CN115508565B - Use of Cilp1 as a biomarker for renal fibrosis - Google Patents
Use of Cilp1 as a biomarker for renal fibrosis Download PDFInfo
- Publication number
- CN115508565B CN115508565B CN202211288045.4A CN202211288045A CN115508565B CN 115508565 B CN115508565 B CN 115508565B CN 202211288045 A CN202211288045 A CN 202211288045A CN 115508565 B CN115508565 B CN 115508565B
- Authority
- CN
- China
- Prior art keywords
- kidney
- fibrosis
- cilp
- cilp1
- kidney fibrosis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000000090 biomarker Substances 0.000 title claims abstract description 33
- 201000002793 renal fibrosis Diseases 0.000 title claims description 29
- 206010023421 Kidney fibrosis Diseases 0.000 claims abstract description 43
- 210000005084 renal tissue Anatomy 0.000 claims abstract description 33
- 230000014509 gene expression Effects 0.000 claims abstract description 23
- 210000002966 serum Anatomy 0.000 claims abstract description 22
- 238000003745 diagnosis Methods 0.000 claims abstract description 12
- 210000004926 tubular epithelial cell Anatomy 0.000 claims abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 230000003827 upregulation Effects 0.000 claims description 2
- 230000003176 fibrotic effect Effects 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 7
- 238000012360 testing method Methods 0.000 abstract description 6
- 238000011282 treatment Methods 0.000 abstract description 6
- 230000002596 correlated effect Effects 0.000 abstract description 4
- 238000013399 early diagnosis Methods 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000001514 detection method Methods 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 3
- 239000000104 diagnostic biomarker Substances 0.000 abstract 1
- 238000004393 prognosis Methods 0.000 abstract 1
- 238000010166 immunofluorescence Methods 0.000 description 28
- 206010016654 Fibrosis Diseases 0.000 description 26
- 230000004761 fibrosis Effects 0.000 description 26
- 241000699670 Mus sp. Species 0.000 description 19
- 208000020832 chronic kidney disease Diseases 0.000 description 16
- 230000005855 radiation Effects 0.000 description 15
- 210000003734 kidney Anatomy 0.000 description 13
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 13
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 12
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 12
- 241000700159 Rattus Species 0.000 description 11
- 208000017169 kidney disease Diseases 0.000 description 10
- 238000010186 staining Methods 0.000 description 10
- 210000000626 ureter Anatomy 0.000 description 10
- 238000005516 engineering process Methods 0.000 description 9
- 208000022831 chronic renal failure syndrome Diseases 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 206010061481 Renal injury Diseases 0.000 description 6
- 208000037806 kidney injury Diseases 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 210000003205 muscle Anatomy 0.000 description 6
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 4
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 4
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 4
- 238000011529 RT qPCR Methods 0.000 description 4
- 210000000683 abdominal cavity Anatomy 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000010219 correlation analysis Methods 0.000 description 4
- 230000000875 corresponding effect Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 101150039623 Clip1 gene Proteins 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 238000008157 ELISA kit Methods 0.000 description 3
- 102000046299 Transforming Growth Factor beta1 Human genes 0.000 description 3
- 101800002279 Transforming growth factor beta-1 Proteins 0.000 description 3
- 210000003855 cell nucleus Anatomy 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 210000002744 extracellular matrix Anatomy 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 230000002285 radioactive effect Effects 0.000 description 3
- 230000002861 ventricular Effects 0.000 description 3
- 206010002091 Anaesthesia Diseases 0.000 description 2
- 101710181683 Cartilage intermediate layer protein 1 Proteins 0.000 description 2
- 102100026622 Cartilage intermediate layer protein 1 Human genes 0.000 description 2
- 206010028594 Myocardial fibrosis Diseases 0.000 description 2
- 235000011609 Pinus massoniana Nutrition 0.000 description 2
- 241000018650 Pinus massoniana Species 0.000 description 2
- 102000009618 Transforming Growth Factors Human genes 0.000 description 2
- 108010009583 Transforming Growth Factors Proteins 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 230000037005 anaesthesia Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 2
