CN117783533A - Application of blood biomarkers as diagnostic markers and therapeutic efficacy testing markers for uremic calciphylaxis - Google Patents

Abstract

The invention discloses a blood biological marker for the diagnosis and efficacy detection of uremia with calciphylaxis, that is, calcific uremic arteriolopathy (CUA): thrombospondin-1 (THBS- 1) Combined with transforming growth factor-β1 (TGF-β1). The invention found that THBS‑1 is the core protein in the regulatory network of differential proteins in the plasma of CUA patients and uremic patients; in addition, among other differential proteins in the blood, the protein abundance of latent transforming growth factor beta-binding protein 1 (LTBP1) is higher in the plasma of CUA patients. Medium to high. Through a follow-up study of CUA patients who successfully received human amniotic mesenchymal stem cells (hAMSCs) treatment, plasma proteomic analysis found that the levels of THBS‑1 and LTBP1 both decreased after treatment, and the change trends of the two were highly consistent; further enzyme-linked Immunosorbent (ELISA) testing verified this trend. The present invention discovered for the first time that THBS-1 combined with TGF-β1 can be used as a hematological biomarker for early and accurate diagnosis and therapeutic efficacy detection of CUA.

Description

Blood biological markers as diagnostic and therapeutic markers for uremic calciphylaxis Application of ambition

Technical field

The present invention belongs to the field of prevention and treatment of uremic calciphylaxis, and particularly relates to the application of blood biological markers as markers for diagnosis and efficacy detection of uremic calciphylaxis.

Background technique

Calciphylaxis, also known as calcific uremic arteriolopathy (CUA) when associated with uremia, is a rare disease caused by cutaneous microvascular obstruction caused by thrombosis and vascular calcification. It is common in end-stage kidney disease (ESKD), especially in patients with diabetes and secondary hyperparathyroidism (SHPT). Among hemodialysis patients, the incidence of calciphylaxis is 0.04% to 4%.

The typical clinical manifestations of calciphylaxis are progressive, painful, necrotic skin lesions, which are common in fat-rich areas such as the abdomen, thighs, breasts, and buttocks. The early manifestations of skin lesions include: purpura, subcutaneous nodules, reticular erythema, light purple nodules, and bullous hemorrhages; without active and effective treatment, the lesions rapidly develop into foul-smelling necrotic ulcers covered with black scabs, presenting mummy-like gangrene of the legs and fingers. Once ulcers form, the patient's 6-month survival rate is only 20%. Difficult-to-heal wounds lead to multiple infections, and the resulting sepsis is the primary cause of death from the disease.

The pathological characteristics of calciphylaxis are calcification of the middle layer of blood vessels, intimal proliferation, microthrombosis, and interstitial calcification changes. Affected blood vessels are mostly found in the dermis and subcutaneous adipose tissue, with an average diameter of 100 μm (range 40-600 μm). Histopathological features of the skin are the gold standard for the diagnosis of calciphylaxis. Early and accurate diagnosis of calciphylaxis is difficult due to the difficulty of performing repeated skin biopsies and the risk of new ulcers and infections.

In the past decade, proteomic analysis has become a very powerful tool to discover unidentified proteins in different physiological or pathological states, thanks to the tremendous development of sample preparation and liquid chromatography mass spectrometry. Plasma is most often used for clinical diagnostics because it is easily accessible and rich in proteins “sampled” from every tissue and organ. These proteins reflect the pathophysiological state of the body and may contain biomarkers of disease and treatment response. The dynamic properties of plasma can reflect protein changes in the circulation system in space and time to better understand the molecular events in disease progression. Isobaric mass labeling by mass spectrometry, such as isobaric tags and tandem mass tags for relative and absolute quantification, has the advantages of high sensitivity, high resolution, and good reproducibility. Advanced proteomic technologies enable rapid plasma proteome analysis in a high-throughput manner, which has opened up new avenues for scientific research. However, there is no proteomic analysis of CUA to study biomarkers of calciphylaxis.

Contents of the invention

The purpose of the present invention is to address the shortcomings of the existing technology and provide the application of blood biological markers as markers for the diagnosis and efficacy detection of uremic calciphylaxis.

In order to achieve the above object, the present invention adopts the following technical solution: blood biological markers for diagnosis and efficacy detection of uremic calciphylaxis, and the blood biological markers are THBS-1 combined with TGF-β1.

The invention discloses an application of a preparation for detecting biological markers of uremic calciphylaxis in blood in preparing a reagent for diagnosing and detecting therapeutic efficacy of uremic calciphylaxis, wherein the blood biological markers are THBS-1 combined with TGF-β1.

The detection preparation detects the level of THBS-1 combined with TGF-β1 in the blood of uremic patients with calciphylaxis through ELISA. Compared with uremic patients without calciphylaxis, the level is significantly increased, so as to judge that uremic patients are accompanied by calciphylaxis. .

