protein CTSB in peripheral blood. The method is simple to use, short in time, low in cost, stable and reliable in detection results, and suitable for large-scale detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the gene network connection diagram of the PPI combined with the EcCentricity algorithm for single-cell transcriptome;

[0015] Figure 2 This is a cell proliferation analysis diagram of the siNC group, si-CTSB group, oeNC group, and oe-CTSB group in Example 2;

[0016] Figure 3 This is a diagram of cell tubule formation analysis of the siNC group, si-CTSB group, oeNC group, and oe-CTSB group in Example 3;

[0017] Figure 4 This is an analysis chart of CTSB expression levels in the normal group, DFU group, and DFU of different Wanger grades detected by ELISA. DETAILED DESCRIPTION

[0018] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0019] Example 1

[0020] Mining genes or their expression products related to DFU vasculopathy by single-cell sequencing

[0021] The implementation steps are as follows:

[0022] Single-cell sequencing

[0023] The single-cell transcriptome gene expression datasets GSE165816 and GSE223964 (including foot ulcer skin samples from 9 DFU patients and foot skin samples from 4 normal people) were downloaded from the GEO database. KEGG enrichment analysis of single-cell transcriptomes was performed, and the clusterProfiler software package was used to identify genes significantly enriched in the vascular endothelial cells of DFU. It was found that the expression of CTSB in DFU patients was significantly lower than that in normal skin, and the gene was specifically expressed in vascular endothelial cells. The enriched gene connection network obtained by combining the PPI of single-cell transcriptomes with the EcCentricity algorithm was found to have a high PPI connectivity in the gene network enriched in DFU and is a secreted protein (see Figure 1 ).

[0024] Table 1 CTSB gene enrichment analysis results

[0025]

[0026] Example 2

[0027] CCK8 experiment verifies that CTSB expression is associated with the proliferation of endothelial cells in DFU wound vessels

[0028] The implementation steps are as follows:

[0029] Experimental groups:

[0030] si-CTSB group: CTSB gene was knocked down in vascular endothelial cells. The interference sequence was purchased from Suzhou Genema Gene Co., Ltd.;

[0031] siNC group: control group for knocking down the target gene in vascular endothelial cells. The interference sequence was purchased from Suzhou Genema Gene Co., Ltd.;

[0032] oe-CTSB: CTSB gene was overexpressed in endothelial cells. This expression vector was purchased from Guangzhou Dongze Biotechnology Co., Ltd.

[0033] oeNC group: control group with overexpression of target gene in vascular endothelial cells. The expression vector was purchased from Guangzhou Dongze Biotechnology Co., Ltd.

[0034] The vascular endothelial cells transduced by the siNC group, si-CTSB group, oeNC group, and oe-CTSB group were inoculated in 96-well plates at 5000 cells per well, and 100 μL of high-glucose culture medium was added to each well. The plates were cultured in a 37°C, 5% CO2 incubator, and the culture plates were taken out at 0h, 24h, 48h, 72h, and 96h of culture, and CCK8 solution 5mg / mL, 10μL / well was added. After incubation for 2h, the absorbance (OD) value of each well was measured at 450nm. The larger the OD value, the more living cells there are and the stronger the cell activity. The interval time was set as the horizontal axis and the absorbance value was set as the vertical axis to draw a CCK8 curve. Through experiments, it was found that the cell proliferation rate of the si-CTSB group was significantly slower than that of the siNC group in a high-glucose environment, which confirmed that the knockdown of CTSB can inhibit the proliferation of vascular endothelial cells in the DFU wound vessels. Through experiments, it was found that the cell proliferation rate of the oe-CTSB group was significantly faster than that of the oeNC group under high glucose conditions, which confirmed that the overexpression of CTSB can promote the proliferation of vascular endothelial cells in the DFU wound blood vessels (see Figure 2 ).

