Application of p55 gamma as aortic dissection treatment target

Abstract

The invention discloses application of p55 gamma as an aortic dissection treatment target, and belongs to the technical field of biological medicine. The p55 gamma overexpression inhibits the formation of BAPN-induced mouse aortic dissection and the degradation of elastic fibers; and the vascular smooth muscle cell knock-down p55 gamma mouse promotes the formation of BAPN-induced aortic dissection and the degradation of elastic fibers. In mechanism, p55 gamma plays a role by maintaining phenotypic transformation of vascular smooth muscle cells, and p55 gamma is knocked down to promote transformation of the vascular smooth muscle cells from a contraction type to a synthetic type. According to the invention, p55gamma is used as a target for screening drugs for preventing and / or treating aortic dissection, so that the drugs obtained by screening can effectively prevent and / or treat aortic dissection, and a new target is provided for treatment of aortic dissection.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of p55γ as a therapeutic target for aortic dissection. Background Art

[0002] Aortic dissection (AD), also known as aortic dissecting aneurysm, is a serious cardiovascular emergency. It is a life-threatening cardiovascular disease caused by tearing of the aortic intima or bleeding in the aortic wall, and separation of the aortic wall (dissection). The disease develops rapidly and is critical. If it is not diagnosed and treated in time, it can cause death in a short period of time, or blood enters the media to form true and false lumens, leading to ischemia and hypoxia of major distal organs, such as cerebral ischemia causing stroke, renal artery involvement or renal ischemia causing acute renal failure, gastrointestinal ischemia causing intestinal necrosis and stress ulcers / bleeding, etc., with very high mortality or disability rates. At present, except for surgical intervention, there is no effective drug to prevent the occurrence of aortic dissection and block the rupture of aortic dissection. Surgical treatment is difficult and expensive, and the mortality and disability rates of surgical treatment are high. Therefore, it is very important to actively study its causes and pathological mechanisms and find therapeutic targets for aortic dissection to effectively prevent and treat the disease. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide an application of p55γ as a target in screening drugs for preventing and / or treating aortic dissection, so that the screened drugs can effectively prevent and / or treat aortic dissection, providing a new target for treating aortic dissection.

[0004] Another object of the present invention is to provide a use of a p55γ activator in the preparation of a drug for preventing and / or treating aortic dissection.

[0005] Another object of the present invention is to provide a drug for preventing and / or treating aortic dissection.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an application of p55γ as a target in screening drugs for preventing and / or treating aortic dissection.

[0008] Preferably, the p55γ includes p55γ gene or p55γ protein.

[0009] Preferably, the overexpression of p55γ inhibits the occurrence of aortic dissection, and the low expression of p55γ promotes the occurrence of aortic dissection.

[0010] The present invention also provides an application of a p55γ activator in preparing a medicine for preventing and / or treating aortic dissection.

[0011] Preferably, the activator comprises a p55γ gene activator or a p55γ protein activator.

[0012] Preferably, the p55γ gene activator includes a substance capable of promoting the expression of the p55γ gene.

[0013] Preferably, the p55γ protein activator includes a substance that can enhance the activity of the p55γ protein.

[0014] The present invention also provides an application of p55γ protein in preparing a medicine for preventing and / or treating aortic dissection.

[0015] The present invention also provides a drug for preventing and / or treating aortic dissection, comprising p55γ protein and / or p55γ activator, and a pharmaceutically acceptable carrier.

[0016] Preferably, the activator comprises a p55γ gene activator or a p55γ protein activator.

[0017] Beneficial effects of the present invention:

