CN113667735A - Application of ALKBH5 in early diagnosis, risk assessment or prognosis degree prediction of diabetic retinopathy - Google Patents
Application of ALKBH5 in early diagnosis, risk assessment or prognosis degree prediction of diabetic retinopathy Download PDFInfo
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Abstract
本发明涉及糖尿病视网膜病变检测技术领域，尤其涉及一种ALKBH5在糖尿病视网膜病变的早期诊断、风险评估或预后程度预测中的应用。本发明提供了检测ALKBH5蛋白的分子在制备用于糖尿病视网膜病变的早期诊断、风险评估或预后程度预测的试剂盒或检测试剂中的用途。可实现对DR的早期诊断，特别是在出现视网膜明显病变之前进行早期筛查、评估DM患者出现DR的风险，进行早期干预与宣教。以及评估已经发生DR的患者，其疾病进展的风险和预后情况。The invention relates to the technical field of diabetic retinopathy detection, in particular to the application of ALKBH5 in early diagnosis, risk assessment or prognosis degree prediction of diabetic retinopathy. The present invention provides the use of a molecule for detecting ALKBH5 protein in the preparation of a kit or a detection reagent for early diagnosis, risk assessment or prognosis prediction of diabetic retinopathy. Early diagnosis of DR can be achieved, especially early screening before obvious retinal lesions, assessment of the risk of DR in DM patients, and early intervention and education. As well as assessing the risk and prognosis of disease progression in patients who have developed DR.
Description
Technical Field
The invention relates to the technical field of diabetic retinopathy detection, in particular to application of ALKBH5 in early diagnosis, risk assessment or prognosis degree prediction of diabetic retinopathy.
Background
Diabetes Mellitus (DM) is a common endocrine disease in China, the prevalence rate of the DM is on the rising trend year by year along with the increase of population, aging, economic development and change of living habits, the total number of DM patients in the world is estimated to reach 3.66 hundred million by 2030, and China is expected to become the country with the most DM patients. Histopathological changes caused by DM can affect multiple target organs and cause multiple complications, wherein Diabetic Retinopathy (DR) is one of the most common complications of DM and is the first blindness-causing disease of people of working age. In China, 75% of DM patients have DR within 15-20 years after diagnosis, which causes serious influence on the vision of the patients. Therefore, it is very important to deeply explore early clinical diagnosis and treatment methods of DR. In the aspect of diagnosis, current screening methods include fundus photography, optical coherence tomography, fluorescein fundus angiography, and the like.
In the aspect of DR diagnosis, current examination and diagnosis methods such as fundus examination, OCT, and the like are often discovered only when the disease progresses to a certain extent and the fundus is diseased, and these examination methods are also difficult to assess the risk of DR occurring in DM patients at an early stage, when microvascular disease has not yet occurred, and to assess the risk of progression of patients who have already developed DR. The limitations of early diagnosis methods also lead to the limitation of the efficacy of treatment regimens, and many current treatment methods, including laser photocoagulation, intravitreal injection of anti-vegf, vitreoretinal surgery, etc., are aimed at the severe and proliferative DR with existing lesions, and do not fundamentally prevent the occurrence and development of DR.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an application of ALKBH5 in early diagnosis, risk assessment or prognosis degree prediction of diabetic retinopathy.
In order to achieve the purpose, the invention adopts the technical scheme that: provides the application of a molecule for detecting ALKBH5 protein in preparing a kit or a detection reagent for early diagnosis, risk assessment or prognosis degree prediction of diabetic retinopathy.
The inventor of the invention researches and discovers that ALKBH5 expression in peripheral blood mononuclear cells of a DR patient is abnormally up-regulated, and the expression of a series of downstream inflammation-related genes is reduced through m6A methylation modification, so that local inflammation of retina is mediated to be generated, and the progress of a DR pathological process is finally caused.
As a preferred embodiment of the use according to the invention, the kit or the detection reagent is used for the detection of a peripheral blood sample.
As a preferred embodiment of the use according to the invention, the kit or the detection reagent is used for the detection of the level of ALKBH5 expression of peripheral blood mononuclear cells in a peripheral blood sample.
As a preferable embodiment of the application, the molecule for detecting ALKBH5 protein is a molecule capable of specifically detecting whether ALKBH5 protein is expressed or not.