Furthermore, the test preparation detects the level of THBS-1 combined with TGF-β1 in the blood of patients with uremic calciphylaxis. After treatment, it is significantly lower than before treatment, so as to judge the efficacy of the treatment plan used.

A reagent for diagnosis or efficacy detection of uremic calciphylaxis, including: a preparation for detecting the expression of THBS-1 combined with TGF-β1.

A diagnostic or efficacy detection kit for uremic calciphylaxis, which includes the above-mentioned diagnostic reagent.

The present invention uses a deep proteomics method to compare the differential proteins in the plasma of uremic patients without calciphylaxis and uremic patients with calciphylaxis, and conducts follow-up research on CUA patients who have successfully received human amniotic mesenchymal stem cell therapy. , observe the change trend of differential proteins; further verify the blood samples of patients with uremia without calciphylaxis, patients with uremia with calciphylaxis, and patients with uremia with skin ulceration and infection but without calciphylaxis. It aims to provide a basis for searching for hematological biomarkers of CUA, achieving early and accurate diagnosis of the disease, exploring the occurrence and development mechanism of the disease, seeking new treatment targets, and guiding precise diagnosis and treatment.

Description of drawings

Figure 1 is an example of the healing process photos of buttock wounds treated with human amniotic mesenchymal stem cells in patients with calciphylaxis (A), (B) skin wounds (area: 12cm×9cm) before hAMSCs treatment, (C), (D), (E) Shows the healing status of skin wounds after 4, 12, and 15 months of treatment.

Figure 2 is a biological function diagram of THBS1.

Figure 3 is a schematic diagram of THBS-1 hydrolyzing LAP-LTBP protein to activate TGF-β1.

Detailed ways

In order to enable those in the technical field to better understand the solutions of the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only These are part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts should fall within the scope of protection of this application.

It should be noted that the terms "comprising" and "having" and any variations thereof in the description and claims of this application and the above-mentioned drawings are intended to cover non-exclusive inclusion, for example, a series of steps or units. The processes, methods, systems, products or devices are not necessarily limited to those steps or units expressly listed, but may include other steps or units not expressly listed or inherent to the processes, methods, products or devices.

From September 2018 to April 2023, a total of 4 cases of patients with uremia and calciphylaxis were accumulated. Due to the rapid progression of the disease, these 4 patients required conventional treatments including infection control, nutritional support, anemia management, chronic kidney disease-mineral bone metabolism disorder (CKD-MBD) management, and intravenous sodium thiosulfate (STS). , wound care, pain management and anticoagulation were ineffective. After multidisciplinary consultation, he received rescue treatment with human amniotic membrane mesenchymal stem cells and achieved good therapeutic effects (Figure 1).

A total of 10 age- and gender-matched patients with uremia without calciphylaxis during the same period were included according to the inclusion and exclusion criteria. Basic information was obtained, and blood samples were collected and sent for routine laboratory indicator monitoring. In addition, plasma samples from patients with uremia and calciphylaxis were collected and sent for proteomic analysis. For 34-year-old female patient 1 with severe CUA, plasma samples were collected before hAMSCs treatment, 3 days, 2 weeks, 1 month and 15 months after hAMSCs treatment and submitted for proteomic analysis.

All 4 CUA patients underwent skin biopsy to confirm the diagnosis of calciphylaxis before receiving hAMSCs treatment. Skin tissues were obtained for hematoxylin-eosin staining (HE).

This study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University (Jiangsu Provincial People's Hospital) (2018-QT-001, 2020-QT-01, 2020-QT-09, 2020-SCR-03). All patients signed informed consent in accordance with the Declaration of Helsinki.

Using high peak removal combined with labeled quantitative proteomics, we identified a total of 1,619 plasma proteins in plasma samples from three patients with calciphylaxis and 10 patients with uremia. By differential analysis of the protein expression matrix between the two groups of patients, a total of 20 significantly changed differential proteins were identified. Among them, thrombospondin-1 (THBS-1 or Tsp-1) was upregulated, with the highest fold change among the differential proteins.

We used proteomics to detect the expression changes of 20 differential proteins in the plasma of a CUA patient before hAMSCs treatment, 3 days, 2 weeks, 1 month and 15 months after treatment, and found that the protein abundance of THBS-1 and LTBP1 showed a highly consistent change trend after hAMSCs treatment, that is, the expression levels of both were the highest before treatment, and rapidly decreased after 3 days of treatment, the lowest after 1 month of treatment, and increased after 15 months of hAMSCs treatment. This suggests that THBS-1 and LTBP1 may play a synergistic role in the occurrence and development of the disease, and their rapid decline after hAMSCs treatment may be closely related to the effectiveness of stem cell therapy.