- 229960002327 chloral hydrate Drugs 0.000 description 2
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000010894 electron beam technology Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000002439 hemostatic effect Effects 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000001678 irradiating effect Effects 0.000 description 2
- 210000000244 kidney pelvis Anatomy 0.000 description 2
- 230000002107 myocardial effect Effects 0.000 description 2
- 208000010125 myocardial infarction Diseases 0.000 description 2
- 239000000101 novel biomarker Substances 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 238000011552 rat model Methods 0.000 description 2
- 238000013517 stratification Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 208000003017 Aortic Valve Stenosis Diseases 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 206010002906 aortic stenosis Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 229940109239 creatinine Drugs 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 206010061989 glomerulosclerosis Diseases 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 238000002595 magnetic resonance imaging Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000651 myofibroblast Anatomy 0.000 description 1
- 238000010827 pathological analysis Methods 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses an application of Cilp as a kidney fibrosis biomarker, and relates to the technical field of kidney fibrosis diagnosis and treatment. Clinical tests, various animal experiments and cell experiments prove that Cilp < 1 > and kidney fibrosis are obviously positively correlated, and Cilp < 1 > can be used as a noninvasive diagnostic biomarker for kidney fibrosis, and has high sensitivity and high specificity; the invention provides an application of Cilp as a kidney fibrosis biomarker in diagnosing kidney fibrosis products, and provides a basis for early diagnosis of kidney fibrosis by developing a related Cilp1 detection kit and the like and detecting relative expression levels of Cilp1 in kidney tissues, tubular epithelial cells and serum of a patient suffering from kidney fibrosis. Meanwhile, by using Cilp1 disclosed by the invention as a kidney fibrosis biomarker, kidney fibrosis and severity thereof can be predicted through Cilp1, and guidance and reference can be provided for kidney fibrosis treatment, diagnosis and prognosis.
Description
Technical Field
The invention relates to the technical field of diagnosis and treatment of renal fibrosis, in particular to an application of Cilp < 1 > as a renal fibrosis biomarker.
Background
Renal fibrosis is a major common pathological link in the progression of chronic kidney disease (chronic KIDNEY DISEASE, CKD) caused by various causes, including glomerulosclerosis and interstitial fibrosis of the kidney, and is characterized by intrinsic cell damage to the kidney, inflammatory cell infiltration, activation and proliferation of myofibroblasts and fibroblasts, accumulation of extracellular matrix (collagen fibers, fibronectin, laminin), intrinsic cell loss of the kidney, tubular atrophy collapse, and rarefaction of blood vessels, ultimately leading to destruction of the normal structure of the kidney. Renal fibrosis marks irreversible kidney damage, so early diagnosis of renal fibrosis and treatment for etiology is critical to delay CKD progression.
At present, imaging methods such as ultrasonic imaging and magnetic resonance imaging are often used for evaluating renal fibrosis of patients, but the technologies can only diagnose the renal fibrosis from a macroscopic level, microscopic lesion information of inflammatory reaction period of the renal fibrosis can not be captured sharply, and the biomarker can evaluate or quantitatively measure biological and pathological processes of certain diseases and can be used as a treatment target of the diseases, and the biomarker participates in each link of the disease process, and the level of the biomarker changes along with the activity or progress degree of the disease process, so that the biomarker for the renal fibrosis is clear, and is helpful for early and accurate diagnosis of the renal fibrosis and targeted treatment.
Liu Qiuyu et al, research progress of renal fibrosis diagnosis biomarkers [ J ]. Molecular biomedical science, 2019, report that the diagnosis markers comprise kidney injury markers, inflammatory and pro-fibrosis factor markers, microRNAs markers and novel biomarkers, and are screened out to be highly sensitive and specific, thereby being expected to replace classical renal pathological diagnosis.