[0018] The present invention found that overexpression of p55γ (PIK3R3) inhibited BAPN-induced aortic dissection and degradation of elastic fibers in mice; while mice with p55γ knockdown in vascular smooth muscle cells promoted BAPN-induced aortic dissection and degradation of elastic fibers. Mechanistically, p55γ exerts its effects by regulating the phenotypic transition of vascular smooth muscle cells. p55γ maintains the contractile phenotype of vascular smooth cells, while knockdown of p55γ promotes the transition of vascular smooth muscle cells from contractile to synthetic types. The present invention uses p55γ as a target for screening drugs for the prevention and / or treatment of aortic dissection, so that the screened drugs can effectively prevent and / or treat aortic dissection, providing a new target for the treatment of aortic dissection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1The expression level of p55γ in patients with aortic dissection and mice with aortic dissection model; A: Schematic diagram of the construction method of mouse aortic dissection model; B: Comparison of the top nine KEGG pathways enriched by all differentially expressed genes in RNA-seq of aortic tissues of control mice and aortic dissection model mice; C: Expression level of p55γ in the aortic media and adventitia in the GSE database (GSE232911) of RNA-seq of patients with aortic dissection and healthy subjects; D: Western Blotting detection results of p55γ in aortic tissues of patients with aortic dissection and healthy subjects and quantification of protein expression level; E: Changes in the mRNA expression level of p55γ in aortic tissues of mice with aortic dissection model; F: Western Blotting detection results of p55γ in aortic tissues of mice with aortic dissection model and quantification of protein expression level; G: Immunostaining results of aortic tissues of mice with aortic dissection model and quantification of α-SMA and the expression level of p55γ in smooth muscle cells labeled with α-SMA.

[0020] Figure 2 The effect of vascular smooth muscle-specific knockdown of p55γ on aortic dissection; A: Schematic diagram of the construction scheme of vascular smooth muscle-specific knockdown of p55γ mice; B: Identification of the genotype of vascular smooth muscle-specific knockdown of p55γ mice; C: Schematic diagram of the construction method of vascular smooth muscle-specific knockdown of p55γ mice aortic dissection model; D: Representative images of Doppler ultrasound detection of the degree of dilation of the ascending aorta of mice and quantification of the degree of dilation of the ascending aorta; E: Representative images of mouse aortic dissection under a stereomicroscope; F: p55γ SMKO Survival curve analysis of the same littermate control mice after 4 weeks of BAPN feeding and sterile water feeding; G: HE and EVG staining results of ascending aorta tissue of mice and statistical results of the degree of elastic fiber degradation in the tunica media; H: mRNA expression levels of SM22α, α-SMA, MMP2 and MMP9 in mouse aorta; I: Western blotting detection results and protein expression level quantification of SM22α, α-SMA, MMP2 and MMP9 in mouse aorta;

[0021] Figure 3 The effect of overexpression of p55γ on aortic dissection; A: Representative images of the dilation of the ascending aorta detected by Doppler ultrasound and quantitative dilation of the ascending aorta; B: Representative images of aortic dissection in mice under stereomicroscope; C: p55γ TGSurvival curve analysis of control mice and littermates after BAPN feeding and sterile water feeding for 4 weeks; D: HE and EVG staining results of ascending aorta tissue of mice and statistical results of elastic fiber degradation in the tunica media; E: mRNA expression levels of SM22α, α-SMA, MMP2 and MMP9 in mouse aorta; F: Western blotting detection results and protein expression level quantification of SM22α, α-SMA, MMP2 and MMP9 in mouse aorta;

[0022] Figure 4 The effect of overexpression of p55γ on the contractile phenotype of vascular smooth muscle cells; A: the mRNA expression level of p55γ after HASMC overexpression of p55γ; B: Western blot of p55γ after HASMC overexpression of p55γ Blotting results and quantitative protein expression levels; C: mRNA expression levels of α-SMA and SM22α after HASMC overexpression of p55γ and TGF-β stimulation; D: Western blotting results and quantitative protein expression levels of α-SMA and SM22α after HASMC overexpression of p55γ and TGF-β stimulation; E: mRNA expression level of p55γ after HASMC knockdown of p55γ; F: Western blotting results and quantitative protein expression levels of p55γ after HASMC knockdown of p55γ; G: mRNA expression levels of α-SMA and SM22α after HASMC knockdown of p55γ and TGF-β stimulation; H: Western blotting results and quantitative protein expression levels of α-SMA and SM22α after HASMC knockdown of p55γ and TGF-β stimulation;

[0023] Figure 5 The effect of overexpression of p55γ on Smad2 expression; A: p55γ after BAPN feeding TG RNA-seq of aortic tissues of WT and BAPN mice; B: p55γ TG Differential gene pathway enrichment in aorta tissues of WT and BAPN mice; C: p55γ TG A: Genes with differential expression in the TGF-β and Notch pathways in RNA-seq of HASMC and WT mice; D: Western blotting results and quantification of Smad2 protein expression levels after HASMC overexpressing p55γ and stimulated with TGF-β; E: Western bloting results and quantification of Smad2 protein expression levels after HASMC knockdown of p55γ and stimulated with TGF-β. DETAILED DESCRIPTION

[0024] The present invention provides an application of p55γ as a target in screening drugs for preventing and / or treating aortic dissection.