As a preferred embodiment of the use of the present invention, the molecule capable of specifically detecting whether or not the ALKBH5 protein is expressed is a nucleic acid or a protein.
In a preferred embodiment of the use of the present invention, the protein is an antibody.
As a preferred embodiment of the use according to the invention, the antibody is a polyclonal antibody or a monoclonal antibody.
The invention also provides a kit for early diagnosis, risk assessment or prognosis degree prediction of diabetic retinopathy, wherein the kit comprises a molecule for detecting ALKBH5 protein.
As a preferred embodiment of the kit of the present invention, the kit further comprises: the kit comprises a 96-well plate precoated with an ALKBH5 antibody, a 96-well plate coating, an ALKBH5 protein standard, a standard diluent, a biotin labeled antibody diluent, horseradish peroxidase labeled avidin, a horseradish peroxidase labeled avidin diluent, a TMB substrate color developing solution, a stop solution, a washing solution and an ALKBH5 detection value corresponding index table.
The invention has the beneficial effects that:
(1) early diagnosis of DR is achieved, particularly early screening before apparent retinopathy occurs.
(2) And evaluating the risk of DR of DM patients, and performing early intervention and propaganda.
(3) Patients who have developed DR are assessed for risk and prognosis of disease progression.
Detailed Description
To more clearly illustrate the technical solutions of the present invention, the following embodiments are further described, but the present invention is not limited thereto, and these embodiments are only some examples of the present invention.
Peripheral blood mononuclear cells are important immune cells in human bodies, and the functional change and the change of molecular expression of the peripheral blood mononuclear cells are frequently used for indicating the occurrence and the development of disease states. Many studies have already confirmed that the abnormal function of peripheral blood mononuclear cells or the change of the expression of certain marker genes is an important early-stage disease marker and can be used for early diagnosis and treatment of clinical diseases.
m6A methylation modification is an important RNA expression regulation mechanism, and mainly means that a hydrogen atom connected to the 6 th nitrogen atom of RNA adenine is replaced by a methyl group, so that multiple layers of RNA expression, shearing, translation and the like are influenced, and the methylation modification is widely involved in the differentiation and functions of cells. As an RNA demethylase, ALKBH5 acts primarily to "erase" the m6A methylation modification in the gene, thereby reducing the level of m6A methylation modification of mRNA.
Example 1
The inventor of the invention researches and discovers that ALKBH5 expression in peripheral blood mononuclear cells of a DR patient is abnormally up-regulated, and the expression of a series of downstream inflammation-related genes is reduced through m6A methylation modification, so that local inflammation of retina is mediated to be generated, and the progress of a DR pathological process is finally caused.
The specific experimental process is as follows:
(1) we first extracted peripheral blood of DR patients and used normal human peripheral blood as a control, mononuclear cells therein were isolated using ficoll method, followed by peripheral blood mononuclear cell RNA extraction by trizol method and reverse transcription, and ALKBH5 expression and inflammation-related gene expression were detected by quantitative PCR method.
(2) The RNA is extracted by the method, the methylation modification level of m6A of the inflammation related gene is detected by RIP experiment, ALKBH5 is knocked out/over-expressed in peripheral blood mononuclear cells by RNAi technology and over-expression technology, and the methylation modification level and the expression level of m6A of the inflammation related gene are further detected.
Example 2
We further explored the value of m6A modification levels and the expression levels of akbh 5 in peripheral blood mononuclear cells for early diagnosis and prognostic assessment in DR patients. By preparing an ELISA kit, the methylation level of m6A and the expression level of ALKBH5 of peripheral blood mononuclear cells of a DR rat model are detected, and the result shows that compared with normal rats, the methylation level of m6A in the peripheral blood mononuclear cells of the DR rat begins to reduce at 1 week after modeling and is reduced most obviously at 8 weeks; whereas the ALKBH5 expression began to increase 1 week after modeling and peaked at 4 weeks, the DR rat retina was not observed for lesions until 8 weeks after modeling. In addition, the level of m6A methylation correlates negatively with its severity of retinopathy. The specific experimental process is as follows:
(1) preparation of DR rat model
Male SPF-grade SD rats for 8 weeks were adaptively bred for 1 week before modeling. Fasting for 16h before modeling of a diabetes group, weighing fasting body weight, taking blood from tail vein to determine blood sugar concentration, carrying out one-time intraperitoneal injection of streptozotocin STZ (65mg/kg), detecting random blood sugar by using a steady blood sugar tester after 72h, 96h and 120h, determining body weight, and observing general conditions such as body type and hair color change. The blood sugar of each group is measured by taking tail vein blood, the blood sugar is more than or equal to 16.7mmol/L for 3 times continuously, and a typical patient with more than three and one less symptoms appears, so that the diabetes model can be determined.
