CN107299112A - A kind of inflammatory molecule（PSGL‑1）The structure of the Anomalous lipid metablism model of missing and its application - Google Patents

Abstract

The invention discloses the construction and application of an abnormal lipid metabolism model lacking an inflammatory molecule (PSGL‑1); the construction method of the model is: based on the abnormal lipid metabolism model-LDLR gene knockout mouse, namely: LDLR‑ ^{/ ‑Mouse} , modify the PSGL‑1 gene in its genome, block the expression of PSGL‑1 protein, and obtain a transgenic animal model with PSGL‑1 deletion combined with lipid metabolism disorder – LDLR ^‑/‑ , PSGL‑1 ^‑/‑ small mouse. The study found that after 12 weeks of high-fat diet induction, compared with LDLR ^‑/‑ mice, the newly constructed LDLR ^‑/‑ , PSGL‑1 ^‑/‑ mice had lower levels of total cholesterol and low-density lipoprotein in serum, and higher Density lipoprotein content increased; LDLR ^‑/‑ , PSGL‑1 ^‑/‑ mice had significantly reduced hepatic lobular steatosis, and significantly reduced the area of atherosclerotic plaques in the aorta and aortic root. This model provides a new research vehicle for systematically studying the relationship between inflammatory molecules and lipid metabolism, and finding new drug targets and therapeutic methods.

Description

Construction of an abnormal lipid metabolism model lacking an inflammatory molecule (PSGL-1) and its application

technical field

The present invention relates to a construction method and application of a transgenic animal model, in particular to a construction method and application of a novel transgenic animal model lacking inflammatory molecule (PSGL-1) combined with abnormal lipid metabolism.

Background technique

With the change of people's diet and living habits, the incidence of metabolic diseases is increasing year by year. Abnormal lipid metabolism can lead to diseases such as hypertension and atherosclerosis, which seriously threaten human health. Studies have shown that inflammatory molecules are closely related to the occurrence and development of abnormal lipid metabolism, and inflammation runs through the entire process of disease occurrence and development. However, the current research on inflammation and lipid metabolism is only limited to the level of cells and clinical tissues, and lacks dynamic overall research. A mouse tool model of inflammatory molecules combined with dyslipidemia. Low-density lipoprotein receptor gene knockout (LDLR ^-/- ) mice are a classic model for studying abnormal lipid metabolism, and adhesion molecule knockout mouse models are often used to study molecular mechanisms related to diseases and inflammation. This project intends to use the inflammatory molecule ((PSGL-1)) deficient mice of the existing genetic engineering mouse platform to cross with LDLR ^-/- mice with abnormal lipid metabolism, aiming to obtain the lipid The metabolic abnormal model provides a new research carrier for systematically studying the relationship between inflammatory molecules and lipid metabolism, searching for new drug targets and treatment methods, and provides a demonstration for the in-depth development of genetically engineered mouse models.

Transgenic animals have emerged as the most effective animal experimental models. Compared with ordinary experimental mice, genetically engineered mice have incomparable advantages: the occurrence of diseases is naturally formed, which can simulate the in vivo occurrence process of human diseases, and the research results are more real and reliable; dynamic continuous observation can overcome the clinical Study limitations. LDLR ^-/- genetically engineered mice are a classic mouse model of abnormal lipid metabolism. Knocking out the low-density lipoprotein receptor gene results in LDLR functional defects, increased plasma LDL-C levels, and induces atherosclerosis and Hepatic steatosis, etc. The mechanism of disease formation is very similar to clinical. A large amount of evidence shows that lipid metabolism diseases such as atherosclerosis are essentially inflammatory diseases, and the expression of inflammatory molecules is closely related to blood lipid disorders and aortic lesions. However, there is still a lack of good models to study the relationship between inflammation and lipid metabolism disorders. (PSGL-1) is an important adhesion molecule. The interaction between P-selectin/(PSGL-1) mediates intercellular reactions such as neutrophil-platelet and neutrophil-endothelial cells, activates platelets, promotes Thrombosis is involved in endothelial injury, mediates inflammatory cell adhesion and lipid deposition, and at the same time (PSGL-1) can transduce extracellular signals, promote the activation of leukocytes and make them adhere stably. The P-selectin/(PSGL-1) interaction plays an important role in the formation and stability of atherosclerosis (AS), suggesting its significance to cardiovascular diseases. We plan to cross these two kinds of mice to obtain (PSGL-1) ^-/- , LDLR ^-/- mouse models, providing researchers with a useful tool model suitable for studying the relationship between inflammation and metabolic diseases At the same time, it provides a demonstration for the in-depth development of genetically engineered mouse innovative models, which has good application value and social significance.