Cartilage intermediate layer protein 1 (CARTILAGE INTERMEDIATE LAYER protein 1, cilp 1) is an extracellular matrix protein that inhibits the effects of myocardial interstitial fibrosis, and can provide a new strategy for intervention in myocardial fibrosis progression; the literature reports that Cilp is an antagonist of Transforming Growth Factor (TGF) - β, an extracellular matrix (ECM) protein involved in myocardial fibrosis signaling. Cilp1 RNA was up-regulated in the expression in animal models of Left Ventricular (LV) pressure overload and left ventricular myocardial infarction, and Cilp protein levels were significantly elevated in myocardial tissue of patients with aortic valve stenosis or myocardial infarction; cilp1, which is a potential role for novel biomarkers of RV and LV pathological remodeling, is associated with RV maladaptation and ventricular arterial uncoupling in pulmonary arterial high pressure environments, but no study of Cilp in relation to renal fibrosis has been reported in the prior art.
Disclosure of Invention
The invention aims to provide the application of Cilp1 as the kidney fibrosis biomarker, which proves that Cilp and kidney fibrosis are obviously positively correlated, and Cilp1 can be used as a noninvasive diagnosis biomarker for kidney fibrosis and has high sensitivity and high specificity.
In order to achieve the technical purpose and the technical effect, the invention is realized by the following technical scheme:
use of Cilp1 as a biomarker for renal fibrosis.
Further, the biomarker Cilp1 is upregulated relative expression levels of Cilp1 in renal tissue, tubular epithelial cells, and patient serum in a subject with renal fibrosis relative to a subject with no renal fibrosis.
The invention Cilp is used as a kidney fibrosis biomarker, and the serum of normal people and chronic kidney disease (1-5 phase) patients is collected, and the ELISA kit is used for detecting the content of Cilp1 in the serum;
comparing the difference between the chronic renal failure patient and the healthy human serum Cilp, wherein the serum concentration of the chronic renal failure patient Cilp is higher than that of the normal person according to the ELISA result of the serum Cilp1 of the chronic renal failure patient and the healthy human; as a result, cilp1 was confirmed as an evaluation index.
The invention Cilp is used as a kidney fibrosis biomarker, the collected kidney tissues of a patient meeting the standard (stage 1-5) are subjected to Masson staining to detect the kidney fibrosis degree, the expression condition of Cilp1 in the kidney is detected by an immunofluorescence technology, and TGF-beta 1 is used as a fibrosis index to evaluate the prediction condition of Cilp;
Masson (Masson stain) of kidney tissue of kidney disease patient, calculate the fibrosis percentage, reflect the degree of tissue fibrosis; performing a correlation analysis corresponding to the immunofluorescence intensity of patient Cilp, correlation coefficient r= 0.3063;
Meanwhile, the area (%) of Masson in kidney tissue of kidney disease patient was compared with Cilp a immunofluorescence intensity, further verifying Cilp a as a kidney fibrosis biomarker.
The application of Cilp of the invention 1 as a kidney fibrosis biomarker, the targeted establishment of an animal model for verification, through UFO rat test, comprises the following steps:
s1: constructing a rat model of a false operation group and a UUO test group;
The method specifically comprises the following steps: after the rats are adaptively fed for one week, 5% chloral hydrate is injected into the abdominal cavity of two groups of rats for anesthesia, skin and muscle are sequentially cut along the median line of the abdomen, kidneys and ureters are dissociated, the middle section part of the left ureter is supported by tissue forceps, the muscles and the skin are conventionally sutured in a sham operation group, the ureter is clamped by UFO group hemostatic forceps, the proximal renal pelvis section of the left ureter is ligated at two ends by 4-0 silk threads for two times, the ureter is cut off, and then the muscles and the skin are conventionally sutured;
S2: after 21 days, the rats are sacrificed, kidney tissue specimens are collected, the kidney fibrosis degree is observed by HE and Masson staining, the expression condition of Cilp < 1 > in the kidney is detected by immunofluorescence technology, and the prediction condition of Cilp < 1 > is evaluated by taking TGF-beta 1 as a fibrosis index;
comparing the area (%) of Masson in UFO rat kidney tissue with Cilp < 1> immunofluorescence intensity, the immunofluorescence result shows that Cilp < 1> fluorescence intensity increases with the increase of fibrosis degree;
a radioactive kidney injury mouse assay comprising:
s1: 30C 57 mice were divided into 3 groups of 10 mice each, each divided into a normal control group, a10 gy radiation dose group and a 20gy radiation dose group according to the weight-stratification random number table method.
