CN111308088A - Biomarkers for vascular injury in chronic kidney disease - Google Patents
Biomarkers for vascular injury in chronic kidney disease Download PDFInfo
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- CN111308088A CN111308088A CN202010120173.2A CN202010120173A CN111308088A CN 111308088 A CN111308088 A CN 111308088A CN 202010120173 A CN202010120173 A CN 202010120173A CN 111308088 A CN111308088 A CN 111308088A
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Abstract
The invention relates to a biomarker for chronic kidney disease vascular injury, in particular to application of fibrinogen 1(Fibrillin1) in serum to the biomarker for chronic kidney disease vascular injury. The invention provides application of fibrinogen 1 in serum as a biomarker of chronic kidney diseases, and also provides a kit for dynamically evaluating the vascular injury of chronic kidney diseases of a subject, which can early warn the vascular injury of chronic kidney diseases, can be used as a target for evaluating curative effect and prognosis, and has important significance for screening, diagnosing, treating and prognosing cardiovascular complications of chronic kidney diseases.
Description
Technical Field
The invention relates to a biomarker for chronic kidney disease vascular injury, in particular to application of fibrinogen 1(Fibrillin1) in serum to the biomarker for chronic kidney disease vascular injury.
Background
The increasing incidence of chronic kidney disease worldwide has become a major public health problem worldwide. Epidemiological survey results in china show that the incidence of chronic kidney disease is as high as 10%, with a significant proportion of patients progressing to end stage renal disease. End-stage renal disease patients need to rely on dialysis or kidney transplantation for a long time to maintain life, placing a huge burden on the patients individuals, families and society.
Chronic kidney disease progression is often accompanied by capillary rarefaction, characterized by a decrease in capillary density due to loss of endothelial cells. The vascular endothelial cells are a layer of flat cells positioned between the vascular wall and the blood flow, are both induction cells and effector cells, can not only sense information such as inflammatory signals, hormone levels, shear stress, pressure and the like in the blood, but also play an important role in maintaining vascular tension, normal blood flow, inflammatory repair and vascular proliferation by secreting various vasoactive substances. The functional change of vascular endothelial cells is closely related to the occurrence and development of chronic kidney diseases. Many studies have shown that thinning of capillaries in patients with chronic kidney disease may be associated with impaired repair processes following vascular injury. In recent years, research shows that the loss of peritubular capillary vessels is the most important factor for hypoxia and tubulointerstitial injury in the later stage of chronic kidney diseases. Therefore, monitoring the function of the vascular endothelial cells can provide a diagnosis and prognosis reference for the disease progression of patients with chronic kidney diseases.
In addition, the incidence and the fatality rate of cardiovascular diseases of chronic kidney disease patients are obviously increased, the cardiovascular diseases are the first cause of death of renal failure patients, and the incidence of the cardiovascular diseases of the chronic kidney disease patients is 10-30 times higher than that of general people. There is increasing evidence that cardiovascular disease caused by dysfunction of vascular endothelial cells is a common complication in patients with chronic renal disease and is the most important cause of death in patients.
Disclosure of Invention
The invention aims to provide a biomarker for chronic renal disease vascular injury, which is helpful for dynamic assessment of chronic renal disease vascular injury.
In a first aspect of the invention there is provided the use of fibrinogen 1 in serum as a biomarker for chronic kidney disease.
In a second aspect of the invention, a kit for dynamically assessing chronic renal disease vascular injury in a subject is provided. The kit comprises: an affinity reagent that selectively binds to fibrillar protein 1 in serum; and a container comprising serum.
Preferably, in the kit of the present invention, the affinity reagent is an antibody.
In a third aspect of the invention, another kit for dynamically assessing chronic renal vascular injury in a subject is provided. The kit comprises: a capture reagent to detect the biomarker fibrillarin 1 in a biological sample; and combinations of solid supports with corresponding capture reagents and signal-generating materials.
According to a further feature of the kit of the present invention, the capture reagent is at least one aptamer or antibody.
According to a further feature of the kit of the present invention, the kit further comprises one or more of the following reagents for processing a serum sample: solubilization buffer, detergent or buffer.