Contents of the invention

The purpose of the present invention is to provide a method for constructing a novel mouse model of inflammatory molecule deletion combined with lipid metabolism disorder.

Another object of the present invention is to provide the application of the above animal model.

The technical scheme that the present invention takes is:

In the (PSGL-1) ^-/- , LDLR ^-/- mouse model constructed in the present invention, the loss of (PSGL-1) protein expression does not affect the normal life of the mice.

The inventor found through research that the transgenic engineering animal LDLR ^-/- , (PSGL-1) ^-/- mice was fed a high-fat diet for 12 weeks, compared with LDLR ^-/- mice, LDLR ^-/- , (PSGL-1 ) ^-/- mice serum total cholesterol and low-density lipoprotein content decreased, high-density lipoprotein significantly increased; found by HE staining of the liver, compared with LDLR ^-/- mice, LDLR ^-/- ,( PSGL-1) ^-/- mice hepatic lobular fatty degeneration was significantly reduced, and the total cholesterol and triglycerides in the liver were also significantly reduced; it was found by oil red O staining of the aorta and aortic root, compared with LDLR ^-/- In mice, LDLR ^-/- , (PSGL-1) ^-/- mice had significantly reduced plaque deposition in the aorta and aortic root. Studies have shown that the reduction of (PSGL-1) protein expression can alleviate the abnormal state of lipid metabolism in LDLR ^-/- mice. This model can be effectively used to study the relationship between inflammatory molecules and lipid metabolism, and can be used for the inflammatory mechanism of metabolic diseases Research.

Description of drawings

Figure 1 is a schematic diagram of the structure and combination of (PSGL-1);

Figure 2 is a mouse identification diagram;

Figure 3 is the blood lipid measurement of LDLR ^-/- and LDLR ^-/- , (PSGL-1) ^-/- mice;

Figure 4 is the liver HE staining and liver lipid detection of LDLR ^-/- and LDLR ^-/- , (PSGL-1) ^-/- mice;

Fig. 5 is the statistical analysis of aortic oil red staining and plaque area of LDLR ^-/- and LDLR ^-/- , (PSGL-1) ^-/- mice;

Fig. 6 is the statistical analysis of oil red staining and plaque area of the aortic root of LDLR ^-/- and LDLR ^-/- , (PSGL-1) ^-/- mice.

detailed description

1 Experimental principle

P-selectin glycoprotein ligand-1 ((PSGL-1)) is an adhesion molecule that expresses sialylated and fucosylated glycans discovered in the early 1990s and belongs to type I transmembrane proteins. (PSGL-1) can not only identify and capture leukocytes from the blood during the initial adhesion process, but also act as a signal transduction molecule to transmit signals into the cells to activate leukocytes (Figure 1). It is now known that (PSGL-1) has a regulatory role in the inflammatory response. The model constructed by the present invention focuses on the effect of (PSGL-1) deletion on lipid metabolism and atherosclerotic plaque formation in LDLR ^-/- mice.

2 Experimental methods

2.1 Crossbreed expansion of genetically engineered mice Take LDLR ^-/- and (PSGL-1) ^-/- for crossbreeding to obtain F1 generation (PSGL-1) ^+/- ; LDLR ^+/- mice; F1 generation mice after crossbreeding , 4 different genotype mice can be obtained, namely: (PSGL-1) ^+/- ; LDLR ^-/- , (PSGL-1) ^-/- ; LDLR ^+/- , (PSGL-1) ^+/+ ; LDLR ^-/- (LDLR ^-/- mice) and (PSGL-1) ^-/- ; LDLR ^-/- , the first two genotypes of mice can be used to continue crossbreeding, the latter two genotypes are small The mice were used as control mice and experimental mice, respectively. Mice were born normally during the hybridization process. There are no more than four birds in each cage, and they are bred at a ratio of male: female = 1:3 or 1:2.