S2: after mice are adaptively fed for one week, normal control mice are not interfered normally, a single 10gy radiation dose group (10 gy) and a single 20gy radiation dose group are respectively used for irradiating the abdominal cavity of the mice by an electron beam, and after 2 months, the two groups of mice are sacrificed;
s3: the kidney tissue of the mice is collected, the kidney fibrosis degree is observed by HE and Masson staining, the expression condition of Cilp < 1 > in the kidney is detected by immunofluorescence technology, and the prediction condition of Cilp < 1 > is evaluated by taking TGF-beta 1 as a fibrosis index.
Firstly, calculating Masson (Pinus massoniana staining) of kidney tissues of each group of mice, and calculating fibrosis percentage and fibrosis degree of reaction tissues; correlation analysis was performed corresponding to immunofluorescence intensity of mouse kidney tissue expression Cilp 1:
the two have correlation, and the correlation coefficient R=0.686;
Comparing the area (%) of Masson in kidney tissue of the mice with radiation kidney injury with Cilp <1> immunofluorescence intensity, and the immunofluorescence result shows that Cilp <1> fluorescence intensity increases with the increase of fibrosis degree;
Cilp1 was validated for use as a biomarker for renal fibrosis.
The application of Cilp of the invention as a kidney fibrosis biomarker, the renal tubular epithelial cell line (HK-2) is subjected to induced fibrosis (epithelial mesenchymal transdifferentiation) modeling through TGF-beta 1, and compared with Cilp1 expression conditions in a normal cell group and an induced intervention group, and Western blotting and qPCR technologies are used for detection.
It is another object of the present invention to provide the use of Cilp1 as a biomarker for renal fibrosis in the diagnosis of a renal fibrosis product;
The diagnostic kidney fibrosis product includes, but is not limited to, a reagent, a kit.
A kit for diagnosing renal fibrosis, the kit comprising reagents for detecting an up-regulation of the relative expression level of Cilp a in renal tissue, tubular epithelial cells, and patient serum.
It is another object of the present invention to provide the use of Cilp a in predicting renal fibrosis and its severity.
The invention has the beneficial effects that:
The invention provides the application of Cilp as the kidney fibrosis biomarker, and the invention proves that Cilp and kidney fibrosis are obviously positively correlated, and Cilp1 can be used as a noninvasive diagnosis biomarker for kidney fibrosis, and has high sensitivity and high specificity;
The Cilp is provided as the application of the kidney fibrosis biomarker in diagnosing kidney fibrosis products, and the basis is provided for early diagnosis of kidney fibrosis by detecting the relative expression level of Cilp1 in kidney tissues, renal tubular epithelial cells and patient serum.
Of course, it is not necessary for any one product to practice the invention to achieve all of the advantages set forth above at the same time.
Drawings
FIG. 1 shows ELISA results for serum Cilp of the chronic renal failure patient group and the healthy human group according to example 1 of the present invention;
FIG. 2 is an analysis of the correlation between the area percent (%) of Masson and Cilp immunofluorescence intensity in kidney tissue of a patient with kidney disease according to example 2 of the present invention;
FIG. 3 shows the results of the immunofluorescence intensities of Masson's area (%) and Cilp's area (%) in kidney tissue of a patient with kidney disease according to example 2 of the present invention;
FIG. 4 shows the results of the immunofluorescence intensities of Masson's area (%) and Cilp1 in kidney tissue of UFO rats according to example 3 of the present invention;
FIG. 5 shows the results of the immunofluorescence intensity of areas (%) and Cilp of Masson in kidney tissue of a radiation-damaged mouse according to example 4 of the present invention;
FIG. 6 is an analysis of the correlation between the area percent (%) of Masson and Cilp immunofluorescence intensity in kidney tissue of a radiation-damaged mouse according to example 4 of the present invention;
FIG. 7 shows Western blotting results of Clip1 protein in HK-2 cells according to example 4 of the present invention;
FIG. 8 shows the qPCR result of Clip1 gene expression in HK-2 cells according to example 4 of the present invention;
Detailed Description
In order to more clearly describe the technical scheme of the embodiment of the present invention, the embodiment of the present invention will be described in detail below with reference to the accompanying drawings.