According to a further feature of the kit of the present invention, the kit further comprises one or more of the following reagents: buffer solution, blocking agent, mass spectrum matrix material, antibody capture agent, positive control sample and negative control sample.
A fourth aspect of the invention provides a method for dynamically assessing chronic renal disease vascular injury in a subject. The method comprises the following steps: measuring the level of fibrillar protein 1 in a serum sample obtained from said subject; and correlating the measurement with a chronic renal disease vascular injury state; the measurement comprises the following steps: the presence or absence of fibrillar protein 1 was detected, and the amount of fibrillar protein 1 was quantified.
Experiments prove that the fibril protein 1 in serum can be used as a biomarker for the vascular injury of the chronic kidney disease. The inventor carries out the detection of the expression level of the fibril protein 1 in the blood of normal healthy people and patients with different stages of chronic kidney diseases; and the human umbilical vein endothelial cells cultured in vitro are treated with the recombinant protein of the fibril protein 1, and the influence on the proliferation and the apoptosis of the human umbilical vein endothelial cells is observed. The results show that: compared with normal healthy people, the level of the fibril protein 1 in the serum of a patient with chronic kidney disease is obviously increased, is in negative correlation with glomerular filtration rate (eGFR), and is in positive correlation with serum creatinine, urea nitrogen and cystatin C, so that the expression increase of the fibril protein 1 in the serum of the patient with chronic kidney disease, particularly the patient with middle and late stages is generally shown, and the level of the fibril protein 1 in the serum can predict the occurrence and development of the chronic kidney disease. In addition, in vitro experiments show that after the human umbilical vein endothelial cells are treated by the recombinant protein of the fibril protein 1, the proliferation of the human umbilical vein endothelial cells can be inhibited, the expression of proliferation related protein can be inhibited, the apoptosis and the expression of apoptosis related protein can be promoted, and the fibril protein 1 can cause the damage of the endothelial cells and can be used as a pathogenic factor of endothelial damage.
Therefore, the level of fibrinogen 1 in serum is closely related to the course of chronic kidney disease, and the fibrinogen 1 can cause damage to endothelial cells and become a pathogenic factor of endothelial damage. Therefore, the fibrillar protein 1 in the serum is used as a biomarker of the chronic kidney disease vascular injury, can early warn the vascular injury of the chronic kidney disease, is used as a target point for evaluating the curative effect and prognosis, and has important significance for screening, diagnosing, treating and prognosing the cardiovascular complications of the chronic kidney disease.
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FIG. 1 shows the measurement of fibril protein 1 levels in serum of normal healthy persons and chronic renal disease patients by ELISA.
Figure 2 is a statistical plot of correlation analysis of fibril protein 1 levels in serum with glomerular filtration rate, serum creatinine, urea nitrogen, cystatin C.
FIG. 3 is a graph showing the effect of different concentrations of fibrillin1 recombinant protein on the proliferative activity of human umbilical vein endothelial cells. Wherein, fig. 3A is a 24-hour statistical chart; fig. 3B is a 48 hour statistical plot.
FIG. 4 is an immunoblot of the recombinant fibrillar protein 1 against human umbilical vein endothelial cell proliferation-associated protein. Wherein, fig. 4A is a representative result of an immunoblot assay; FIGS. 4B-E are statistical graphs of all results.
FIG. 5 is the effect of fibrillin1 recombinant protein on apoptosis of human umbilical vein endothelial cells. Wherein, fig. 5A is a representative result of the control group; fig. 5B is a representative result of the fibrillin1 stimulated group; FIG. 5C is a statistical chart of all results.
FIG. 6 is an immunoblot of the effect of fibrillin1 recombinant protein on apoptosis of human umbilical vein endothelial cells. Wherein, fig. 6A is a representative result of an immunoblot assay; FIGS. 6B-I are statistical graphs of all results.
Detailed Description
The invention will now be further described, by way of example only, with reference to the accompanying drawings.
The first embodiment is as follows: fibril protein 1 level in serum is related to the occurrence and development of chronic kidney disease
1. Collecting a blood sample: extracting fasting venous blood of normal healthy people and chronic nephrosis patients, numbering and marking, centrifuging, collecting supernatant, packaging and storing in a refrigerator at-80 deg.C.
2. The experimental process comprises the following steps: the ELISA method measures fibril protein 1 levels in each group of sera.
3. The experimental results are as follows: as shown in figure 1, fibrillar protein 1 levels in serum of chronic kidney disease patients were significantly elevated compared to normal healthy persons and correlated with the severity of chronic kidney disease.
4. And (4) conclusion: fibrillar protein 1 levels in serum are associated with the development of chronic kidney disease.
Example two: the level of fibril protein 1 in serum is negatively correlated with glomerular filtration rate of patients with chronic kidney disease, and positively correlated with creatinine, urea nitrogen and cystatin C
1. Collecting a blood sample: collecting fasting venous blood of normal healthy people and chronic nephrosis patients, numbering and marking, centrifuging, collecting supernatant, packaging and storing in a refrigerator at-80 deg.C.
2. The experimental process comprises the following steps: detecting the level of fibrillar protein 1 in each group of serum by an ELISA method; detecting the levels of creatinine, urea nitrogen and cystatin C in blood by a biochemical automatic instrument; and calculating the glomerular filtration rate according to the ages, heights, weights and sexes of normal healthy people and chronic kidney disease patients. And performing correlation analysis of the two by using statistical analysis software.
3. The experimental results are as follows: as shown in figure 2, the level of fibrillar protein 1 in serum is negatively correlated with glomerular filtration rate in patients with chronic kidney disease, and positively correlated with creatinine, urea nitrogen, cystatin C.
Example three: fibril protein 1 recombinant protein inhibits the proliferative activity of human umbilical vein endothelial cells
1. Experimental materials:
cell: human umbilical vein endothelial cells.
Culture solution: DMEM high-glucose medium containing 10% fetal bovine serum.
The culture conditions are as follows: 5% CO at 37 ℃2An incubator.
2. Experimental treatment: preparing single cell suspension with 10% fetal bovine serum cell culture solution, inoculating cells into 96-well culture plate with volume of 100 μ l per well and cell density of 10 per well4～105Each group is provided with 6 compound holes, and the culture plate is placed in an incubator for culture (5% CO)2At 37 ℃ C. And when the adherent growth and fusion of the cells reach 50-60%, replacing a serum-free DMEM high-sugar culture medium, and continuously culturing for 24 hours to synchronize all groups of cells and enter a resting period. After the cells enter a resting period, cell supernatant is discarded, different reagents of 100 microliter are added into each hole according to the intervention conditions of 112.5 ng/ml of fibril protein, 125ng/ml of fibril protein, 150ng/ml of fibril protein, 1100ng/ml of fibril protein and 1200ng/ml of fibril protein of a control group, the culture plate is placed in an incubator to be continuously cultured, and each group is cultured for 24 and 48 hours respectively. Mu.l of MTT reagent (5mg/ml) was added to each well and incubation was continued for 4 hours. The culture was terminated and the culture medium in the wells was carefully discarded. Adding 150ul dimethyl sulfoxide into each well, and placing on a shaking bed to shake at low speed for 10min to fill the crystalsAnd (4) dissolving. The Optical Density (OD) at 490nm of each well was measured with a microplate reader, and the OD490 values of the blank tubes were averaged to zero, and the average OD490 value of the parallel wells was taken as the optical density value of each experimental group. The cell growth inhibition rate was defined as (control OD490 value-experimental OD490 value)/(control OD490 value-blank OD490 value) × 100%.
3. The experimental results are as follows: as shown in FIG. 3, different concentrations of the fibrillin1 recombinant protein inhibited the proliferation of human umbilical vein endothelial cells.
Example four: fibril protein 1 recombinant protein for inhibiting expression of human umbilical vein endothelial cell proliferation related protein
1. Experimental materials:
cell: human umbilical vein endothelial cells.
Culture solution: DMEM high-glucose medium containing 10% fetal bovine serum.
The culture conditions are as follows: 5% CO at 37 ℃2An incubator.
