HSL gene, HSL gene in-vivo delivery preparation, preparation and application in atherosclerosis
By "grafting" the HSL gene into smooth muscle foam cells and achieving targeted delivery to vascular smooth muscle cells, the problem of difficulty in inhibiting plaques formed in atherosclerosis in the existing technology is solved, and effective inhibition of plaque formation is achieved. and reduce plaque area.
Patent Information
- Application Number
- CN202411360204.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-27
- Publication Date
- 2025-01-14
AI Technical Summary
The prior art is difficult to effectively inhibit the regression and shrinkage of plaques formed in atherosclerosis, and can only slow down the progress of the lesions.
By "grafting" the HSL gene into smooth muscle foam cells, HSL gene in vivo delivery preparations are prepared to achieve targeted delivery of HSL genes to vascular smooth muscle cells, and overexpression of HSL improves cholesterol ester hydrolysis and inhibits smooth muscle cell migration.
Effectively inhibits the formation of arterial plaques in atherosclerotic mice, significantly reduces the area of plaques in aortic cross-sections, improves cholesterol metabolism and inhibits the proliferation and migration of smooth muscle cells.
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Figure CN119303114A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine, and specifically relates to an HSL gene, an HSL gene in vivo delivery preparation, and its preparation and application in atherosclerosis. Background Art
[0002] Atherosclerosis is a chronic inflammatory cardiovascular and cerebrovascular disease caused by lipid metabolism disorders, with excessive accumulation of cholesterol carried by low-density lipoprotein in the arterial wall as its pathological basis. Acute myocardial infarction and stroke that occur after atherosclerotic plaques fall off have become important fatal and disabling diseases. Therefore, finding effective treatment measures is a major issue that needs to be solved in the current field of cardiovascular and cerebrovascular diseases.
[0003] The formation of foam cells is a pathological hallmark of the entire atherosclerotic disease. In past studies, macrophages have been considered the main source of foam cells. Recent studies have revealed that more than 50% of foam cells in atherosclerotic plaques come from vascular smooth muscle cells. Under the stimulation of cholesterol load and inflammation, vascular smooth muscle cells proliferate and migrate to the intima, where some cells transform into a foam cell phenotype similar to macrophages and form atherosclerosis.
[0004] At present, lipid-lowering drugs mainly reduce the uptake of plasma lipoproteins and reduce the production of foam cells, which can only slow down the progression of lesions. However, the most important treatment for atherosclerosis is to make the formed plaques disappear and shrink. The present invention aims to provide a gene drug for treating atherosclerosis by "grafting" the key lipolytic enzyme HSL of adipocytes into smooth muscle foam cells. Summary of the invention
[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an HSL gene, an HSL gene in vivo delivery preparation, and its preparation and application in atherosclerosis.
[0006] To achieve the above object, the technical solution adopted by the present invention is:
[0007] An application of HSL gene in preparing medicine for treating atherosclerosis.
[0008] The HSL gene is a human HSL gene; the nucleotide sequence of the human HSL gene is shown in SEQ ID No.1.
[0009] The HSL gene is a mouse HSL gene; the nucleotide sequence of the mouse HSL gene is shown in SEQ ID No.2.
[0010] The present invention also includes an HSL gene in vivo delivery preparation, which includes an HSL gene plasmid and an in vivo delivery vector encapsulating the HSL gene plasmid.
[0011] The HSL gene plasmid is a human HSL gene plasmid; the preparation method of the human HSL gene plasmid is to use YOE-LV005 as a vector to insert the full-length human HSL gene sequence.
[0012] The HSL gene plasmid is a mouse HSL gene plasmid; the preparation method of the mouse HSL gene plasmid is to use pCMV as a vector to insert the full-length mouse HSL gene sequence.
[0013] The in vivo delivery carrier is an encapsulation layer composed of ionizable lipid, auxiliary phospholipid, pegylated lipid, cholesterol and targeting material; preferably, the ionizable lipid is SM-102, the auxiliary phospholipid is DSPC, the pegylated lipid is DMG-PEG 2000, and the targeting material is DSPE-PEG-cRGD (fk). The molar ratio of ionizable lipid, auxiliary phospholipid, pegylated lipid, cholesterol and targeting material is 50:10:1.5:38.5:1%.