Example 1
Analysis of clinical trial data
Serum of normal people and chronic kidney disease (stage 1-5) patients is collected, and the content of Cilp1 in the serum is detected by an ELISA kit; serum content of Cilp1 was measured by ELISA kit by collecting serum from normal and chronic kidney disease (stage 1-5) patients 20 times, and the results are shown in FIG. 1:
In fig. 1, the abscissa indicates grouping information (healthy population group and chronic renal failure patient group); serum concentration along the ordinate Cilp; the Clip1 content was different between the two groups.
Comparing the difference of Cilp1 in the serum of the chronic renal failure patient group and the serum Cilp of the healthy people group, the ELISA result of the serum Cilp of the chronic renal failure patient group shows that the serum concentration of Cilp1 of the chronic renal failure patient is higher than that of the healthy people; as a result, cilp1 was confirmed as an evaluation index.
Example 2
In this example, 12 kidney disease patients meeting the criteria were selected;
Detecting kidney fibrosis degree by Masson staining of collected kidney tissues of patients with chronic kidney diseases (1-5 phases) meeting the standard, detecting Cilp expression conditions of the kidney by immunofluorescence technology, and evaluating Cilp1 prediction conditions by taking TGF-beta 1 as fibrosis indexes and combining with clinical indexes (indexes such as blood creatinine, blood urea nitrogen and the like);
The following table is a collection of renal patient data:
TABLE 1 renal disease patient data Condition
As shown in fig. 2, masson (Masson stain) of kidney tissue of kidney disease patient, the fibrosis percentage was calculated, and the degree of fibrosis of the tissue was reflected; performing a correlation analysis corresponding to the immunofluorescence intensity of patient Cilp, correlation coefficient r= 0.3063;
As shown in fig. 3, the areas (%) of Masson in kidney tissue of the kidney disease patients were compared with Cilp immunofluorescence intensities of the above patients numbered 2, 6, 12;
As can be seen from the left side view of fig. 3, the Masson staining results for three patients indicated that the degree of fibrosis was sequentially increasing (the above patients numbered 2,6, 12 in order from top to bottom);
as can be seen from the right side view of fig. 3, immunofluorescence results indicate that Cilp a increases in fluorescence intensity with increasing fibrosis, DAPI in the right side view: a cell nucleus; cilp1: green; TGF-. Beta.1: red; merge: cilp1 with TGF-beta 1 (patient above numbered 2, 6, 12 in order from top to bottom);
based on analysis of the correlation of the area percent (%) of Masson in kidney tissue of kidney disease patients with Cilp a. The comparison of the area (%) of Masson in kidney tissue of kidney disease patients with Cilp a. It can be seen that Cilp a was further validated as a kidney fibrosis biomarker.
