CN116004803A - Application of gene detection reagent in preparation of kit for diagnosing diabetes combined coronary heart disease - Google Patents

Abstract

The invention provides the application of a gene detection reagent in the preparation of a kit for diagnosing diabetes complicated with coronary heart disease, which belongs to the technical field of biomedicine. The detection reagent provided by the present invention is a reagent for detecting the expression level of LncRNA-AC141930.2. By detecting the expression level of LncRNA-AC141930.2, it is possible to detect early whether type 2 diabetes patients are complicated with coronary heart disease. At the same time, the siRNA provided by the present invention can effectively reduce the senescence of human microvascular endothelial cells caused by high glucose and can reduce the tube-forming ability of human microvascular endothelial cells caused by high glucose, so it can be used to prepare a drug for treating diabetes combined with coronary heart disease. drug.

Description

Application of a gene detection reagent in the preparation of a kit for diagnosing diabetes combined with coronary heart disease

This case is a divisional application. The invention name of the original application is: a drug for the treatment of type 2 diabetes combined with coronary heart disease. The filing date of the original application is: 2022-02-15, and the application number of the original application is: CN202210136598.1 .

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to the application of a gene detection reagent in the preparation of a kit for diagnosing diabetes complicated with coronary heart disease.

Background technique

Type 2 diabetes is a common metabolic disease, and its prevalence is increasing year by year. At present, type 2 diabetes is no longer just a disease of the rich in developed countries, and developing countries including China have also become the hardest hit areas of type 2 diabetes. Type 2 diabetes generally causes damage to blood vessels throughout the body, because the blood vessels involved are irreversible throughout the body. Therefore, type 2 diabetes has become an important killer of modern diseases.

Sustained increase in blood sugar is a prominent feature of type 2 diabetes patients. Long-term high blood sugar makes type 2 diabetes patients more likely to suffer from cardiovascular disease than non-diabetics. Diabetes combined with coronary heart disease is the cause of death or fatal cardiovascular complications. main cause of disability. Type 2 diabetes combined with coronary heart disease is a type of diabetic coronary heart disease, which is based on type 2 diabetes and leads to coronary atherosclerosis or changes in coronary artery function, narrowing and blockage of the blood cavity of the heart, or ischemia and hypoxia of the heart Even necrotizing heart disease. Therefore, it is necessary to realize early diagnosis and early treatment of type 2 diabetes, so as to improve the quality of life of patients.

At the same time, existing studies have found that long-term abnormal glucose and lipid metabolism leads to endothelial dysfunction, so effectively reducing endothelial damage caused by high glucose will help the treatment of type 2 diabetes combined with coronary heart disease.

Contents of the invention

The problem to be solved by the present invention is to provide a medicine for treating type 2 diabetes combined with coronary heart disease and a marker for diagnosing type 2 diabetes combined with coronary heart disease.

In order to solve the above problems, the present invention provides the following technical solutions:

A medicine for treating type 2 diabetes combined with coronary heart disease, the medicine for treating type 2 diabetes combined with coronary heart disease contains LncRNA-AC141930.2 siRNA and a drug carrier.

Further, the drug carrier is one or more of cholesterol, liposome, and nanoparticle;

Further, the drug can be administered orally or parenterally. For parenteral administration, it can be administered through intravenous injection, intranasal injection, local injection, intracerebroventricular injection, spinal injection, subcutaneous injection, intraperitoneal injection, Administration by means of transdermal administration or the like.

Further, the transcript sequence of the LncRNA-AC141930.2 is shown in SEQ ID NO.1, and the sequence of the siRNA is shown in SEQ ID NO.4 and SEQ ID NO.5.

Application of siRNA of LncRNA-AC141930.2 in the preparation of drugs for treating diabetes complicated with coronary heart disease.

Further, the transcript sequence of the LncRNA-AC141930.2 is shown in SEQ ID NO.1, and the sequence of the siRNA is shown in SEQ ID NO.4 and SEQ ID NO.5.

Application of siRNA of LncRNA-AC141930.2 in the preparation of biological agents for inhibiting cell senescence caused by high glucose.

Further, the transcript sequence of the LncRNA-AC141930.2 is shown in SEQ ID NO.1, the sequence of the siRNA is shown in SEQ ID NO.4 and SEQ ID NO.5, and the cells are human microvascular endothelial cells .

