Application of p55γ as a therapeutic target for aortic dissection

Abstract

The present invention discloses the application of p55γ as a therapeutic target for aortic dissection, belonging to the technical field of biomedicine. Overexpression of p55γ inhibits the formation of aortic dissection and the degradation of elastic fibers induced by BAPN in mice; while knockdown of p55γ in vascular smooth muscle cells promotes the formation of aortic dissection and the degradation of elastic fibers induced by BAPN. Mechanistically, p55γ plays a role by maintaining the contractile phenotype of vascular smooth muscle, and knockdown of p55γ promotes the transformation of vascular smooth muscle cells from the contractile type to the synthetic type. The present invention uses p55γ as a target for 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 the treatment of aortic dissection.

Description

Technical Field

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

[0002] Aortic dissection (AD) is a severe cardiovascular emergency and a life-threatening cardiovascular disease caused by the tearing of the aortic intima or bleeding within the aortic wall, resulting in the separation (dissection) of the aortic wall. This disease develops rapidly and is critically ill. If not diagnosed and treated promptly, it can lead to the death of the patient in the short term, or due to blood entering the media, forming true and false lumens, resulting in ischemia and hypoxia of distal vital organs, such as cerebral ischemia causing stroke, renal artery involvement or renal ischemia causing acute renal failure, gastrointestinal ischemia causing intestinal necrosis and stress ulcer / bleeding, etc. The fatality rate or disability rate is very high. Currently, in addition to 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, costly, and has a high mortality and disability rate. Therefore, actively studying its pathogenesis and pathological mechanism and finding therapeutic targets for aortic dissection are very important for effectively preventing and treating this 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 the treatment of aortic dissection.

[0004] Another purpose of the present invention is to provide an application of a p55γ activator in the preparation of drugs for preventing and / or treating aortic dissection.

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

[0006] In order to achieve the above-mentioned invention purposes, 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 the p55γ gene or the p55γ protein.

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

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

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

[0012] Preferably, the p55γ gene activator includes a substance that can promote 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 the p55γ protein in the preparation of a medicament for preventing and / or treating aortic dissection.

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

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

[0017] Advantages of the present invention:

[0018] The present invention discovers that overexpression of p55γ (PIK3R3) inhibits BAPN-induced aortic dissection formation and elastic fiber degradation in mice; while knockdown of p55γ in vascular smooth muscle cells promotes BAPN-induced aortic dissection formation and elastic fiber degradation. Mechanistically, p55γ plays a role by regulating the phenotypic transformation of vascular smooth muscle cells. p55γ maintains the contractile phenotype of vascular smooth muscle cells, while knockdown of p55γ promotes the transformation of vascular smooth muscle cells from the contractile type to the synthetic type. The present invention uses p55γ as a target for screening medicaments for preventing and / or treating aortic dissection, enabling the screened medicaments to effectively prevent and / or treat aortic dissection, and providing a new target for the treatment of aortic dissection. Description of the Drawings

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

[0020] Figure 2 The effect of vascular smooth muscle-specific knockdown of p55γ on the occurrence of aortic dissection; where A: Schematic diagram of the construction plan of mice with vascular smooth muscle-specific knockdown of p55γ; B: Genotype identification of mice with vascular smooth muscle-specific knockdown of p55γ; C: Schematic diagram of the construction method of the aortic dissection model in mice with vascular smooth muscle-specific knockdown of p55γ; D: Representative images of Doppler ultrasound detection of the degree of ascending aortic dilation in mice and quantification of the degree of ascending aortic vascular dilation; E: Representative images of mouse aortic dissection under a stereomicroscope; F: p55γ SMKO and survival curve analysis of littermate control mice after 4 weeks of BAPN feeding and sterile water feeding; G: Results of HE and EVG staining of the ascending aortic tissues of mice and statistical results of the degree of medial elastic fiber degradation; H: mRNA expression levels of SM22α, α-SMA, MMP2, and MMP9 in the mouse aorta; I: Results of Westernbloting detection of SM22α, α-SMA, MMP2, and MMP9 in the mouse aorta and quantification of protein expression levels;

[0021] Figure 3 The effect of overexpressing p55γ on the occurrence of aortic dissection; where A: Representative images of Doppler ultrasound detection of the degree of ascending aortic dilation in mice and quantification of the degree of ascending aortic vascular dilation; B: Representative images of mouse aortic dissection under a stereomicroscope; C: p55γ TGAnalysis of the survival curves of littermate control mice and mice fed with BAPN and sterile water for 4 weeks; D: HE and EVG staining results of mouse ascending aorta tissues and statistical results of the degradation degree of medial elastic fibers; E: mRNA expression levels of SM22α, α-SMA, MMP2 and MMP9 in mouse aorta; F: Western blotting detection results of SM22α, α-SMA, MMP2 and MMP9 in mouse aorta and quantification of protein expression levels;

[0022] Figure 4 Effects of overexpressing p55γ on the contractile phenotype of vascular smooth muscle cells; among them, A: mRNA expression level of p55γ after overexpressing p55γ in HASMC; B: Western blotting detection results of p55γ after overexpressing p55γ in HASMC and quantification of protein expression levels; C: mRNA expression levels of α-SMA and SM22α after overexpressing p55γ in HASMC and stimulating with TGF-β; D: Western blotting detection results of α-SMA and SM22α after overexpressing p55γ in HASMC and stimulating with TGF-β and quantification of protein expression levels; E: mRNA expression level of p55γ after knocking down p55γ in HASMC; F: Western blotting detection results of p55γ after knocking down p55γ in HASMC and quantification of protein expression levels; G: mRNA expression levels of α-SMA and SM22α after knocking down p55γ in HASMC and stimulating with TGF-β; H: Western blotting detection results of α-SMA and SM22α after knocking down p55γ in HASMC and stimulating with TGF-β and quantification of protein expression levels;

[0023] Figure 5 Effects of overexpressing p55γ on the expression of Smad2; among them, A: RNA-seq of aorta tissues of p55γ TG and WT mice after BAPN feeding; B: Differential gene pathway enrichment of aorta tissues of p55γ TG and WT mice after BAPN feeding; C: Genes with differential expression in the TGF-β and Notch pathways in the RNA-seq of p55γ TG and WT mice after BAPN feeding; D: Western blotting detection results of Smad2 after overexpressing p55γ in HASMC and stimulating with TGF-β and quantification of protein expression levels; E: Western blotting detection results of Smad2 after knocking down p55γ in HASMC and stimulating with TGF-β and quantification of protein expression levels. Detailed implementation manners

[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 implementable embodiment, the human p55γ gene sequence number is NCBI GENE ID: 8503. The expression of the p55γ gene or protein is down-regulated in both the mice with BAPN-induced aortic dissection model and the patients with aortic dissection. The mice with specific knockdown of p55γ in vascular smooth muscle cells showed more obvious ascending aortic dilation and increased mortality, and the number of broken elastic fibers in the media layer increased significantly. At the same time, it could decrease the expression levels of the indicators related to the contractile phenotype of smooth muscle cells and increase the expression levels of the indicators related to the synthetic pro-inflammatory phenotype of smooth muscle cells, indicating that knockdown of p55γ could promote the formation of BAPN-induced aortic dissection in mice. While the mice overexpressing p55γ showed lower ascending aortic dilation and mortality, and the rupture of elastic fibers in the media layer was significantly reduced. At the same time, it could significantly improve the down-regulation of the expression of α-SMA and SM22α and the up-regulation of the expression of MMP2 and MMP9 induced by BAPN, indicating that overexpression of p55γ inhibited the formation of BAPN-induced aortic dissection in mice. Mechanistically, p55γ plays a role by maintaining the contractile phenotype of vascular smooth muscle cells. p55γ inhibits the transformation of vascular smooth muscle cells from the contractile type to the synthetic type, while knockdown of p55γ promotes this phenomenon, which provides a new target for the treatment of aortic dissection.