[0025] In the present invention, the p55γ includes the p55γ gene or the p55γ protein. As an embodiment, the human p55γ gene sequence number is NCBI GENE ID: 8503. The p55γ gene or protein is downregulated in both BAPN-induced aortic dissection model mice and patients with aortic dissection. Mice with p55γ knockdown in vascular smooth muscle cells showed more obvious ascending aortic dilatation and increased mortality, and the number of media elastic fiber fractures increased significantly. At the same time, the expression level of indicators related to the contractile phenotype of smooth muscle cells was reduced, and the expression level of indicators related to the pro-inflammatory phenotype of smooth muscle synthesis was increased, indicating that p55γ knockdown can promote BAPN-induced aortic dissection in mice. Mice overexpressing p55γ showed lower ascending aortic dilatation and mortality, and the media elastic fiber fracture was significantly reduced. At the same time, the expression downregulation of α-SMA and SM22α induced by BAPN and the upregulation of MMP2 and MMP9 were significantly improved, indicating that p55γ overexpression inhibited BAPN-induced aortic dissection in mice. Mechanistically, p55γ exerts its effects by maintaining the contractile phenotype of vascular smooth muscle cells. p55γ inhibits the conversion of vascular smooth muscle cells from contractile to synthetic types, while knocking down p55γ promotes this phenomenon, which provides a new target for the treatment of aortic dissection.

[0026] In the present invention, as an implementable method, by detecting the effect of the drug on the expression level of p55γ, a drug capable of preventing and / or treating aortic dissection is screened; if the drug can increase the expression level of p55γ gene and / or protein, it has the effect of preventing and / or treating aortic dissection.

[0027] The present invention also provides an application of a p55γ activator in preparing a medicine for preventing and / or treating aortic dissection.

[0028] In the present invention, the activator includes a p55γ gene activator or a p55γ protein activator. The p55γ gene activator includes a substance that can promote the expression of the p55γ gene. The present invention does not specifically limit the type of the substance that can promote the expression of the p55γ gene, including but not limited to nucleic acid molecules, nucleic acid constructs, inorganic compounds or organic compounds. The nucleic acid molecules include p55γ gene, p55γ gene-specific microRNA, nucleic acid molecules that activate the p55γ gene promoter, etc. The nucleic acid construct is a gene fragment containing the above-mentioned nucleic acid molecule, which can express the nucleic acid molecule. The p55γ protein activator includes a substance that can increase the activity of the p55γ protein. The present invention does not specifically limit the type of the substance that can increase the activity of the p55γ protein, and it can be conventionally selected according to actual needs.

[0029] The present invention also provides a use of p55γ protein in preparing a drug for preventing and / or treating aortic dissection. The p55γ protein can maintain the contractile phenotype of vascular smooth cells and inhibit the occurrence of aortic dissection. The present invention has no special restrictions on the source of the p55γ protein, which can be purchased through conventional commercial channels or prepared by conventional methods.

[0030] The present invention also provides a drug for preventing and / or treating aortic dissection, comprising p55γ protein and / or p55γ activator, and a pharmaceutically acceptable carrier.

[0031] In the present invention, the pharmaceutically acceptable carrier includes, but is not limited to, water, saline, buffer, glycerol, ethanol, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should match the dosage form of the drug. The activator includes a p55γ gene activator or a p55γ protein activator. The p55γ gene activator includes a substance that can promote the expression of the p55γ gene. The present invention does not specifically limit the type of the substance that can promote the expression of the p55γ gene, including but not limited to nucleic acid molecules, nucleic acid constructs, inorganic compounds or organic compounds. The nucleic acid molecules include p55γ genes, p55γ gene-specific microRNAs, nucleic acid molecules that activate the p55γ gene promoter, and the like. The nucleic acid construct is a gene fragment containing the above-mentioned nucleic acid molecules, which can express the nucleic acid molecules. The p55γ protein activator includes a substance that can increase the activity of the p55γ protein. The present invention does not specifically limit the type of the substance that can increase the activity of the p55γ protein, and it can be conventionally selected according to actual needs. The present invention has no particular limitation on the dosage form of the drug, and may be an injection, oral preparation (tablet, capsule, oral solution), transdermal preparation, sustained-release preparation, etc. The present invention has no particular limitation on the mode of administration, and may conventionally select injection, oral administration, application, etc. according to the dosage form of the drug and actual needs.