Example 3
UUUO rat test
S1: constructing a rat model of a false operation group and a UUO test group;
The method specifically comprises the following steps: after the rats are adaptively fed for one week, 5% chloral hydrate is injected into the abdominal cavity of two groups of rats for anesthesia, skin and muscle are sequentially cut along the median line of the abdomen, kidneys and ureters are dissociated, the middle section part of the left ureter is supported by tissue forceps, the muscles and the skin are conventionally sutured in a sham operation group, the ureter is clamped by UFO group hemostatic forceps, the proximal renal pelvis section of the left ureter is ligated at two ends by 4-0 silk threads for two times, the ureter is cut off, and then the muscles and the skin are conventionally sutured;
S2: after 21 days, the rats are sacrificed, kidney tissue specimens are collected, the kidney fibrosis degree is observed by HE and Masson staining, the expression condition of Cilp < 1 > in the kidney is detected by immunofluorescence technology, and the prediction condition of Cilp < 1 > is evaluated by taking TGF-beta 1 as a fibrosis index;
As shown in fig. 4, the area (%) of Masson in UUO rat kidney tissue was compared with Cilp immunofluorescence intensity: as can be seen from fig. 4, the area (%) of Masson in kidney tissues of the normal Control group (Control) and the UUO model group (UUO) was compared with Cilp a1 immunofluorescence intensity, and the immunofluorescence result showed that Cilp a1 fluorescence intensity increased with increasing fibrosis degree. (DAPI: nucleus; cilp: green; TGF-. Beta.1: red; merge: cilp1 is displayed overlapping TGF-. Beta.1).
In fig. 4, control is a normal group, UUO is a model group, and corresponds to the upper and lower contents in sequence, and DAPI is performed simultaneously: a cell nucleus; cilp1: green; TGF-. Beta.1: red; merge: cilp1 was co-stained with TGF- β1, and the results also demonstrated that immunofluorescence results indicated that Cilp1 fluorescence intensity increased with increasing fibrosis;
the use of Cilp1 as a biomarker for renal fibrosis was verified.
Example 4
Radioactive kidney injury mouse test
S1: 30C 57 mice were divided into 3 groups of 10 mice each, each divided into a normal control group, a10 gy radiation dose group and a 20gy radiation dose group according to the weight-stratification random number table method.
S2: after mice are adaptively fed for one week, normal control mice are not interfered normally, a single 10gy radiation dose group (10 gy) and a single 20gy radiation dose group are respectively used for irradiating the abdominal cavity of the mice by an electron beam, and after 2 months, the two groups of mice are sacrificed;
s3: the kidney tissue of the mice is collected, the kidney fibrosis degree is observed by HE and Masson staining, the expression condition of Cilp < 1 > in the kidney is detected by immunofluorescence technology, and the prediction condition of Cilp < 1 > is evaluated by taking TGF-beta 1 as a fibrosis index.
Firstly, calculating Masson (Pinus massoniana staining) of kidney tissues of each group of mice, and calculating fibrosis percentage and fibrosis degree of reaction tissues; correlation analysis was performed corresponding to immunofluorescence intensity of mouse kidney tissue expression Cilp 1:
as shown in fig. 5, the two have correlation, and the correlation coefficient r=0.686;
area (%) of Masson in kidney tissue of mice with radiation kidney injury was compared with Cilp a immunofluorescence intensity as shown in fig. 6:
The radioactive kidney injury mice are divided into a normal Control group (Control), a 10gy radiation dose group (10 gy), and a 20gy radiation dose group (20 gy);
By DAPI: a cell nucleus; cilp1: green; TGF-. Beta.1: red; merge: cilp 1A and TGF-beta 1 co-staining experiments sequentially comprise a normal group, a 10gy radiation dose group (10 gy) and a 20gy radiation dose group (20 gy) from top to bottom, and the correlation of the radiation doses can be obtained through the results, and meanwhile, the immunofluorescence result shows that the fluorescence intensity of Cilp A increases with the increase of the fibrosis degree;
the use of Cilp1 as a biomarker for renal fibrosis was verified.