Application of siRNA of LncRNA-AC141930.2 in the preparation of biological preparations for inhibiting the reduction of cell angiogenesis ability caused by high glucose.

Further, the transcript sequence of the LncRNA-AC141930.2 is shown in SEQ ID NO.1, the sequence of the siRNA is shown in SEQ ID NO.4 and SEQ ID NO.5, and the cells are human microvascular endothelial cells .

Application of a reagent for detecting the expression level of LncRNA-AC141930.2 in the preparation of a kit for diagnosing diabetes combined with coronary heart disease.

Further, the transcript sequence of the LncRNA-AC141930.2 is as described in SEQ ID NO.1, the reagent is the PCR primer of LncRNA-AC141930.2, and the sequence of the PCR primer is as SEQ ID NO.2 and SEQ ID Shown in NO.3.

The beneficial effects of the present invention are:

The siRNA of LncRNA-AC141930.2 provided by the present invention can reduce the aging of human microvascular endothelial cells caused by high glucose and can reduce the tube-forming ability of human microvascular endothelial cells caused by high glucose, so it can be used to prepare and treat type 2 diabetes Drugs for coronary heart disease. At the same time, the LncRNA-AC141930.2 provided by the present invention can be used as a marker to diagnose patients with diabetes and coronary heart disease.

Description of drawings

Figure 1 The expression difference of LncRNA-AC141930.2 in different groups;

Figure 2 ROC curve between healthy control group and type 2 diabetes combined with coronary heart disease group;

Figure 3 ROC curve between type 2 diabetes group and type 2 diabetes combined with coronary heart disease group;

Figure 4 ROC curve between coronary heart disease group and type 2 diabetes combined with coronary heart disease group;

Figure 5 Effect of high glucose on the RNA level of LncRNA-AC141930.2 in human microvascular endothelial cells;

The interference effect of the si-lnc designed in Fig. 6 of the present invention;

Figure 7 Effect of si-lnc on cell senescence of human microvascular endothelial cells induced by high glucose;

Fig. 8 Effect of si-lnc on tube-forming ability of human microvascular endothelial cells induced by high glucose.

Detailed ways

In order to clearly illustrate the technical characteristics of this solution, the content of the present invention will be further described in detail below through specific implementation methods. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. According to common technical knowledge and conventional means in this field, without departing from the above-mentioned basic technical idea precursor of the present invention, other various forms of modification, replacement or change can also be made. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Example 1

(1) Collect peripheral blood from normal people (30 cases, healthy control group), peripheral blood from patients with type 2 diabetes (30 cases, type 2 diabetes group), peripheral blood from patients with coronary heart disease (30 cases, coronary heart disease group) 2 Peripheral blood of patients with type 2 diabetes mellitus and coronary heart disease (30 cases, type 2 diabetes mellitus and coronary heart disease group), there was no significant difference in age and sex between the groups, and the patients voluntarily participated in this study and filled out the informed consent form;

(2) Diagnostic criteria for diabetes: fasting blood glucose ≥ 7.0mmol L ^-1 , or random blood glucose ≥ 11.1mmol L ^-1 ; diagnostic criteria for coronary heart disease: coronary angiography with at least one large epicardial vessel stenosis ≥ 50;

(3) Diagnostic criteria for diabetes mellitus complicated with coronary heart disease: fasting blood glucose ≥7.0mmol L-1, or random blood glucose ≥11.1mmol L-1 and at least one large epicardial vessel stenosis ≥50% by coronary angiography;

(4) Exclusion criteria: the diabetic group excluded patients with diabetic nephropathy, diabetic retinopathy, hypertension, peripheral arteriosclerosis, who were taking hypoglycemic, lipid-lowering, blood pressure-lowering and insulin therapy;

History of acute and chronic infection, liver and kidney insufficiency, severe cerebrovascular accident, malignant tumor, rheumatism, immune system disease, hyperthyroidism, gout and pituitary disease;

Female patients need to exclude the possibility of lactation and pregnancy;

patients with mental illness;

(5) The early morning fasting venous blood of the subjects selected for the experiment was collected, centrifuged at 3000r/min for 15min, and then the supernatant was taken to obtain the sample serum.

Example 2

RNA extraction from samples

(1) Take 200ul sample serum and add it to a centrifuge tube, add 200ul RNA lysate, shake and mix for 30s;

(2) After standing at room temperature for 5 minutes, centrifuge at 12,000 rpm for 10 minutes, and pipette the supernatant into a new centrifuge tube;

(3) Add 200ul of chloroform, shake for 15s, place at room temperature for 5min, centrifuge at 12000rpm for 15min, and transfer the upper aqueous phase to a new centrifuge tube;

(4) Add an equal volume of isopropanol, mix well and let stand for 10 minutes, then centrifuge at 12000 rpm for 10 minutes, discard the supernatant, and keep the precipitate;

(5) Add 70% ethanol prepared with 500ul pre-cooled DEPC water to dissolve the precipitate, centrifuge at 8000 rpm for 5min, repeat once, discard the supernatant, dry at room temperature for 10min, add DEPC water to obtain RNA.

Example 3

reverse transcription reaction

(1) The reverse transcription process was performed with reference to the PrimeScript ^TM RT reagent Kit kit from TaKaRa Company;

(2) configure the following reaction system:

Reagent Usage amount 5×PrimeScript Buffer(for Real Time) 2.0μl PrimeScript RT Enzyme Mix I 0.5μl Oligo dT Primer(50μM)*1 0.5μl Random 6mers(100μM)*1 0.5μl Total RNA 500ng RNase Free dH2O Up to 10μl

(3) Carry out the reverse transcription reaction on the PCR instrument according to the following conditions:

37°C for 15min*3; 85°C for 5s; 4°C for 5min.

Example 4

PCR reaction

(1) PCR reaction refers to TaKaRa company TB Premix Ex Taq ^TM II kit steps to operate;

(2) configure the following reaction system:

(3) react according to the following reaction conditions:

Step1 95°C 30s, Step2 95°C 5s, 60°C 30s 40cycle;

(4) Design and synthesize the primer sequence of LncRNA-AC141930.2 to be detected in the present invention:

(5) The results obtained are shown in Figure 1.

Compared with the healthy control group, the expression level of LncRNA-AC141930.2 in the type 2 diabetes group was 0.93±0.53, the difference was not statistically significant, and the expression level of LncRNA-AC141930.2 in the coronary heart disease group was 1.10±0.54, the difference was not significant. Statistically significant, the expression level of LncRNA-AC141930.2 in the type 2 diabetes combined with coronary heart disease group was 2.40, the difference was statistically significant, at the same time, compared with the type 2 diabetes group and the coronary heart disease group, the difference was also statistically significant .

According to the results in Figure 1, draw the ROC curve of type 2 diabetes combined with coronary heart disease group and healthy control group, draw the ROC curve of type 2 diabetes combined with coronary heart disease group and type 2 diabetes group, draw the type 2 diabetes combined with coronary heart disease group and coronary heart disease The ROC curve of the group, the obtained curves are shown in Figure 2, Figure 3 and Figure 4, respectively, where the AUC value in Figure 2 is 0.9180, the AUC value in Figure 3 is 0.9278, and the AUC value in Figure 4 is 0.8971, It can be seen that when LncRNA-AC141930.2 is used as a diagnostic marker, it can effectively distinguish the healthy control group, the type 2 diabetes group and the combined coronary heart disease group.

Example 5

Effect of high glucose on the expression of LncRNA-AC141930.2 in human microvascular endothelial cells (HMEC-1)

(1) Inoculate HMEC-1 cells in a 6-well plate and place them in a cell incubator for cultivation;

(2) When the cell density reaches more than 85%, add ordinary DMEM medium to the control group, add DMEM medium containing 30mmol/L D-glucose to experimental group a, and add DMEM medium containing 25mmol/L mannitol to experimental group b , set 3 repetitions for each group;

(3) After 48 hours of cell culture, RNA was extracted to detect the expression of LncRNA-AC141930.2 in different groups.

The experimental results are shown in Figure 5, where the expression level of LncRNA-AC141930.2 in experimental group a was 3.68±0.52, the difference was statistically significant, and the expression level of LncRNA-AC141930.2 in experimental group b was 0.98±0.10, the difference was not significant Statistical significance. The above results indicate that high glucose can increase the expression of LncRNA-AC141930.2 in human microvascular endothelial cells, and the increased expression is not caused by osmotic pressure.