(2) Detection of m6A methylation level and ALKBH5 expression level in DR rats and normal rats
Rat serum is collected by a tail breaking method, peripheral blood mononuclear cells are separated according to the method, RNA is extracted for reverse transcription, the ALKBH5 expression level is detected by a fluorescent quantitative PCR method, and the total m6A methylation level is detected by an m6A methylation detection kit.
Example 3
We further collected peripheral blood mononuclear cells from DR patients, and examined their m6A methylation level and ALKBH5 expression level, and the results suggest that m6A methylation level of DR patients is significantly reduced and ALKBH5 expression is significantly increased, and is significantly correlated with the severity of retinopathy, compared to normal controls and DM patients without DR. These results suggest that the m6A methylation modification level and the ALKBH5 expression level of peripheral blood mononuclear cells can be used as early diagnosis and prognostic evaluation indicators of DR. The specific experimental process is as follows:
screening normal persons, DM patients without DR and DM patients with DR, separating peripheral blood mononuclear cells according to the method, extracting RNA of the peripheral blood mononuclear cells, performing reverse transcription, detecting the expression level of ALKBH5 by a fluorescence quantitative PCR method, and performing correlation analysis on the evaluation of retinopathy of the patients.
Example 4 m6A methylation modification detection kit and preparation thereof
(1) Composition of m6A methylation modification detection kit
The kit comprises a 96-well plate, a 96-well plate coating, an m6A binding antibody, an m6A binding solution, an m6A modified fragment standard, a standard diluent, a biotin labeled antibody diluent, horseradish peroxidase labeled avidin, a horseradish peroxidase labeled avidin diluent, a TMB substrate color developing solution, a stop solution, a washing solution and an m6A methylation detection numerical value corresponding index table.
(2) Preparation steps of m6A kit
Coating of 96-well plates: carbonate buffer solution with pH 9.6 was prepared, 500ul of m6A binding solution was added, and 1ml of the test sample was added simultaneously, and the mixture was incubated at 4 ℃ overnight. The well was discarded and washed 3 times with wash solution for 3 minutes each.
Preparing standard substance diluent, biotin-labeled antibody diluent and horseradish peroxidase-labeled avidin diluent: 100ml of PBS buffer was aspirated, 0.1g BSA was added, and well mixed.
Preparing a washing solution: 1000ml PBS buffer, add 1ml Tween solution, fully mix.
Fourthly, stop solution: preparing a 2mol/L sulfuric acid solution, and standing at room temperature for later use.
Example 5 ALKBH5 detection kit and preparation thereof
(1) Composition of ALKBH5 detection kit
The kit comprises a 96-well plate precoated with an ALKBH5 antibody, a 96-well plate coating, an ALKBH5 protein standard, a standard diluent, a biotin labeled antibody diluent, horseradish peroxidase labeled avidin, a horseradish peroxidase labeled avidin diluent, a TMB substrate color developing solution, a stop solution, a washing solution and an ALKBH5 detection value corresponding index table.
(2) Preparation steps of ALKBH5 kit
Coating of 96-well plate antibodies: carbonate buffer pH 9.6 was prepared, and the akbh 5 antibody was diluted to 5ug/ml, 0.2ml was added per well, and incubated at 4 ℃ overnight. The well was discarded and washed 3 times with wash solution for 3 minutes each.
Preparing standard substance diluent, biotin-labeled antibody diluent and horseradish peroxidase-labeled avidin diluent: 100ml of PBS buffer was aspirated, 0.1g BSA was added, and well mixed.
Preparing a washing solution: 1000ml PBS buffer, add 1ml Tween solution, fully mix.
Fourthly, stop solution: preparing a 2mol/L sulfuric acid solution, and standing at room temperature for later use.
Example 6 methods for detecting m6A methylation modifications and ALKBH5
(1) 5ml of peripheral blood of a DR patient is extracted, ficoll solution is added, centrifugation is carried out for 15min at 10000 Xg, a middle white membrane layer is left, and the cracking is carried out by a repeated liquid nitrogen freeze thawing method.