[0014] The present invention also includes a method for preparing the HSL gene in vivo delivery system, comprising the following steps: using a syringe to respectively absorb a lipid organic phase and an aqueous phase containing an HSL gene plasmid, controlling the flow rate to inject into a microfluidic chip for mixing, and stopping collecting liquid after the aqueous phase solution in the syringe flows out to obtain the lipid nanoparticles; the aqueous phase includes an HSL gene plasmid and a citric acid buffer.
[0015] The present invention also includes an application of the HSL gene in vivo delivery preparation for preparing a drug for treating atherosclerosis.
[0016] Compared with the prior art, the present invention has the following beneficial effects:
[0017] Foam cell-like adipocytes have strong lipid storage and lipid metabolism capabilities and are the main energy depot of the human body. However, adipocytes do not undergo pathological changes in cell foaming due to the large accumulation of lipid droplets, and can maintain physiological functions normally. Hormone-sensitive lipase (HSL), encoded by the L IPE gene, plays a key role in lipid metabolism in adipocytes. Therefore, in combination with the lipolysis mechanism of adipocytes, the present invention prepares a plasmid of the HSL gene. In cell experiments, the overexpression of HSL can improve intracellular cholesterol ester hydrolysis, increase the expression of free cholesterol transport receptors, and inhibit smooth muscle cell migration by transfecting the plasmid into vascular smooth muscle cells. Animal experiments have shown that the HSL gene in vivo delivery preparation prepared by the present invention can achieve targeted delivery of the HSL gene to blood vessels, and can effectively inhibit the formation of arterial plaques in mice with atherosclerotic disease by injecting the HSL gene in vivo delivery preparation prepared by the present invention into the tail vein. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 Infographic of homo-HSL plasmid and mus-HSL plasmid;
[0019] Figure 2 Western blotting to detect the overexpression efficiency of homo-HSL and mus-HSL plasmids transfected into smooth muscle cells (HAS, VSM);
[0020] Figure 3 Oil red staining confirmed that overexpression of HSL significantly inhibited the accumulation of lipid droplets in smooth muscle cells;
[0021] Figure 4 Nile red staining confirmed that overexpression of HSL significantly inhibited the accumulation of lipid droplets in smooth muscle cells;
[0022] Figure 5 Total cholesterol (TC) and free cholesterol (FC) content analysis chart;
[0023] Figure 6 Western bl ot experiments showed that HSL overexpression increased the expression level of ABCA1 / ABCG1 in smooth muscle cells;
[0024] Figure 7 The results of the scratch test and CCK-8 test showed that HSL overexpression greatly weakened the proliferation and migration ability of smooth muscle cells induced by high fat;
[0025] Figure 8 Diagram of the construction of HSL gene in vivo delivery system HSL-P l asmi d@LNP-c(RGDfk);
[0026] Fig. 9 Graph showing the therapeutic effect of the constructed HSL-P l asmid@LNP-c(RGDfk) on atherosclerotic mice. DETAILED DESCRIPTION
[0027] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and the best embodiments.
[0028] Unless otherwise specified, the drugs and reagents involved in the examples are all common commercially available products; the experimental operations involved in the examples are all carried out according to routine operations in the art unless otherwise specified.
[0029] Example 1
[0030] Construct homo-HSL (human HSL gene) and mus-HSL (mouse HSL gene) overexpression plasmids and verify the HSL overexpression efficiency at the cell level
[0031] 1. Construct a plasmid overexpressing homo-HSL; Figure 1 As shown in A, YOE-LV005 was used as a vector to insert the full-length human HSL gene sequence. The detailed information is as follows:
[0032] Gene name: homo-HSL; Gene ID: 3991; Transcript: NM_005357.4; Gene sequence is shown in SEQ ID NO: 1; Sequence length: 3231 bp; Vector name: YOE-LV005.
[0033] The sequence of the present invention is SEQ ID NO: 1:
[0034]
[0035] 2. Construct a plasmid overexpressing mus-HSL; Figure 1 As shown in B, the full length of the mouse HSL gene sequence was inserted using pCMV as a vector. The detailed information is as follows:
[0036] Gene name: mus-HSL; Gene ID: 16890; Transcript: NM_010719.5; The gene sequence is shown in SEQ ID NO: 2; Sequence length: 2409 bp; Vector name: pCMV.
[0037] The sequence of the present invention is SEQ ID NO:2:
[0038]
[0039] 3. Transfect homo-HSL or empty vector plasmid into human vascular smooth muscle cells (HAS) and mus-HSL or empty vector plasmid into mouse arterial smooth muscle cells (VSM) using transfection reagent Lipofectamine 2000 (#166T; Invitrogen). After 48 hours, harvest the cells to verify the overexpression efficiency. Figure 2 As shown, homo-HSL and mus-homo can significantly increase the expression level of HSL in smooth muscle cells.