2. Experimental treatment: inoculating human umbilical vein endothelial cells with good growth state in 6-well plate to obtain cells with density of 1 × 105And/ml. And (3) when the cells adhere to 60-70%, discarding the culture solution, washing for 2 times by using a PBS solution, continuously culturing by using a serum-free culture solution for 24 hours, and keeping the cells in a synchronized state. According to the intervention conditions of a control group, a 10% fetal calf serum group and a 10% fetal calf serum + fibrillar protein 150ng/ml group, the proteins are collected after continuous culture for 48 hours, and a Westernblot experiment is carried out.
3. The experimental results are as follows: as shown in FIG. 4, fibrillin1 inhibited the expression of PCNA, Cyclin D1, c-Fos, c-Myc proteins associated with human umbilical vein endothelial cell proliferation.
Example five: fibril protein 1 recombinant protein promotes apoptosis of human umbilical vein endothelial cells
1. Experimental materials:
cell: human umbilical vein endothelial cells.
Culture solution: DMEM high-glucose medium containing 10% fetal bovine serum.
The culture conditions are as follows: 5% CO at 37 ℃2An incubator.
2. Experimental treatment: adjusting the number of human umbilical vein endothelial cells in logarithmic growth phase to 2 × 105Per ml, seeded in 6-well plates, 1ml per well volume. After the cells adhere to the wall overnight, the cells are stimulated according to grouped intervention conditions and placed in an incubator to be cultured for 48 hours continuously. The cells were harvested by digestion with 0.25% pancreatin solution without EDTA (ethylenediaminetetraacetic acid). The cells were washed twice with pre-chilled PBS (4 ℃), 1500g/rpm, centrifuged for 5min, and the cells were collected in an EP tube. Mu.l of Binding Buffer was added to the EP tube to suspend the cells. Add 5. mu.l Annexin V-FITC into the EP tube, mix well, add 5. mu.l propidium iodide into the EP tube, mix well. The EP tube was left at room temperature in the dark and protected from light (dark room), and the reaction was carried out for 5 to 15 minutes. And (3) within 1 hour of adding the detection kit, carrying out cell apoptosis observation and quantity detection on a flow cytometer. The experiment was repeated 3 times.
3. The experimental results are as follows: as shown in figure 5, fibrillar protein 1 promotes apoptosis of human umbilical vein endothelial cells.
Example six: fibril protein 1 recombinant protein for promoting expression of human umbilical vein endothelial cell apoptosis related protein
1. Experimental materials:
cell: human umbilical vein endothelial cells.
Culture solution: DMEM high-glucose medium containing 10% fetal bovine serum.
The culture conditions are as follows: 5% CO at 37 ℃2An incubator.
2. Experimental treatment: inoculating human umbilical vein endothelial cells with good growth state in 6-well plate to obtain cells with density of 1 × 105And/ml. And (3) when the cells adhere to 60-70%, discarding the culture solution, washing for 2 times by using a PBS solution, continuously culturing by using a serum-free culture solution for 24 hours, and keeping the cells in a synchronized state. And (4) continuously culturing for 48h according to the intervention conditions of the control group and the fibril protein group of 150ng/ml, collecting the protein, and performing Western blot experiment.
3. The experimental results are as follows: as shown in FIG. 6, fibrillar protein 1 promoted the expression of Fas, FasL, FADD, P53, PARP-1, Bax, Caspase 3, ET-1 protein in human umbilical vein endothelial cells.
In conclusion, the level of fibrillar protein 1 in serum is positively correlated with the occurrence, development and course of chronic kidney disease. The fibril protein 1 can inhibit the proliferation of human umbilical vein endothelial cells and promote the apoptosis of the human umbilical vein endothelial cells. Thus, fibrillar protein 1 levels in serum can be used as a biomarker for dynamically assessing the progression of vascular injury in chronic renal disease.
Example seven: reagent kit
The level of fibrillin1 in serum can be detected by using a suitable kit, such as the kit for dynamically evaluating the vascular damage of chronic renal disease in a subject provided by the present invention.
In one embodiment, the kit of the present invention comprises: an affinity reagent that selectively binds to fibrillar protein 1 in serum; and a container comprising serum.
Preferably, the affinity reagent is an antibody.
The kit may contain one or more detectable labels known, such as fluorescent labels and the like.