[0032] In the present invention, as an implementable embodiment, the method for treating aortic dissection includes achieving the treatment of aortic dissection by increasing the expression level of p55γ gene and / or protein; the method for increasing the expression level of p55γ gene and / or protein includes using gene amplification, gene editing and other technologies to specifically promote the expression of p55γ or using drugs containing p55γ gene and / or protein activators and p55γ protein.

[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0035] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0036] Example 1

[0037] (1) Screening of differentially expressed genes

[0038] First, a mouse model of aortic dissection was established: 3-week-old wild-type C57 mice were fed with 3-aminopropionitrile fumarate (BAPN) solution (concentration of 0.25%, 2.5 g BAPN per 1 L sterile water, Sigma-Aldriich, A3134-25G) in drinking water. After 4 weeks of feeding, they were killed and recorded as the WT+BAPN group. 3-week-old wild-type C57 mice of the same litter that were fed without BAPN in drinking water for 4 weeks were used as controls and recorded as the WT group. The model construction process is as follows Figure 1 As shown in A. The RNA-seq of aortic tissues of aortic dissection model mice (WT+BAPN) and control mice (WT) was analyzed, and the results are shown in Figure 1 As shown in B, the PI3K-AKT pathway was found to be significantly enriched through KEGG pathway enrichment. We then analyzed the GSE database (GSE232911) of patients with aortic dissection, and the results were as follows Figure 1 As shown in middle C, p55γ was found to be downregulated in both the media and adventitia of aortic dissection.

[0039] (2) Expression level of p55γ in aortic tissue of patients with aortic dissection

[0040] To confirm the changes of p55γ in aortic dissection, the protein expression level of p55γ in aortic tissues of patients with aortic dissection (AD) and healthy subjects was detected. Figure 1 As shown in D. The results showed that the protein expression of p55γ was downregulated in AD patients.

[0041] (3) Expression level of p55γ in aortic tissue of mice with aortic dissection

[0042] The same method as above was used to construct an aortic dissection model mouse, which was recorded as the BAPN group. Three-week-old wild-type C57 mice of the same age that were fed with drinking water without BAPN for 4 weeks were used as the control, recorded as the Water group. The entire aortic tissue was obtained from the two groups of mice, and the ascending aorta segment was fixed and then stained for aortic sectioning. Immunofluorescence staining was used to detect the expression of p55γ in aortic smooth muscle cells. After the remaining aortic tissue was quickly frozen in liquid nitrogen, part of it was used to extract RNA, and qPCR was used to detect the mRNA level of p55γ; part of it was used to extract protein, and Western Blotting was used to detect the protein level of p55γ. The results are shown in Figure 1 As shown in EG.

[0043] The results showed that the mRNA and protein levels of p55γ were downregulated in the aortic tissue of the aortic dissection model mice. Immunofluorescence results showed that α-SMA in the aorta was downregulated after BAPN induction; because α-SMA is a typical contractile protein of vascular smooth muscle cells in the media, the expression of p55γ in vascular smooth muscle cells marked by a-SMA was detected, and it was found that p55γ was downregulated in smooth muscle cells in the media.

[0044] Example 2

[0045] (1) p55γ knockdown in vascular smooth muscle cells-specific mice SMKO Construction: In order to explore the function of p55γ in aortic dissection, a mouse p55γ knockdown gene was constructed in vascular smooth muscle cells. SMKO . First, according to the mp55γ genome structure and protein function conservative region, Exon4 was found. Exon4 is a public exon, located in the p55γ protein function conservative region: SH2_nSH2_p85_like, and the number of bases in the Exon4 protein coding region is 181bp, which is not a multiple of 3. After conditional deletion of this exon, the SH2_nSH2_p85_like domain will be destroyed, and the mRNA will be re-spliced ​​to form a new mRNA, which will cause a frameshift mutation and protein inactivation. Therefore, it was decided to insert FloxP sites on both sides of Exon4. The mpik3r3-FloxP mice were mated with the Cre mice that specifically expressed SM22α in vascular smooth muscle cells, so that the Exon4 exon of mpik3r3 was deleted, mpik3r3 would not be translated or would undergo a frameshift mutation, and the mpik3r3 protein would be inactivated, thereby achieving the purpose of conditional knockout of the mpik3r3 gene, and obtaining the mouse p55γ with vascular smooth muscle cell-specific knockdown of p55γ. SMKO The construction process is as follows Figure 2As shown in A. p55γ f / f Mice served as controls.