(2) A standard sample was taken from the kit, centrifuged at 10000rpm for 1min, dissolved in 1ml of sample diluent, and diluted 7 times (15.6-1000ng) by the double dilution method.
(3) 100ul of standard or test sample (ALKBH5 kit) or m6A antibody solution (m6A kit) is added into each well, covered with a film, and incubated for 2h at room temperature in a shaking table.
(4) Discard the liquid, add washing solution 200ul per well, wash 3 times, each for 1 min.
(5) And dissolving and diluting the biotin-labeled antibody by using a biotin-labeled antibody diluent, adding 100ul of the diluted biotin-labeled antibody into each hole, and incubating for 1h at room temperature in a shaking table.
(6) The same as the step (4).
(7) And (3) dissolving and diluting the horseradish peroxidase-labeled avidin by using a horseradish peroxidase-labeled avidin diluent, adding 100ul of avidin into each hole, and incubating for 1h at room temperature in a shaking table.
(8) The same as the step (4).
(9) 100ul of TMB substrate developing solution is added into each well, and the wells are incubated for 30min at room temperature in a dark place.
(10) Stop solution was added to 50ul per well.
(11) The optical density of each hole is detected by a microplate reader at the wavelength of 450nm, and the actual numerical value is calculated by fitting a standard curve.
And (4) analyzing results:
(1) detection range: 0.1-100 ng/ml.
(2) Specificity: has no cross reaction with other RNA methylation modification, m6A related enzyme and the like.
(3) And (3) recovery rate:
| sample(s) | Recovery Range (%) | Average recovery (%) |
| Serum (n ═ 10) | 100-106 | 103 |
| Plasma (n ═ 10) | 82-93 | 88 |
| Cell culture supernatant (n ═ 10) | 85-94 | 90 |
(4) Linearity:
(5) the accuracy is as follows: the intra-batch difference CV is less than 6.4 percent, and the inter-batch difference CV is less than 5.8 percent
(6) Results of the study
TABLE 1 index corresponding to measured values of m6A
TABLE 2 ALKBH5 test values correspondence index
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (9)
1. Use of a molecule for detecting ALKBH5 protein in preparation of a kit or a detection reagent for early diagnosis, risk assessment or prognosis degree prediction of diabetic retinopathy.
2. Use according to claim 1, wherein the kit or detection reagent is for the detection of a peripheral blood sample.
3. The use according to claim 2, wherein the kit or detection reagent is for the detection of the level of ALKBH5 expression of peripheral blood mononuclear cells in a peripheral blood sample.
4. The use of claim 1, wherein the molecule for detecting ALKBH5 protein is a molecule capable of specifically detecting whether ALKBH5 protein is expressed.
5. The use according to claim 4, wherein the molecule capable of specifically detecting the expression of the ALKBH5 protein is a nucleic acid or a protein.
6. Use according to claim 5, wherein the protein is an antibody.
7. The use according to claim 6, wherein the antibody is a polyclonal antibody or a monoclonal antibody.
8. A kit for early diagnosis, risk assessment or prognosis degree prediction of diabetic retinopathy, wherein the kit comprises a molecule for detecting the ALKBH5 protein.
9. The kit of claim 8, further comprising: the kit comprises a 96-well plate precoated with an ALKBH5 antibody, a 96-well plate coating, an ALKBH5 protein standard, a standard diluent, a biotin labeled antibody diluent, horseradish peroxidase labeled avidin, a horseradish peroxidase labeled avidin diluent, a TMB substrate color developing solution, a stop solution, a washing solution and an ALKBH5 detection value corresponding index table.
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| CN118360388A (en) * | 2024-06-14 | 2024-07-19 | 中南大学湘雅二医院 | Cyclic RNA and application of m6A methylation modification thereof |
| CN119716058A (en) * | 2024-12-12 | 2025-03-28 | 复旦大学附属眼耳鼻喉科医院 | Use of a substance that specifically binds to NSE protein or its encoding gene in the preparation of ARN diagnostic products or prognostic products |
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| CN119716058A (en) * | 2024-12-12 | 2025-03-28 | 复旦大学附属眼耳鼻喉科医院 | Use of a substance that specifically binds to NSE protein or its encoding gene in the preparation of ARN diagnostic products or prognostic products |
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