Example 5
The renal tubular epithelial cell line (HK-2) is subjected to induced fibrosis (epithelial mesenchymal transdifferentiation) modeling through TGF-beta 1, and the expression condition of Cilp protein in a normal cell group and an induced intervention group is compared, and Western blotting and qPCR technologies are used for detection;
As shown in FIG. 7, HK-2 cell western blotting results:
The 4 th-5 th generation HK-2 (renal tubular epithelial cells) is divided into two groups, a control group and a TGF-beta 1 induction group, the control group is not treated, the TGF-beta group is induced by 2ng/ml, 5ng/ml, 10ng/ml and 20ng/ml TGF-beta 1 cytokines for 24 hours, and the protein expression amounts of the two groups Cilp are calculated;
As the TGF- β1 concentration increases, the amount of Cilp1 expressed increases gradually compared to the normal control group;
as shown in FIG. 8, HK-2 cell qPCR results:
The 4 th to 5 th generation HK-2 (renal tubular epithelial cells) are divided into two groups, a control group and a TGF beta 1 induction group, the control group is not treated, the TGF beta 1 group is induced by 10ng/ml TGF beta 1 cytokine for 24 hours, and the mRNA expression quantity of the two groups Cilp1 is calculated;
compared with the normal control group, the mRNA expression quantity of Cilp1 is higher than that of the normal control group, and the mRNA expression quantity of the two groups of Cilp1 is different and has statistical significance (P is less than or equal to 0.05).
The invention provides the application of Cilp as the kidney fibrosis biomarker, and the invention proves that Cilp and kidney fibrosis are obviously positively correlated, and Cilp1 can be used as a noninvasive diagnosis biomarker for kidney fibrosis, and has high sensitivity and high specificity;
The Cilp is provided as the application of the kidney fibrosis biomarker in diagnosing kidney fibrosis products, and the basis is provided for early diagnosis of kidney fibrosis by detecting the relative expression level of Cilp1 in kidney tissues, renal tubular epithelial cells and patient serum.
The preferred embodiments of the invention disclosed above are intended only to assist in the explanation of the invention. The preferred embodiments are not exhaustive or to limit the invention to the precise form disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.
Claims (5)
- Use of cilp1 as a biomarker for renal fibrosis in the diagnosis of renal fibrosis products.
- 2. Use of Cilp1 as claimed in claim 1 as a kidney fibrosis biomarker in the diagnosis of a kidney fibrosis product, wherein: the diagnostic kidney fibrosis product includes, but is not limited to, a reagent, a kit.
- 3. A kit for diagnosing renal fibrosis, characterized in that: the kit comprises reagents for detecting up-regulation of Cilp expression levels in fibrotic kidney tissue, tubular epithelial cells and patient serum.
- Use of cilp1 in the preparation of a product for predicting renal fibrosis and its severity.
- 5. Use of Cilp a1 according to claim 4 for the preparation of a product for predicting renal fibrosis and its severity, wherein: by detecting the relative expression levels of Cilp a in kidney tissue, tubular epithelial cells and patient serum, a comparison was made with patients with kidney fibrosis at each stage.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211288045.4A CN115508565B (en) | 2022-10-20 | 2022-10-20 | Use of Cilp1 as a biomarker for renal fibrosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211288045.4A CN115508565B (en) | 2022-10-20 | 2022-10-20 | Use of Cilp1 as a biomarker for renal fibrosis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115508565A CN115508565A (en) | 2022-12-23 |
| CN115508565B true CN115508565B (en) | 2024-11-15 |
Family
ID=84509526
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211288045.4A Active CN115508565B (en) | 2022-10-20 | 2022-10-20 | Use of Cilp1 as a biomarker for renal fibrosis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115508565B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN120420375B (en) * | 2025-07-09 | 2025-09-05 | 成都医学院 | A traditional Chinese medicine composition for preventing and/or treating vascular calcification in chronic kidney disease, and its preparation method and use |