[0040] Example 2
[0041] Effect of HSL gene plasmid on vascular smooth muscle cell foaming: The vascular smooth muscle cell foaming disease model was constructed by pre-treating HAS and VSM cells with 80 μg / mL oxidized low-density lipoprotein (ox-LDL). After 48 hours of pre-treatment, the HSL plasmid was transfected into the smooth muscle cells and cultured for another 48 hours. The results were analyzed by oil red (#G1262, Solarbio) staining ( Figure 3 ), Nile red (#I N0170, Sol arbi o) staining ( Figure 4 ), total cholesterol (TC) / free cholesterol (FC) content ( Figure 5 ), cholesterol reverse transporter receptor ABCA1 and ABCG1 levels ( Figure 6 ) and cell proliferation and migration ability ( Figure 7-Figure 8 ) and other indicators were used to evaluate the reversal effect of HSL gene on the progression of smooth muscle cell foaming.
[0042] 1. The results of oil red and Nile red staining showed that compared with the control group, a large number of lipid droplets aggregated and formed foam cells in smooth muscle cells after 48 hours of ox-LDL pretreatment, while overexpression of HSL significantly inhibited the accumulation of lipid droplets and the formation of foam cells in HAS cells and VSM cells ( Figure 3-Figure 4 ).
[0043] 2. Collect the HAS cells and VSM cells treated above, add 500 μL of isopropanol to extract lipids, collect the suspension after ultrasonic emulsification, and calculate and analyze the total cholesterol (TC) and free cholesterol (FC) content according to the instructions of the total cholesterol determination kit. The results showed that overexpression of HSL significantly enhanced the ability of cholesterol ester hydrolysis in smooth muscle cells, significantly reduced the total cholesterol content, and increased free cholesterol ( Figure 5 ).
[0044] 3. Free cholesterol is transported through the cell membrane cholesterol reverse transport protein receptors ABCA1 and ABCG1 to enter the liver for metabolism. Western blotting experiments showed that HSL overexpression increased the expression level of ABCA1 / ABCG1 in smooth muscle cells ( Figure 6 ).
[0045] 4. Abnormal proliferation and migration of smooth muscle cells plays an important role in the formation of atherosclerotic plaques. The results of the scratch test and CCK-8 experiment showed that HSL overexpression greatly weakened the proliferation and migration ability induced by high fat ( Figure 7 ).
[0046] Based on the above results, it is shown that the homo-HSL and mus-HSL recombinant overexpression plasmids prepared by the present invention can significantly reverse the foaming process of smooth muscle cells induced by high fat.
[0047] Example 3
[0048] Construction of HSL gene delivery system HSL-P l asmid@LNP-c(RGDfk) in vivo
[0049] 1. Lipid-ethanol solution preparation: Weigh the corresponding amount of each material (SM-102 molar ratio 50, DSPC molar ratio 10, DMG-PEG2000 molar ratio 1.5, cholesterol molar ratio 38.5, DSPE-PEG-cRGD (fk) molar ratio 1%), add ethanol and ultrasonically dissolve them fully, then mix them, add ethanol to make up to the corresponding volume, vortex mix, and pass through a 0.2 μm filter membrane.
[0050] 2. Plasmid-citrate buffer preparation: HSL-P l asmid will be dissolved in 0.05M citrate buffer to form an aqueous phase.
[0051] 3. LNP preparation: Connect the syringes containing "lipid ethanol" and "buffer" to the sample introduction tube respectively, and install them on the syringe pump. Adjust the syringe model and flow rate (2mL / min) on the control panel, click Run, and collect the outflowing liquid with a collection tube. After the sample is collected, dilute the ethanol concentration to less than 1% with 30 volumes of PBS solution containing 10% sucrose.
[0052] 4. Detection and characterization: Detect encapsulation efficiency and measure LNP particle size and potential.
[0053] The results are as follows Figure 8 As shown, the particle size of HSL-P l asmid@LNP-c(RGDfk) is 167.1nm, the average PDI is 0.1538, the nucleic acid encapsulation efficiency is 96.7%, and the potential is -2.252mV.
[0054] Example 4
[0055] The constructed HSL-P l asmi d@LNP-c (RGDfk) has a therapeutic effect on atherosclerotic mice. In order to simulate atherosclerotic disease, the present invention uses the commonly used ApoE - / - The animal model was established using mice.