2.2 The blood lipid measurement adopts Zhongsheng Beikong blood lipid determination kit, and the reaction volume is reduced by 5 times at the same time when measuring the blood lipid of mice. TC, TG and HDL-C concentrations were measured directly. The measurement method is: prepare each reaction working solution according to the requirements, the TC and HDL reaction working solutions are the same, but the TG reaction working solution is different from the former two. When measuring HDL, it is necessary to mix the serum sample and precipitant at a ratio of 1:1 in advance, and after standing at room temperature for 30 minutes, centrifuge at 3000rpm for 30 minutes. The supernatant was used for measurement. Dilute the standard products of TC, TG and HDL by 2 times, 4 times, 8 times, 16 times and 32 times respectively. At the same time, 200 μL of the corresponding working solution was added to each well. Add each diluted standard to the corresponding well, and add each serum sample correspondingly. The standard and sample addition volumes are 2 μL (TC), 2 μL (TG) and 5 μL (HDL-C) respectively. solution as a blank control well. After reacting at 37°C for 10 minutes, read at a wavelength of 490nm, and count the OD values separately. The detection of LDL-C is different from the above three detection methods. Pipette 3 μL each of the standard and sample, then add the R1 reaction solution in the kit, mix well, put it in a 37°C oven and incubate for 5 minutes, then measure the absorbance with a microplate reader at a wavelength of 570nm to obtain the OD ₁ value. Then add the R2 reaction solution in the kit, mix well, put it in a 37°C oven and incubate for 5min, and then measure the absorbance with a microplate reader at a wavelength of 570nm to obtain the OD ₂ value. The final calculated OD value=OD ₂ −OD ₁ , and finally calculated the final concentration according to the standard curve.

2.3 HE staining Paraffin sections were dewaxed, stained with hematoxylin for 1.5 minutes, bluish for 30 minutes, stained with eosin for 1.5 seconds, overnight at 37°C, and sealed. The structural changes of each tissue were observed and photographed under a microscope.

2.4 Oil red staining experiment of vascular plaques After the mice were perfused, the aorta was separated along the mouse spine, up to the arterial arch at the heart orifice, down to the thoracoabdominal aorta, that is, the branch of the abdominal aorta to the two mouse kidneys. The isolated artery was fixed vertically on a plastic square with a black background, and the adipose tissue stripped from the outer wall of the aorta was separated and stripped under a stereomicroscope. It is necessary to press the blood vessel slightly flat with the tweezers while using the other tweezers to tear the fat strips along the direction of the blood vessels. After the fat lumps on the outer wall of the blood vessels are separated, the blood vessels are cut open with fine scissors. Because the artery is a curved structure, it generally needs to be cut along the arch, spread out in the shape of a branch and spread on a filter paper of a relatively large size. Fix with 4% paraformaldehyde for about 10min, rinse slightly with PBS and transfer to 1.5ml EP tube with filter paper to be stained. The dissected aorta was removed from the filter paper and transferred to a 1.5ml EP tube filled with 60% isopropanol for rinsing for about 30s. Then transfer to a 1.5ml EP tube filled with 60% Oil-Red-O, and then place the EP tube on a rotary machine for 1-1.5 hours of rotation and dark staining. After staining, transfer the blood vessel to another EP tube filled with 1ml of 60% isopropanol, and use a rotary machine to decolorize for 1 minute (if the background is dark, the decolorization time can be extended appropriately). Spread the decolorized blood vessels on a glass slide, and then put the whole slide on a table with a dark background and take pictures with an ordinary Canon camera.

2.5 Oil red staining experiment of aortic root The heart of the mouse was taken out and embedded for frozen section (if it could not be sliced immediately, it should be stored in a -80°C refrigerator), and serial sections of the aortic root were collected. Some sections were briefly rinsed with PBS to elute the frozen embedding medium. After rinsing with 60% isopropanol for 20-30 seconds, transfer to 60% Oil Red O staining. After 30 min of dark staining, rinse briefly with 60% isopropanol for 40 sec to elute background staining. After 3 minutes of nuclear staining with hematoxylin, wash twice with PBS, 5 minutes each time. After rinsing, the slides were dried in a 37°C oven. The slides were sealed with ordinary glue and photographed under an Olympus optical microscope. The captured photos were placed in Image pro Plus software to count the area of atherosclerotic plaque in the mice. The average value of the plaque area of 8 consecutive slices of each mouse was the area value of atherosclerosis in the mouse.

3 Experimental results

3.1 Identification results of genetically engineered mice by hybridization and population expansion: Tail clipping, numbering, DNA extraction, PCR amplification, agarose electrophoresis, LDLR mice can see a band at 350bp, and wild-type mice can see a band at 167bp; (PSGL-1) mice can see a band at 500bp, and wild-type mice can see a band at 140bp. It can be seen from Fig. 2 that 1#-7# mice are LDLR-/- mice; 8#-11# are (PSGL-1) ^-/- ; LDLR ^-/- double knockout mice. (Note that the Mark strips are 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp from top to bottom)

3.2 Blood lipid measurement results As shown in Figure 3, the total cholesterol and low-density lipoprotein contents in the serum of LDLR ^-/- , (PSGL-1) ^-/- mice were reduced, and high-density lipoprotein was significantly increased.

3.3 HE staining The results of HE staining and liver lipid measurement in mouse liver are shown in Figure 4. In the liver lobules of LDLR ^-/- mice, hepatic edema and large fat vacuole degeneration appeared, while LDLR ^-/- ,(PSGL-1) ^-/- double-knockout mice showed reduced hepatic edema, and LDLR ^-/- ,(PSGL-1) ^-/- double-knockout mice had significant changes in total cholesterol and triglycerides in the liver reduce.

3.4 Oil red staining experiment of vascular plaque The results of the oil red staining experiment of aortic plaque are shown in Figure 5. The absence of (PSGL-1) protein can reduce the deposition of aortic atherosclerotic plaque in mice with statistically significant significance (P≤0.01).

3.5 Oil red staining experiment of the aortic root The results of the oil red staining experiment of the aortic root are shown in Figure 6. The absence of (PSGL-1) protein can reduce the deposition of atherosclerotic plaque in the aortic root of mice.

4 Experimental conclusions

After being induced by high-fat diet for 12 weeks, compared with LDLR-/- mice, the newly constructed LDLR-/-, (PSGL-1)-/- mice decreased the serum levels of total cholesterol and low-density lipoprotein, and the high-density lipoprotein Lipoprotein content increased; LDLR-/-, (PSGL-1)-/- mice had significantly reduced hepatic lobular fatty degeneration, and significantly reduced the area of atherosclerotic plaques in the aorta and aortic root. It shows that the reduction of (PSGL-1) protein expression can alleviate the abnormal state of lipid metabolism in LDLR-/- mice, and this model can be effectively used to study the relationship between inflammatory molecules and lipid metabolism, and can be used for the research of the inflammatory mechanism of metabolic diseases .

Claims (4)

1. a kind of construction method of the Anomalous lipid metablism model of inflammatory molecule (PSGL-1) missing, it is characterised in that：Using low close Spend lipoprotein receptor gene knockout LDLR^-/-Mouse carries out genetic modification engineering technology knockout (PSGL-1) gene so that (PSGL- 1) the expression contents reduction of albumen, obtains a kind of new LDLR^-/-,(PSGL-1)^-/-Anomalous lipid metablism mouse model.

2. the construction method of the Anomalous lipid metablism model of inflammatory molecule (PSGL-1) missing according to claim 1, it is special Levy and be：In the case of normal diet, the growing of Anomalous lipid metablism mouse, the bodily form and daily row of (PSGL-1) missing All not show exception.

3. the construction method of the Anomalous lipid metablism model of Hu inflammatory molecule (PSGL-1) missing according to claim 2, it is special Levy and be：In the case of high fat is induced 12 weeks, LDLR is contrasted^-/-Mouse, LDLR^-/-,(PSGL-1)^-/-Total courage is consolidated in mice serum The content reduction of alcohol and low-density lipoprotein, hdl concentration rise；Lobuli hepatis steatosis substantially mitigates；Sustainer There is obvious reduction with the plaque area of aortic root.

4. the construction method of the Anomalous lipid metablism model of inflammatory molecule ((PSGL-1)) missing according to claim 3, it is special Levy and be：LDLR-/-, (PSGL-1) -/- DNA murine can be used for the zooscopy of lipid metaboli relevant disease, be that system research is scorching The correlation of disease molecule and lipid metaboli, finds new drug targets and a kind of new Study of Support for the treatment of method offer.