[0046] Mouse tail DNA was extracted, and the PCR system was set according to the mouse gene type and primers. Then, agarose gel electrophoresis was used to identify the mouse genotype. Figure 2 As shown in B. It can be seen that p55γ SMKO In mice, both Flox and cre bands are present, while p55γ f / f Only the Flox band appeared, indicating that the mouse with vascular smooth muscle cell-specific knockdown of p55γ was successfully constructed.

[0047] (2) Experimental grouping: 32 3-week-old p55γ SMKO Mice and 36 age-matched wild-type littermates p55γ f / f Mice were used as controls; among them, 18 p55γ SMKO Mice and 22 p55γ f / f At 3 weeks of age, mice were given BAPN (concentration 0.25%) in drinking water for 4 weeks. Figure 2 As shown in C, they are denoted as p55γ SMKO +BAPN group and p55γ f / f +BAPN group; 14 p55γ SMKO Mice and 14 p55γ f / f At 3 weeks of age, mice were fed with sterile water for 4 weeks and were labeled as p55γ SMKO +Water group and p55γ f / f +Water group.

[0048] (3) Experimental measurement: After 4 weeks of feeding, Doppler ultrasound was used to detect the degree of vasodilation of the ascending aorta of the four groups of mice. The whole aorta tissue was taken and photographed under a stereo microscope, and the mortality rate was calculated. The results are as follows: Figure 2 As shown in DF. It can be seen that BAPN induction can increase the dilation of the ascending aorta and the mortality rate; under BAPN induction conditions, compared with the control group (p55γ f / f Compared with mice, p55γ SMKO Mice showed increased ascending aortic dilation and mortality.

[0049] The ascending aortas of the four groups of mice were fixed, embedded in paraffin, and then sectioned. HE staining and EVG staining were performed to compare whether there were any differences in the rupture of vascular elastic fibers. The results are as follows: Figure 2 As shown in G. It can be seen that BAPN induction can significantly increase the breakage of elastic fibers in the middle membrane; under BAPN induction conditions, compared with p55γ f / f Mouse, p55γ SMKOThe rupture of elastic fibers in the tunica media of mice was significantly increased.

[0050] Subsequently, the aortic tissues of the four groups of mice were taken out and quickly frozen in liquid nitrogen to detect the mRNA and protein levels of smooth muscle cell phenotype conversion-related indicators (SM22α, α-SMA, MMP2, and MMP9). The results are as follows Figure 2 As shown in the figure, BAPN induction can reduce the mRNA and protein levels of α-SMA and SM22α, and increase the mRNA and protein levels of MMP2 and MMP9; under BAPN induction conditions, compared with p55γ f / f Mouse, p55γ SMKO The mice had significantly aggravated BAPN-induced downregulation of α-SMA and SM22α and upregulation of MMP2 and MMP9.

[0051] These results indicate that SMC-specific knockdown of p55γ promotes aortic dissection.

[0052] Example 3

[0053] (1) Construction of p55γ overexpressing mice: In order to explore the function of p55γ in aortic dissection, a p55γ transgenic mouse was constructed. TG The specific construction process is as follows: the cDNA of p55γ is cloned into the expression vector to obtain the transgenic p55γ construct. The p55γ construct is then injected into C57BL / 6 mouse one-cell embryos and further hybridized with C57BL / 6 mice to obtain the p55γ transgenic (p55γ TG ) mice. The p55γ TG Mice and their age-matched wild-type (WT) littermates served as controls.

[0054] (2) Experimental groups: 33 3-week-old p55γ TG Mice and 38 wild-type mice from the same littermate as controls; among them, 21 p55γ TG Mice and 26 WT mice were given BAPN (concentration of 0.25%) in drinking water at 3 weeks of age for 4 weeks and recorded as p55γ TG +BAPN group and WT+BAPN group; 12 p55γ TG Mice and 12 WT mice were fed with sterile water for 4 weeks as controls and were denoted as p55γ TG +Water group and WT+Water group.