[0056] The present invention selects ApoE of 6-week-old male - / - Mice (purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.) were used as research models and divided into 3 groups:
[0057] Control group: normal feeding for 18 weeks
[0058] Model group: high-fat feeding (1-8 weeks), injection of normal saline via tail vein (once a week) + high-fat feeding (9-16 weeks), and continued high-fat feeding for 2 weeks (17-18 weeks)
[0059] Treatment group: high-fat feeding (1-8 weeks), tail vein injection of HSL-Plasmid@LNP-c(RGDfk) (once a week, 25μg / mouse / time) + high-fat feeding (9-16 weeks), and high-fat feeding for 2 weeks (17-18 weeks)
[0060] Finally, the mice were killed in vivo, and the arteries of the mice were dissected and separated. HE staining and oil red staining were used to detect the therapeutic effect of the constructed HSL-Plasmid@LNP-c(RGDfk) on atherosclerotic mice. HSL immunohistochemistry was used to evaluate the targeted delivery effect of HSL in the blood vessels in vivo ( Fig. 9 ).
[0061] The results are shown in the figure. HE and oil red staining showed that the aortic lipid deposition in the high-fat diet model group was verified, while the aortic lipid deposition in the treatment group was significantly reduced, and the area of the aortic cross-section plaque was significantly reduced.
[0062] In summary, adipocytes similar to foam cells have strong lipid storage and lipid metabolism capabilities and are the main energy depot of the human body. However, adipocytes do not undergo pathological changes in cell foaming due to the large accumulation of lipid droplets, and can maintain physiological functions normally. Hormone-sensitive lipase (HSL), encoded by the LIPE gene, plays a key role in lipid metabolism in adipocytes. Therefore, in combination with the lipolysis mechanism of adipocytes, the present invention prepares a plasmid of the HSL gene. In cell experiments, the plasmid is transfected into vascular smooth muscle cells. Overexpression of HSL can improve intracellular cholesterol ester hydrolysis, increase the expression of free cholesterol transport receptors, and inhibit smooth muscle cell migration. Animal experiments have shown that the HSL gene in vivo delivery preparation prepared by the present invention can achieve targeted delivery of the HSL gene to blood vessels by tail vein injection, and can effectively inhibit the formation of arterial plaques in mice with atherosclerotic disease.
[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. An application of HSL gene in the preparation of a drug for treating atherosclerosis.
2. The use according to claim 1, characterized in that: The HSL gene is a human HSL gene; the nucleotide sequence of the human HSL gene is shown in SEQ ID No.
1.
3. The use according to claim 1, characterized in that: The HSL gene is a mouse HSL gene; the nucleotide sequence of the mouse HSL gene is shown in SEQ ID No.
2.
4. An HSL gene in vivo delivery system, characterized in that: The invention comprises an HSL gene plasmid and an in vivo delivery vector which encapsulates the HSL gene plasmid.
5. The HSL gene in vivo delivery system according to claim 4, characterized in that: The HSL gene plasmid is a human HSL gene plasmid; the preparation method of the human HSL gene plasmid is to use YOE-LV005 as a vector to insert the full-length human HSL gene sequence.
6. The HSL gene in vivo delivery system according to claim 4, characterized in that: The HSL gene plasmid is a mouse HSL gene plasmid; the preparation method of the mouse HSL gene plasmid is to use pCMV as a vector to insert the full-length mouse HSL gene sequence.
7. The HSL gene in vivo delivery system according to claim 4, characterized in that: The in vivo delivery carrier is an encapsulation layer composed of ionizable lipids, auxiliary phospholipids, pegylated lipids, cholesterol and targeting materials; preferably, the ionizable lipid is SM-102, the auxiliary phospholipid is DSPC, the pegylated lipid is DMG-PEG 2000, and the targeting material is DSPE-PEG-cRGD (fk).
8. A method for preparing the HSL gene in vivo delivery system according to any one of claims 4 to 7, characterized in that: The method comprises the following steps: using a syringe to absorb a lipid organic phase and an aqueous phase containing an HSL gene plasmid respectively, controlling the flow rate to inject into a microfluidic chip for mixing, and stopping collecting liquid after the aqueous phase solution in the syringe flows out to obtain the lipid nanoparticles; the aqueous phase comprises an HSL gene plasmid and a citric acid buffer.
9. Use of the HSL gene in vivo delivery system according to any one of claims 4 to 7, characterized in that: It is used to prepare drugs for treating atherosclerosis.