Example 6

Construction of siRNA for LncRNA-AC141930.2

(1) Design and synthesize the siRNA of LncRNA-AC141930.2, which is named si-lnc for convenience, and the sequence of si-lnc is as follows:

Justice chain: AUUUCAUAAUUGCAUUUGC, SEQ ID NO.4;

Antisense strand: GCAAAUGCAAUUAUGAAAU, SEQ ID NO.5;

(2) Seeding HMEC-1 in a 6-well culture plate, and transfecting si-nC and si-lnc into the cells when the cell density reaches above 85%;

(3) After 48 hours of transfection, cellular RNA was extracted to detect the expression level of LncRNA-AC141930.2.

The experimental results are shown in Figure 6, the si-lnc provided by the present invention can effectively inhibit the expression level of LncRNA-AC141930.2 in HMEC-1 cells, and the inhibition rate is 82.3%.

Example 7

Effect of transfection si-lnc on cell senescence of HMEC-1 induced by high glucose

(1) Inoculate HMEC-1 in a 6-well culture plate, and when the cell density reaches above 85%, the control group: transfect si-NC, and use common DMEM medium for culture, experimental group a: transfect si-NC NC was cultured in DMEM medium containing 30mmol/L D-glucose, experimental group b: transfected with si-lnc and cultured in DMEM medium containing 30mmol/L D-glucose;

(2) After 48 hours of transfection, the medium was removed, and the cells were washed 3 times with PBS;

(3) Add 1ml of cell fixative to fix the cells, and place at room temperature for 20 minutes;

(4) After the fixation, remove the fixative, wash the cells with PBS, add β-galactosidase staining solution, and stain overnight at 37°C;

(5) After staining, remove the staining solution, wash the cells twice with PBS, add 2ml of PBS and take pictures under a microscope to detect the positive rate of cells.

The experimental results are shown in Figure 7. It can be seen that transfection of si-lnc can effectively inhibit cell senescence caused by high glucose.

Example 8

Effect of transfection si-lnc on HMEC-1 tube-forming ability induced by high glucose

(1) Place the Matrigel glue in an ice box one day before the experiment, put it in a 4°C refrigerator, and let the glue melt slowly overnight;

(2) ECM medium and Matrigel glue are mixed according to the ratio of 1:1 to obtain a mixed solution;

(3) In a 48-well plate, add 180 μ of the mixed solution to each well, and place the 48 wells at 37°C for 30 minutes to solidify the gel;

(4) The control group used cells transfected with si-nc and cultured in ordinary medium, experimental group a used cells transfected with si-nc and cultured in DMEM medium containing 30mmol/L D-glucose, and experimental group b used For cells transfected with si-lnc and cultured in DMEM medium containing 30mmol/L D-glucose, 500ul containing 4× ¹⁰⁴ cells were incubated in a 48-well plate at 37°C;

(5) After incubation for 4 hours, take pictures under an inverted microscope, and count the number of branches.

The experimental results are shown in Figure 8. It can be seen that after transfection with si-lnc, the reduced tube-forming ability caused by high glucose can be effectively restored.

Claims (4)

1. The reagent that detects LncRNA-AC141930.2 expression level is in the application in the preparation diagnosis kit of diabetes mellitus combined with coronary heart disease, it is characterized in that, the transcript sequence of described LncRNA-AC141930.2 is as described in SEQ ID NO.1, said Said reagent is the PCR primer of LncRNA-AC141930.2, and the sequence of said PCR primer is as shown in SEQ ID NO.2 and SEQ ID NO.3. 2. A medicine for treating type 2 diabetes combined with coronary heart disease, characterized in that the medicine for treating type 2 diabetes combined with coronary heart disease contains siRNA and a drug carrier of LncRNA-AC141930.2; The transcript sequence of the LncRNA-AC141930.2 is shown in SEQ ID NO.1, and the sequence of the siRNA is shown in SEQ ID NO.4 and SEQ ID NO.5. 3. The application of the siRNA of LncRNA-AC141930.2 in the preparation of biological preparations for inhibiting cell senescence caused by high glucose, characterized in that the transcript sequence of the LncRNA-AC141930.2 is shown in SEQ ID NO.1, the The sequence of the siRNA is shown in SEQ ID NO.4 and SEQ ID NO.5, and the cells are human microvascular endothelial cells. 4. The application of the siRNA of LncRNA-AC141930.2 in the preparation of biological preparations that inhibit the reduction of cell angiogenesis ability caused by high glucose, is characterized in that the transcript sequence of the LncRNA-AC141930.2 is as shown in SEQ ID NO.1 The sequence of the siRNA is shown in SEQ ID NO.4 and SEQ ID NO.5, and the cells are human microvascular endothelial cells.

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