[0035] Example 3

[0036] Tube formation experiment verifies that CTSB expression is associated with tubule formation in endothelial cells in DFU wound vessels

[0037] The implementation steps are as follows:

[0038] Matrigel was added to a 24-well plate at a volume of 50 μL per well, spread evenly and placed at 4°C overnight. The 24-well plate was then placed in a 37°C, 5% CO2 incubator for 1 hour to allow the matrigel to solidify. The vascular endothelial cells transduced by the siNC group, si-CTSB group, oeNC group, and oe-CTSB group were seeded in a 24-well plate at 150,000 cells per well, respectively. 500 μL of high-glucose culture medium was added to each well, and the cells were incubated at 37°C for 24 hours. The tube formation was observed under a confocal microscope and photographed. Through experiments, it was found that the cell proliferation rate of the si-CTSB group was significantly slower than that of the siNC group under a high-glucose environment, which confirmed that the knockdown of CTSB can inhibit the tubule formation of vascular endothelial cells in the DFU wound blood vessels. Through experiments, it was found that the cell proliferation rate of the oe-CTSB group was significantly faster than that of the oeNC group under a high-glucose environment, which confirmed that the overexpression of CTSB can promote the tubule formation of vascular endothelial cells in the DFU wound blood vessels (see Figure 3 ).

[0039] Example 4

[0040] Enzyme-linked immunosorbent assay was used to detect the expression of CTSB in patients

[0041] Specific testing process:

[0042] Dissolve CTSB antigen with 50mM carbonate coating buffer (pH 9.6) to make the antigen concentration 15μg / mL, add 100μL / well to 96-well ELISA plate, place at 4℃ overnight, discard the coating solution the next day, and wash 3 times with PBST. Add 5% BSA and place at 37℃ for 40 minutes. Fill each reaction well with blocking solution during blocking, remove bubbles in each well, and wash 3 times with PBST after blocking. Repeat the above steps and divide the blocked ELISA plate into 3 groups, 17 in each group. The diluted ELISA test serum samples of healthy people and DFU group were added to the ELISA reaction wells, 100μL per well, placed at 37℃ for 40 minutes, and washed 3 times with PBST after completion. Add enzyme-labeled antibody, 100 μL per well, incubate at 37°C for 60 minutes, and wash three times with PBST. Add TMB-urea hydrogen peroxide solution as substrate solution, 100 μL per well, incubate at 37°C in dark for 10 minutes. Add 50 μL of terminator to terminate the reaction and measure the experimental results. Use an enzyme reader to read the data at a wavelength of 450 nm, and calculate the corresponding concentration based on the OD value. Through experiments, it was found that the expression level of CTSB in the DFU patient group was lower than that in the healthy group, and its expression level decreased with the increase of DFU Wanger grade, which further proved that the expression level of CTSB was negatively correlated with the risk of vascular lesions in DFU (see Figure 4 ).

[0043] Table 2 OD value test results of serum samples from healthy group and DFU group

[0044]

[0045] Table 3 OD value test results of serum samples in the DFU group with different Wanger grades

[0046]

[0047] The results of the above examples show that the secretory protein CTSB is significantly enriched in the vascular endothelial cells of the DFU wound surface and is lowly expressed. The expression level of the secretory protein ELSIA is negatively correlated with the Wanger grade of DFU, and the mechanism by which CTSB promotes the pathogenesis of DFU is related to its inhibition of the proliferation and tubule formation of vascular endothelial cells in the patient's wound surface. These results show that the secretory protein CTSB can be used as a marker for the risk prediction of DFU peripheral vascular lesions.

[0048] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a reagent for detecting the content of secretory protein CTSB in a reagent or kit for predicting the risk of vascular lesions in patients with DFU, characterized in that: When the content of secretory protein CTSB in the serum of a DFU patient is significantly lower than the average CTSB content of a healthy person, it is determined that the DFU patient to be tested has a risk of developing vascular disease.

2. The use according to claim 1, characterized in that: The secretory protein CTSB is the secretory protein CTSB in peripheral blood.

3. The use according to claim 1 or 2, characterized in that: Reagents for detecting the content of secretory protein CTSB include ELISA detection reagents.

4. The use according to claim 1, characterized in that: Overexpression of the secretory protein CTSB in a high-glucose environment promotes the proliferation and tubulogenesis of vascular endothelial cells in DFU wound vessels.

5. The use according to claim 1, characterized in that: Knockdown of the secretory protein CTSB in a high-glucose environment inhibits the proliferation and tubule formation of vascular endothelial cells in DFU wound vessels.

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