| CN120927980B (en) * | 2025-10-13 | 2026-01-23 | 成都医学院 | A group of septicemia-specific biomarkers and their applications |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ATE392622T1 (en) * | 2004-08-12 | 2008-05-15 | Hoffmann La Roche | METHOD FOR DIAGNOSIS OF LIVER FIBROSIS |
| US8591459B2 (en) * | 2009-12-21 | 2013-11-26 | Ethicon Endo-Surgery, Inc. | Use of biomarkers and therapeutic agents with surgical devices |
| US20130296642A1 (en) * | 2012-03-24 | 2013-11-07 | The Curators Of The University Of | Biomarkers and method for predicting occurence of ventral hernias |
| US11560594B2 (en) * | 2015-02-05 | 2023-01-24 | Duke University | Methods of detecting osteoarthritis and predicting progression thereof |
| TWI856437B (en) * | 2017-01-20 | 2024-09-21 | 法商賽諾菲公司 | Anti-tgf-beta antibodies and their use |
| CA3165068A1 (en) * | 2020-01-30 | 2021-08-05 | John E. Blume | Lung biomarkers and methods of use thereof |
| CN111840514B (en) * | 2020-08-14 | 2021-07-30 | 武汉大学 | Application of CILP2 in the preparation of drugs for improving cardiac aging and cardiac hypertrophy |
-
2022
- 2022-10-20 CN CN202211288045.4A patent/CN115508565B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| Biomarkers for the detection of renalfibrosis and prediction of renal outcomes:a systematic review;，Sherry G. Mansour;BMC Nephrology;20171231;1-13 * |
| Cartilage intermediate layer protein-1 alleviates pressure overload-induced cardiac fibrosis via interfering TGF-β1 signaling;Cheng-Lin Zhang;Journal of Molecular and Cellular Cardiology;20180210;第116卷;135-144 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115508565A (en) | 2022-12-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5450375B2 (en) | Predictive kidney safety biomarkers and biomarker signatures for monitoring kidney function | |
| CN107099581B (en) | Methods for predicting, diagnosing and treating idiopathic pulmonary fibrosis | |
| Kim et al. | Urinary monocyte chemoattractant protein-1 in renal disease | |
| CN115508565B (en) | Use of Cilp1 as a biomarker for renal fibrosis | |
| Rende et al. | Diagnostic and prognostic biomarkers for tubulointerstitial fibrosis | |
| KR20100128281A (en) | How to classify individuals with cardiovascular disease according to survival prognosis found by measuring levels of biomarker WaiKell-40 | |
| Daniel et al. | Transgelin is a marker of repopulating mesangial cells after injury and promotes their proliferation and migration | |
| CN111500710A (en) | Use of circRNA730 as biomarker for the prediction, diagnosis or prognosis of sepsis | |
| KR101992060B1 (en) | Alzheimer’s disease diagnostic fluid biomarker including the combination of four proteins | |
| CN112961913A (en) | Application of lncRNA in diagnosis and treatment of recurrent abortion | |
| Singh et al. | Biomarkers in acute traumatic brain injury: a systematic review and meta-analysis | |
| CN110016504A (en) | Application of CDR1as in prenatal screening for neural tube defects, products and methods for prenatal screening for neural tube defects | |
| CN115754304A (en) | Use of bone morphogenetic protein-7 as biomarker in sepsis diagnosis, prognosis or therapy monitoring | |
| Ruppert et al. | Molecular signatures and the study of gene expression profiles in inflammatory heart diseases | |
| Baird et al. | Urinary Clusterin is a biomarker of renal epithelial senescence and predicts human kidney disease progression | |
| US8148091B2 (en) | Detection of chronic kidney disease patients or coronary artery disease using bone morphogenic protein-4 | |
| CN118294666A (en) | A biomarker and its application in Parkinson's disease | |
| JP2014128250A (en) | Control factor of neurodegenerative disease | |
| Kikuchi et al. | Urinary and circulating levels of the anti-angiogenic isoform of vascular endothelial growth factor-A in patients with chronic kidney disease | |
| JP6158825B2 (en) | Tenascin C and its use in rheumatoid arthritis | |
| CN114959012B (en) | Product for detecting tendon injury | |
| JP6306124B2 (en) | Tuberculosis testing biomarker | |
| KR102717427B1 (en) | Skin Aging-Related Olfactory Receptor Genes Expressed in Dermal Fibroblasts and Use Thereof | |
| CN117741157A (en) | Application of TMSB4X in early diagnosis of diabetic nephropathy | |
| Sharif et al. | Neutrophil Gelatinase Associated Lipocalin: Is not an Early Marker Inductor for Diabetic Nephropathy in Qatari Population |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |