CN117783533A - Application of blood biological marker as uremia calcification defense disease diagnosis and curative effect detection marker - Google Patents
Application of blood biological marker as uremia calcification defense disease diagnosis and curative effect detection marker Download PDFInfo
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Abstract
The invention discloses a blood biological marker for diagnosing and detecting the curative effect of uremia accompanied with calcification defenses (calciphylaxis), namely calcified uremic arteriolar diseases (calcific uremic arteriolopathy, CUA): thrombospondin-1 (THBS-1) in combination with transforming growth factor-beta 1 (TGF-beta 1). The invention discovers that THBS-1 is a core protein in a plasma difference protein regulation network of CUA patients and uremic patients; furthermore, among other differential proteins in the blood, the protein abundance of latent transforming growth factor beta binding protein 1 (LTBP 1) is elevated in CUA patient plasma. Through follow-up study on CUA patients successfully receiving human amniotic mesenchymal stem cells (hAMSCs), plasma proteomic analysis finds that the levels of THBS-1 and LTBP1 are reduced after treatment, and the change trend of the two is highly consistent; further enzyme-linked immunosorbent assay (ELISA) detection confirmed this trend. The invention discovers that THBS-1 combined with TGF-beta 1 can be used as a blood biomarker for CUA early-stage accurate diagnosis and curative effect detection for the first time.
Description
Technical Field
The invention belongs to the field of prevention and treatment of uremia calcification defense diseases, and particularly relates to application of a blood biological marker as a uremia calcification defense disease diagnosis and curative effect detection marker.
Background
Calcification defense disease (calciphylaxis), which is also known as calcified uremic arterioles (calcific uremic arteriolopathy, CUA) when associated with uremic patients, is a rare disease of cutaneous microvascular obstruction caused by thrombosis and vascular calcification, commonly seen in end-stage renal disease (end-stage kidney disease, ESKD), particularly in patients with diabetes mellitus, secondary hyperparathyroidism (secondary hyperparathyroidism, SHPT). In hemodialysis patients, the incidence of calcification defenses is 0.04% -4%.
Calcification defenses are typically manifested as progressive, painful, necrotic skin lesions, commonly found in fat-rich areas such as abdomen, thighs, breasts and buttocks. Early manifestations of skin lesions were: purpura, subcutaneous nodules, reticuloendotheliosis, purplish induration, bullous bleeding; if no active treatment is effective, the lesions rapidly develop into malodorous necrotic ulcers, which are covered with black scab, and which appear as mummy-like gangrene of legs and fingers. Once ulcers form, the patient's 6 month survival rate is only 20%. Wounds that are difficult to heal lead to multiple infections, and sepsis is the leading cause of death in the disease.
The pathological features of calcification defense disease are calcification of the media layer, intimal proliferation, microthrombosis and altered calcification of the interstitium, the affected vessels are most common in the dermis and subcutaneous adipose tissue, and the average diameter is 100 μm (range 40-600 μm). Skin histopathological features are gold criteria for diagnosing calcification defenses. Repeated skin biopsies are difficult to perform and pose a difficulty in early, accurate diagnosis of calcification defenses due to the risk of new ulcers and infections being initiated.
During the last decade, proteomic analysis has become a very powerful tool for the discovery of unrecognized proteins in different physiological or pathological states due to the tremendous development of sample preparation and liquid chromatography mass spectrometry. Plasma is most commonly used for clinical diagnosis because it is readily available and is rich in proteins "sampled" from each tissue and organ. These proteins reflect the pathophysiological state of the body and may contain biomarkers for disease and therapeutic response. The dynamic nature of plasma can reflect protein changes in the circulatory system in space and time to better understand molecular events in disease progression. Isobaric mass labeling such as relative and absolute quantitative isobaric labels and tandem mass labels through mass spectrometry has the advantages of high sensitivity, high resolution, good reproducibility and the like. Advanced proteome technology enables rapid plasma proteome analysis in a high throughput manner, which opens up a new approach for scientific research. However, there has been no study of proteomic analysis of CUA to develop biological markers of calcification defense disease.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and provides application of a blood biological marker as a uremia calcification defense disease diagnosis and curative effect detection marker.
In order to achieve the above purpose, the present invention adopts the following technical scheme: and (3) a blood biological marker for diagnosing and detecting the curative effect of uremia calcification defense disease, wherein the blood biological marker is THBS-1 combined with TGF-beta 1.
Application of a preparation for detecting a biological marker of uremia calcification defense disease in blood in preparing reagents for diagnosing and detecting the curative effect of uremia calcification defense disease, wherein the biological marker of blood is THBS-1 combined with TGF-beta 1.
The detection preparation detects the level of THBS-1 combined with TGF-beta 1 in the blood of uremic calcification defending patients by ELISA method, and the level is obviously increased compared with that of uremic non-calcification defending patients, so that uremic patients are judged to be accompanied with calcification defending diseases.
Furthermore, the detection preparation can judge that the used treatment scheme has curative effect by detecting the level of THBS-1 combined with TGF-beta 1 in blood of uremic calcification defending patients and significantly reducing the level after treatment compared with the level before treatment.
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.
A kit for diagnosing or detecting the efficacy of uremia calcification defenses, wherein the diagnostic kit comprises the diagnostic reagent.
The invention compares the differential protein in the plasma of uremia non-calcification defensive patients and uremia calcification defensive patients by a deep proteomics method, and observes the variation trend of the differential protein by follow-up study on CUA patients successfully receiving human amniotic mesenchymal stem cell treatment; further, the blood samples of patients with uremia and skin-breaking infection but not calcification defense patients are verified. Aims at providing basis for seeking a blood biomarker of CUA, realizing early and accurate diagnosis of diseases, exploring the occurrence and development mechanism of the diseases, seeking a new therapeutic target and guiding accurate diagnosis and treatment.
Drawings
FIG. 1 is a photograph of the healing process of pedestrian amniotic mesenchymal stem cells of a patient with calcification defensive treatment on buttock wound surface (A), and (B) hAMSCs before treatment on skin wound surface (area: 12cm x 9 cm), and (C), D, and E) are the healing conditions of skin wound surface after treatment for 4, 12, and 15 months in sequence.
Fig. 2 is a biological functional diagram of THBS 1.
FIG. 3 is a schematic representation of the THBS-1 hydrolysis of the LAP-LTBP protein to activate TGF-beta 1.
Detailed Description
In order to make the present application solution better understood by those skilled in the art, the following description will be made in detail and with reference to the accompanying drawings in the embodiments of the present application, it is apparent that the described embodiments are only some embodiments of the present application, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, shall fall within the scope of the present application.
It should be noted that the terms "comprises" and "comprising," and any variations thereof, in the description and claims of the present application and in the foregoing figures, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed or inherent to such process, method, article, or apparatus.
4 cases of uremia and calcification defensive patients are accumulated altogether from 9 in 2018 to 4 in 2023. The 4 patients developed rapidly, and had received salvage treatment of human amniotic mesenchymal stem cells by multidisciplinary consultation, and had achieved good therapeutic effects on conventional treatments including infection control, nutritional support, anemia management, chronic kidney disease-mineral bone metabolic disorder (CKD-MBD) management, intravenous sodium thiosulfate (sodium thiosulfate, STS), wound care, pain management, anticoagulation, etc. (fig. 1).
The patients with uremia matched with the age and sex in the same period are 10 cases without calcification defense according to the standard of the incidence and the discharge. Basic data are obtained, blood samples are reserved for routine laboratory index monitoring, plasma samples of uremia and calcification defenses 2 groups of patients are additionally collected, and proteomics analysis is carried out. For 34-year-old female patient CUA 1, plasma specimens were collected and submitted for proteomic analysis 3 days, 2 weeks, 1 month and 15 months after treatment, respectively, prior to hAMSCs treatment.
The 4 CUA patients underwent definitive diagnosis of calcification defenses by skin biopsy prior to treatment with hAMSCs. Skin tissue was stained with hematoxylin-eosin (HE).
The study was approved by the ethical committee of the first affiliated hospital of the university of Nanjing medical science (Jiangsu province people's hospital) (2018-QT-001, 2020-QT-01, 2020-QT-09, 2020-SCR-03). All patients signed informed consent according to the declaration of helsinki.
In total, 1619 plasma proteins were identified in plasma samples from 3 patients with calcification defense and 10 uremic patients using high kurtosis removal in combination with the labeled quantitative proteomics approach. A total of 20 significantly altered differential proteins were identified by differential analysis of the protein expression matrices for the two groups of patients. Wherein the expression of thrombospondin-1 (THBS-1, THBS-1 or Tsp-1) is up-regulated, and the fold change is highest among the differential proteins.
The protein abundances of THBS-1 and LTBP1 are found to have a highly consistent trend after hAMSCs treatment, namely, the expression levels of the two are highest before the treatment, namely, the expression levels are rapidly reduced after 3 days of the treatment, namely, the expression levels are rapidly reduced after 1 month of the treatment, and the expression levels are slightly increased after 15 months of the hAMSCs treatment, by detecting the expression changes of 20 differential proteins in the plasma of 1 CUA patient by using a proteomics method before the treatment, at the 3 rd day, the 2 weeks, the 1 month and the 15 months after the treatment of the hAMSCs. It is suggested that THBS-1 and LTBP1 may play a synergistic role in the development and progression of disease, and that their rapid decline after treatment with hAMSCs may be closely related to the effectiveness of stem cell therapy.
As a multidomain glycoprotein, THBS-1 performs a variety of 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; promoting the progressive stenosis and hardening of the lumen caused by intimal hyperplasia by inducing migration and proliferation of VSMC; reducing tissue perfusion by inhibiting NO pathway vasoconstriction; by enhancing local thrombosis, vascular occlusion is accelerated, leading to rapid development of ischemic injury. The biological function of THBS-1 is shown in FIG. 2.
LTBP1 has been shown to be an extracellular multi-domain protein critical for TGF- β1 folding, secretion, matrix localization and activation, by interaction with fibrillin and other extracellular matrix (extracellular matrix, ECM) proteins. TGF- β1 is typically present in the form of an inactive large potential complex (large latent complex, LLC) formed by covalent disulfide bonding of the C-terminal mature TGF- β1 peptide and the N-terminal latency-related peptide (latency associated peptide, LAP) to LTBP 1. THBS-1 hydrolyzes LAP-LTBP proteins to dissociate TGF- β1 and mediate activation of TGF- β1 by interacting with the LSKL sequence on TGF- β1-LAP complex via the KRFK sequence thereon, involved in biological processes such as wound healing, cell proliferation, inflammatory response, fibrosis, etc. (see fig. 3).
The study was further included in 16 uremic patients, and none of the age, sex, BMI and levels of blood bone mineral metabolism indicators (including calcium, phosphorus and iPTH) were statistically significantly different from the calcification defensive patient group (n=4). The results showed that the plasma THBS-1 level (5319.67 ± 9835.62) ng/ml was significantly higher in CUA patients than in uremic patients (318.55 ± 431.32) ng/ml (p=0.000). The plasma TGF- β1 level (41979.67 ± 27103.42) pg/ml of CUA patients was significantly higher than the uremic patient level (3551.79 ± 1707.04) pg/ml (p=0.000). Consistent with the trend of abundance of THBS-1 and LTBP1 in the corresponding groups of patients in proteomics.
ELISA test results prove that the levels of blood THBS-1 and TGF-beta 1 of calcified defensive patients 1 and 3 are rapidly reduced 3 days after the first hAMSCs treatment, and the levels of blood THBS-1 and TGF-beta 1 are reduced to the lowest value after 1 month of treatment, which is consistent with the change trend of blood THBS-1 and LTBP1 before and after the treatment of CUA patient 1 observed in proteomics. Notably, in CUA patient 3, plasma THBS-1 and TGF- β1 levels were again significantly elevated half a year after discontinuation of the hAMSCs treatment, exceeding the levels prior to first receiving stem cells, with patient blood THBS-1 and TGF- β1 again exhibiting a downward trend as stage 2 stem cell treatment continued. The study shows that the levels of THBS-1 and TGF-beta 1 in the blood plasma of CUA patients are far higher than those of uremia patients, and the blood plasma can be rapidly reduced after hAMSC treatment, and the dynamic change trend is highly consistent.
We further analyzed patients with uremia accompanied by skin-breach infection and skin pathology confirmed non-calcified defenses, and detected that the blood THBS-1 level was close to that of common uremic patients, significantly lower than that of CUA patients. The combined monitoring of the levels of the THBS-1 and TGF-beta 1 proteins in blood is suggested to have important advantages as a blood biological marker of CUA patients.
It will be appreciated that the invention has been described by way of example only and that modifications may be made while remaining within the scope and spirit of the invention. The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without requiring creative effort by one of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by a person skilled in the art according to the inventive concept shall be within the scope of protection defined 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.
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