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Diabetic retinopathy (DR) metabolic memory detection reagent and application thereof

A technology for diabetic retina and detection reagents, which is applied in the fields of biotechnology and medicine, can solve problems such as unclear functions, achieve good stability, high detection sensitivity, and good clinical application value

Inactive Publication Date: 2014-12-17
陈有信
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PGC1-α has become a new target for the treatment of metabolic diseases such as DM, but the role of PGC1-α in DR and its metabolic memory is still unclear and needs further study

Method used

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  • Diabetic retinopathy (DR) metabolic memory detection reagent and application thereof
  • Diabetic retinopathy (DR) metabolic memory detection reagent and application thereof
  • Diabetic retinopathy (DR) metabolic memory detection reagent and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Experimental Grouping

[0033] Experimental materials and reagents:

[0034] Normal human retinal microvascular endothelial cells (Human retinal microvascular endothelial cells, HRMECs, cAP-0010TM), American Angio-Proteomie Company; Endo-Basal medium (ENDO-Basal medium), ScienCell; Fetal bovine serum (FBS) , ScienCell; Endothelial cell growth supplement (ECGS), ScienCell; Penicillin-Streptomycin (Penicillin-Streptomycin), ScienCell; Bovine plasma fibronectin (Fibronectin, FN), ScienCell; 0.25% trypsin (Trypsin), Poputech; D-Glucose, Sigma; D-Mannitol, Sigma.

[0035] HRMECs were divided into 5 groups according to intervention and treatment factors, and the specific grouping and treatment methods are shown in Table 1.

[0036] Table 1. Experimental grouping and treatment

[0037]

[0038]

[0039] Note: General culture medium = ENDO-Basal medium with a glucose concentration of 5.5mmol / L, no need to prepare; high-sugar culture medium = ENDO-Basal medium...

Embodiment 2

[0040] Example 2 Effect of high glucose on HRMECs cell viability

[0041] experimental method:

[0042] Mix the Buffer and Substrate in Promega’s Cell Titer-Glo kit, store at -20°C, and place it at 4°C in the dark before use to dissolve; after preparing the cells of each group after 7 days of culture, directly mix the cells The solution was added to a 96-well plate, 100 μl / well; oscillated on a shaker for 5 minutes, protected from light; 150 μl of the solution was transferred to a new opaque 96-well plate, and detected by chemiluminescence.

[0043] The cell viability of HRMECs in each group was detected by the Cell Titer-Glo method on the 7th day, and the results of the cell viability assay were expressed in relative fluorescence units (Relative light unit, RLU), as shown in Table 2 and figure 1 . SPSS20.0 software was used to test the normality and homogeneity of variance of the data, and the results indicated that the data of each group obeyed the normal distribution and ...

Embodiment 3

[0050] Example 3 Effect of High Sugar on SOD2 and PGC1-α Gene Transcription

[0051] experimental method:

[0052] After the cells were cultured for 1 day and 7 days, respectively, the expression levels of SOD2 and PGC1-α mRNA in each group were measured by RT-PCR.

[0053] 1. RNA extraction and quality inspection

[0054] (1) Digest the cells with 0.25% trypsin and collect them separately, about 2×10 6 Add 1ml / well of Trizol reagent to the cells in a 6-well plate, pipette repeatedly to completely lyse the cells, and place the above samples at room temperature for 5 minutes;

[0055] (2) Add 0.2ml of chloroform, mix thoroughly, let stand for 3 minutes, centrifuge at 12000rpm at 4°C for 15 minutes, and separate the samples;

[0056] (3) Carefully transfer the upper aqueous phase into another 1.5ml Eppendorf tube without RNase contamination, add 0.5ml isopropanol, mix gently, let stand at room temperature for 10 minutes, centrifuge at 12000rpm at 4°C for 10 minutes, and reser...

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Abstract

The invention belongs to the field of biotechnology and medical science and particularly relates to a diabetic retinopathy (DR) metabolic memory detection reagent and application thereof. According to experimental proof, methylation of locus (+214) in a peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1-alpha) gene is an early diagnosis mark of DR metabolic memory. On the basis of the study, the detection reagent is provided for detecting the diagnosis mark, and a corresponding detection kit is prepared. According to the provided methylation detection reagent, whether methylation of a detected object of the PGC1-alpha gene locus +214 occurs or not and methylation degree can be qualitatively or quantitatively detected, thereby, whether DR metabolic memory and illness risks of the detected object exist or not can be predicted, and the clinical application value is excellent.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine, and in particular relates to a DR metabolic memory detection reagent and application thereof. Background technique [0002] Diabetic retinopathy (Diabetic retinopathy, DR) is the most common and serious eye complication of diabetes mellitus (DM), and is the leading cause of blindness in the working-age population. At present, the number of DM patients in the world is as high as 347 million, nearly 30% of DM patients have different degrees of DR, and the vision of 37.3 million DR patients is threatened. If effective measures are not taken immediately, it is estimated that by 2030, the number of DR patients worldwide will increase to 190 million, and the number of DR patients whose vision is threatened will increase to 56.3 million. Therefore, it is of great significance and urgent to study the occurrence and development of DR and seek new means to effectively control DR. [0003] The p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q2600/154
Inventor 陈有信张古沐阳马楠田蓉张辰茜张梦雨
Owner 陈有信
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