[0055] (3) Experimental measurement: After 4 weeks of feeding, mice were taken and the degree of vasodilation of the ascending aorta was detected by Doppler ultrasound. The whole aorta tissue was taken and photographed under a stereo microscope. The mortality rate of each group was calculated. The results are as follows: Figure 3 As shown in middle AC. It can be seen that BAPN induction can increase ascending aorta dilation and mortality; under BAPN induction conditions, compared with the control group (WT) mice, p55γ TG Ascending aorta dilatation and decreased mortality in mice.

[0056] The ascending aortas of the four groups of mice were fixed, embedded in paraffin, and then sectioned. HE staining and EVG staining were performed to compare whether there were any differences in the rupture of vascular elastic fibers. The results are as follows: Figure 3 As shown in D. It can be seen that BAPN induction can significantly increase the breakage of elastic fibers in the tunica media; under BAPN induction conditions, compared with WT mice, p55γ TG The number of broken layers of elastic fibers in the tunica media of mice was significantly reduced.

[0057] Subsequently, the aortic tissues of the four groups of mice were taken out and quickly frozen in liquid nitrogen to detect the mRNA and protein levels of smooth muscle cell phenotype conversion-related indicators (SM22α, α-SMA, MMP2, and MMP9). The results are as follows Figure 3 As shown in E and F. It can be seen that BAPN induction can reduce the mRNA and protein levels of α-SMA and SM22α, and increase the mRNA and protein levels of MMP2 and MMP9; under BAPN induction conditions, compared with WT mice, p55γ TG The mice significantly alleviated the BAPN-induced downregulation of α-SMA and SM22α and upregulation of MMP2 and MMP9.

[0058] The above results indicate that overexpression of p55γ inhibits the formation of aortic dissection.

[0059] Example 4

[0060] Previous studies have found that p55γ is involved in PDGF-BB-induced smooth muscle cell proliferation, and the loss and phenotypic conversion of smooth muscle cells (HASMC) in aortic dissection is widely demonstrated. In order to verify whether p55γ is involved in the phenotypic conversion of HASMC, aortic smooth muscle cells overexpressing p55γ were constructed.

[0061] (1) Construction of HASMCs overexpressing p55γ: HASMCs were transfected with Ad-p55γ for 48 h to obtain HASMCs overexpressing p55γ (denoted as the Ad-p55γ group); HASMCs were transfected with Ad-Lac for 48 h as a control (denoted as the Ad-Lac group).

[0062] The mRNA and protein levels of p55γ in the two groups of HASMC after transfection were detected. Figure 4 As shown in A and B. It can be seen that the mRNA and protein of p55γ in HASMC transfected with Ad-p55γ are overexpressed, indicating that the construction is successful.

[0063] After the construction was successful, TGF-β was added to the culture medium of Ad-Lac and Ad-p55γ cells, respectively, so that the concentration of TGF-β in the culture medium was 10 ng / mL, and the treatment was carried out for 48 hours to induce the phenotypic transformation of smooth muscle cells. Ad-Lac without TGF-β was used as a control. The mRNA and protein levels of α-SMA and SM22α in the three groups of cells were detected. The results are as follows Figure 4 As shown in C and D. It can be seen that the addition of TGF-β upregulates the mRNA and protein expression levels of HASMC contractile proteins (SM22α, α-SMA); overexpression of p55γ can further promote the upregulation of HASMC contractile protein expression levels induced by TGF-β.

[0064] (2) Construction of HASMC with specific knockdown of p55γ: HASMC were transfected with siRNA (hp55γsi1 and hp55γsi2) to knockdown p55γ, and HASMC with specific knockdown of p55γ were obtained (respectively, denoted as p55γsi1 group and p55γsi2 group); HASMC transfected with Scrambled were used as control (respectively, denoted as Scrambled group); the sense chain sequence of Scrambled was: UUCUCCGAACGUGUCACGUTT (SEQ ID NO: 1), and the antisense chain sequence was: ACGUGACACGUUCGGAGAATT (SEQ ID NO: 2); the sense chain sequence of hp55γsi1 was: GAAGGACAGUUCUGUUUCUTT (SEQ ID NO: 3), and the antisense chain sequence was: AGAAACAGAACUGUCCUUCTT (SEQ ID NO: 4); the sense chain sequence of hp55γsi2 was: GAGAUUCAUGAUAGCAAAATT (SEQ ID NO: 5), and the antisense sequence is: UUUUGCUAUCAUGAAUCUCTT (SEQ ID NO: 6).