In another embodiment, the kit of the present invention comprises: a capture reagent, such as at least one aptamer or antibody, to detect the biomarker fibrillarin 1 in a serum sample; and combinations of solid supports with corresponding capture reagents and signal-generating materials.
The kit may also contain one or more reagents (e.g., solubilization buffer, detergent or buffer) to process the serum sample. Any of the kits described herein can further comprise, for example, buffers, blocking agents, mass spectrometry matrix materials, antibody capture agents, positive control samples, negative control samples.
The kits of the invention may be used with a computer system or software to analyze and report the results of an analysis of a biological sample, to classify an individual from whom the serum sample was obtained as having chronic kidney disease and/or not, or to determine the extent of disease progression in the individual having chronic kidney disease.
Claims (8)
1. Use of fibrinogen 1(Fibrillin1) in serum as a biomarker for chronic kidney disease.
2. A kit for dynamically assessing chronic renal disease vascular injury in a subject, comprising: an affinity reagent that selectively binds to fibrillar protein 1 in serum; and a container comprising serum.
3. The kit of claim 2, wherein: the affinity reagent is an antibody.
4. A kit for dynamically assessing chronic renal disease vascular injury in a subject, comprising: a capture reagent to detect the biomarker fibrillarin 1 in a biological sample; and combinations of solid supports with corresponding capture reagents and signal-generating materials.
5. The kit of claim 4, wherein: the capture reagent is at least one aptamer or antibody.
6. The kit of claim 4, further comprising one or more of the following reagents for processing the serum sample: solubilization buffer, detergent or buffer.
7. The kit of claim 4, further comprising one or more of the following reagents: buffer solution, blocking agent, mass spectrum matrix material, antibody capture agent, positive control sample and negative control sample.
8. A method for dynamically assessing chronic renal disease vascular injury in a subject, comprising the steps of:
measuring the level of fibrillar protein 1 in a serum sample obtained from said subject; and
correlating the measurement with a chronic renal disease vascular injury status; the measurement comprises the following steps: the presence or absence of fibrillar protein 1 was detected, and the amount of fibrillar protein 1 was quantified.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117711618A (en) * | 2023-12-15 | 2024-03-15 | 南方医科大学南方医院 | Protein-based kidney disease occurrence risk prediction system and storage medium |
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| CN106662588A (en) * | 2014-07-22 | 2017-05-10 | 国立研究开发法人产业技术综合研究所 | Hepatocellular carcinoma marker |
| CN107589260A (en) * | 2016-07-07 | 2018-01-16 | 南方医科大学南方医院 | Purposes of the MMP-7 as renal fibrosis and the biomarker of CKD in urine |
| WO2019232512A1 (en) * | 2018-06-01 | 2019-12-05 | Musc Foundatiion For Research Development | Glycan analysis of proteins and cells |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106662588A (en) * | 2014-07-22 | 2017-05-10 | 国立研究开发法人产业技术综合研究所 | Hepatocellular carcinoma marker |
| CN107589260A (en) * | 2016-07-07 | 2018-01-16 | 南方医科大学南方医院 | Purposes of the MMP-7 as renal fibrosis and the biomarker of CKD in urine |
| WO2019232512A1 (en) * | 2018-06-01 | 2019-12-05 | Musc Foundatiion For Research Development | Glycan analysis of proteins and cells |
Non-Patent Citations (3)
| Title |
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| ANURADHA VIVEKANANDAN-GIRI: "Urine Glycoprotein Profile Reveals Novel Markers for Chronic Kidney Disease", 《INTERNATIONAL JOURNAL OF PROTEOMICS》 * |
| MING CHANG HU等: "Klotho Deficiency Causes Vascular Calcification in Chronic Kidney Disease", 《J AM SOC NEPHROL》 * |
| MOHAMMAD AL-HAGGAR等: "A Novel Fibrillin–1 Mutation in an Egyptian Marfan Family: A Proband Showing Nephrotic Syndrome Due to Focal Segmental Glomerulosclerosis", 《SAUDI J KIDNEY DIS TRANSPL》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117711618A (en) * | 2023-12-15 | 2024-03-15 | 南方医科大学南方医院 | Protein-based kidney disease occurrence risk prediction system and storage medium |
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