As a multi-domain glycoprotein, THBS-1 exerts multiple functions by binding to specific proteins in the extracellular matrix and receptors on the cell surface. High levels of THBS-1 antagonize angiogenesis by inducing endothelial cell apoptosis; by inducing the migration and proliferation of VSMC, promoting intimal hyperplasia leading to progressive stenosis and sclerosis of the lumen; by inhibiting the NO pathway, constricting blood vessels and reducing tissue perfusion; by enhancing Local thrombosis forms, accelerates vascular occlusion, and leads to the rapid development of ischemic injury. The biological functions of THBS-1 are shown in Figure 2.

LTBP1 has been confirmed to be an extracellular multi-domain protein that is critical for TGF-β1 folding, secretion, matrix localization and activation through interaction with fibrillin and other extracellular matrix (ECM) proteins. TGF-β1 usually exists in the form of an inactive large latent complex (LLC), which consists of C-terminal mature TGF-β1 peptide and N-terminal latency associated peptide (LAP). A sulfur bond is covalently bound to LTBP1. THBS-1 can hydrolyze LAP-LTBP protein to dissociate TGF-β1, and interacts with the LSKL sequence on the TGF-β1-LAP complex through the KRFK sequence on it, mediating the activation of TGF-β1 and participating in wound healing, Biological processes such as cell proliferation, inflammatory response, and fibrosis (see Figure 3).

This study further included 16 uremia patients, and there were no significant statistical differences in age, gender, BMI and blood bone mineral metabolism indicators (including blood calcium, phosphorus and iPTH) between the calciphylaxis group (N=4). The results showed that the plasma THBS-1 level of CUA patients (5319.67±9835.62) ng/ml was significantly higher than that of uremia patients (318.55±431.32) ng/ml (P=0.000). The plasma TGF-β1 level of CUA patients (41979.67±27103.42) pg/ml was significantly higher than that of uremia patients (3551.79±1707.04) pg/ml (P=0.000). This was consistent with the abundance trend of THBS-1 and LTBP1 in the corresponding groups of patients in proteomics.

ELISA test results confirmed that the blood THBS-1 and TGF-β1 levels of calciphylaxis patients 1 and 3 dropped rapidly 3 days after the first hAMSCs treatment, and dropped to the lowest value after 1 month of treatment, which was consistent with the change trend of blood THBS-1 and LTBP1 observed in proteomics before and after treatment of CUA patient 1. It is particularly noteworthy that the plasma THBS-1 and TGF-β1 levels of CUA patient 3 increased significantly again after the hAMSCs treatment was interrupted for half a year, exceeding the level before the first stem cell treatment. With the continuation of the second stage of stem cell treatment, the patient's blood THBS-1 and TGF-β1 levels showed a downward trend again. This study found that the plasma THBS-1 and TGF-β1 levels of CUA patients were much higher than those of uremic patients, and they could drop rapidly after hAMSC treatment, and their dynamic change trends were highly consistent.

We further analyzed patients with uremia accompanied by skin ulceration and infection, and skin pathology confirmed non-calcific defense disease, and found that their blood THBS-1 levels were close to those of ordinary uremic patients, and significantly lower than those of CUA patients. It is suggested that joint monitoring of blood THBS-1 and TGF-β1 protein levels has important advantages as blood biological markers in CUA patients.

It is to be understood that the invention has been described by way of example only and that modifications may be made within the scope and spirit of the invention. The preferred embodiments of the present invention are described in detail above. It should be understood that those skilled in the art can make many modifications and changes according to the concept of the present invention without creative efforts. Therefore, any technical solutions that can be obtained by those skilled in the art through logical analysis, reasoning or limited testing on the basis of the prior art based on the concept of the present invention should be within the scope of protection determined by the claims.

Claims (6)

1. A blood biological marker for diagnosing and detecting the curative effect of uremia calcification defenses disease is characterized in that: the blood biological marker is THBS-1 combined with TGF-beta 1.

2. The application of a preparation for detecting biological markers of uremia calcification defense disease in blood in preparing reagents for diagnosing and detecting the curative effect of uremia calcification defense disease is characterized in that: the blood biological marker is THBS-1 combined with TGF-beta 1.

3. The use according to claim 2, characterized in that: the detection preparation can be used for judging that uremia has calcification defense disease by detecting the levels of THBS-1 and TGF-beta 1 in blood of uremia calcification defense disease patients, and the levels are obviously increased compared with those of uremia patients without calcification defense disease.

4. The use according to claim 2, characterized in that: the detection preparation can judge that the used treatment scheme has curative effect by detecting the levels of THBS-1 and TGF-beta 1 in blood of uremic patients and obviously reducing the levels after treatment compared with the levels before treatment.

5. A diagnostic or therapeutic test agent for uremic calcification defenses, comprising: and (3) detecting the expression level of THBS-1 combined with TGF-beta 1.

6. A diagnosis or curative effect detection kit for uremia calcification defense disease is characterized in that: the diagnostic kit comprises the diagnostic reagent according to claim 5.