[0065] The mRNA and protein levels of p55γ in the three groups of HASMC after transfection were detected. Figure 4 As shown in E and F. It can be seen that the mRNA and protein expression levels of p55γ in HASMCs with specific knockdown of p55γ (p55γsi1, p55γsi2) decreased, indicating that the construction was successful.

[0066] After the construction was successful, TGF-β was added to the culture medium of HASMC with specific knockdown of p55γ (p55γsi1, p55γsi2) and HASMC without knockdown of p55γ (Scrambled) to make the concentration of TGF-β in the culture medium 10 ng / mL. The cells were treated for 48 hours to induce phenotypic transformation of smooth muscle cells. Scrambled without TGF-β was used as a control. The mRNA and protein expression levels of α-SMA and SM22α in the four groups of cells were detected. The results are shown in Figure 4 As shown in G and H. It can be seen that the addition of TGF-β upregulated the mRNA and protein expression levels of HASMC contractile proteins (SM22α, α-SMA); the knockdown of p55γ inhibited the upregulation of TGF-β-induced smooth muscle cell contractile protein expression levels.

[0067] From this we concluded that p55γ maintains the contractile phenotype of vascular smooth muscle, overexpression of p55γ promotes the maintenance of the contractile phenotype of smooth muscle cells, and knockdown of p55γ promotes the conversion of smooth muscle cells from a contractile phenotype to a synthetic phenotype.

[0068] Example 5

[0069] (1) In order to study the mechanism of p55γ regulating aortic dissection formation, p55γ was taken after 4 weeks of BAPN feeding in Example 3. TG and WT mice, the mouse aorta tissue was dissected, RNA was extracted, and high-throughput sequencing was performed to search for downstream target genes of p55γ. Figure 5 As shown in AC. The results of differential gene pathway enrichment showed that the extracellular matrix interaction and local adhesion pathways were widely enriched. We found that the expression levels of some genes in the TGF-β-Smad pathway and the Nocth pathway changed. Since the TGF-β-Smad2 / 3 pathway is a classic pathway for smooth muscle cells to maintain a contractile phenotype, we detected the changes in the expression content of Smad2 by p55γ and found that p55γ can upregulate the expression level of Smad2.

[0070] (2) In Example 4, the protein expression levels of Smad2 in the three groups of cells were detected using Ad-Lac and Ad-p55γ induced by TGF-β, and Ad-Lac without TGF-β. The results are as follows: Figure 5 As shown in D. It can be seen that TGF-β induction causes an increase in the protein expression level of Smad2 in HASMC, and overexpression of p55γ can further promote the increase in the expression level of Smad2 induced by TGF-β.

[0071] (3) In Example 4, the protein expression levels of smad2 in the p55γsi1, p55γsi2 and Scrambled cells induced by TGF-β and the Scrambled cells without TGF-β were detected. The results are as follows: Figure 5 As shown in E. It can be seen that TGF-β induction causes an increase in the protein expression level of Smad2 in HASMC, and the increase in Smad2 expression level induced by TGF-β can be inhibited by knockdown of p55γ.

[0072] These results indicate that p55γ maintains the contractile phenotype of smooth muscle cells by upregulating the expression of Smad2.

[0073] 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. Application of p55γ as a target in screening drugs for the prevention and / or treatment of aortic dissection.

2. The use according to claim 1, characterized in that: The p55γ includes p55γ gene or p55γ protein.

3. The use according to claim 1, characterized in that: The overexpression of p55γ inhibits the occurrence of aortic dissection, and the low expression of p55γ promotes the occurrence of aortic dissection.

4. Use of p55γ activators in the preparation of drugs for preventing and / or treating aortic dissection.

5. The use according to claim 4, characterized in that: The activator includes a p55γ gene activator or a p55γ protein activator.

6. The use according to claim 5, characterized in that: The p55γ gene activator includes substances that can promote the expression of the p55γ gene.

7. The use according to claim 5, characterized in that: The p55γ protein activator includes substances that can enhance the activity of the p55γ protein.

8. Use of p55γ protein in the preparation of drugs for preventing and / or treating aortic dissection.

9. A drug for preventing and / or treating aortic dissection, characterized in that: The invention comprises p55γ protein and / or p55γ activator, and a pharmaceutically acceptable carrier.

10. The drug according to claim 9, characterized in that The activator includes a p55γ gene activator or a p55γ protein activator.

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