[0026] In the present invention, as an implementable embodiment, drugs for preventing and / or treating aortic dissection are screened by detecting the effect of the drug on the expression level of p55γ; if the drug can increase the expression level of the 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 the preparation of drugs 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 substances that can promote the expression of the p55γ gene. The present invention has no special limitation on the types of substances that can promote the expression of the p55γ gene, including but not limited to nucleic acid molecules, nucleic acid constructs, inorganic compounds, organic compounds, etc. The nucleic acid molecules include the 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 coding nucleic acid molecule and can express the nucleic acid molecule. The p55γ protein activator includes substances that can increase the activity of the p55γ protein. The present invention has no special limitation on the types of substances that can increase the activity of the p55γ protein and can be conventionally selected according to actual needs.

[0029] The present invention also provides an application of the p55γ protein in the preparation of drugs for preventing and / or treating aortic dissection. The p55γ protein can maintain the contractile phenotype of vascular smooth muscle cells and can inhibit the occurrence of aortic dissection. The present invention has no special limitation on the source of the p55γ protein, which can be conventionally purchased through commercial channels or obtained by conventional methods.

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

[0031] In the present invention, the pharmaceutically acceptable carrier includes but not limited to: water, saline, buffer solution, glycerol, ethanol, liposome, lipid, protein, protein-antibody conjugate, peptide substance, cellulose, nanogel, or a combination thereof. The selection of the carrier should match the drug dosage form. The activator includes a p55γ gene activator or a p55γ protein activator. The p55γ gene activator includes substances that can promote the expression of the p55γ gene. The present invention has no special limitation on the types of substances that can promote the expression of the p55γ gene, including but not limited to nucleic acid molecules, nucleic acid constructs, inorganic compounds, organic compounds, etc. The nucleic acid molecules include the 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 coding nucleic acid molecule and can express the nucleic acid molecule. The p55γ protein activator includes substances that can increase the activity of the p55γ protein. The present invention has no special limitation on the types of substances that can increase the activity of the p55γ protein and can be conventionally selected according to actual needs. The present invention has no special limitation on the drug dosage form, which can be an injection, an oral preparation (tablet, capsule, oral liquid), a transdermal agent, a sustained-release agent, etc. The present invention has no special limitation on the administration method, and injection, oral administration, topical application and other methods can be conventionally selected according to the drug dosage form and actual needs.

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

[0033] The technical solutions provided by the present invention will be described in detail below with reference to 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 are conventional methods.

[0035] In the following embodiments, the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0036] Example 1

[0037] (1) Screening of differential genes

[0038] First, a mouse model of aortic dissection was established: 3-week-old wild-type C57 mice were fed with a 3-aminopropionitrile fumarate (BAPN) solution (concentration: 0.25%, 2.5 g BAPN added to every 1 L of sterile water, Sigma-Aldriich, A3134-25G) in drinking water for 4 weeks and then sacrificed, denoted as the WT + BAPN group. 3-week-old littermate wild-type C57 mice fed with drinking water without BAPN for 4 weeks were used as the control, denoted as the WT group. The model construction process is as shown in Figure 1 Figure A. RNA-seq of the aortic tissues of the aortic dissection model mice (WT + BAPN) and the control group mice (WT) was analyzed, and the results are as shown in Figure 1 Figure B. Through KEGG pathway enrichment, it was found that the PI3K-AKT pathway was significantly enriched. Subsequently, we analyzed the GSE database (GSE232911) of aortic dissection patients, and the results are as shown in Figure 1 Figure C. It was found that p55γ was downregulated in both the media and adventitia of aortic dissection.

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

[0040] To confirm the changes of p55γ in aortic dissection, the protein expression levels of p55γ in aortic tissues of aortic dissection (AD) patients and healthy people were detected, and the results are as shown in Figure 1 Figure 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 mouse model of aortic dissection was constructed by the same method as above, denoted as the BAPN group. Three-week-old wild-type C57 mice fed with water without BAPN for 4 weeks were used as the control, denoted as the Water group. The whole aortic tissues of the two groups of mice were taken respectively. After the ascending aortic segments were fixed, aortic sections were stained, and immunofluorescence staining was used to detect the expression of p55γ in aortic smooth muscle cells. The remaining aortic tissues were frozen in liquid nitrogen immediately. One part was used to extract RNA, and qPCR was used to detect the mRNA level of p55γ; the other part was used to extract proteins, and Western Blotting was used to detect the protein level of p55γ. The results are shown in Figure 1 E-G below.

[0043] The results showed that both the mRNA and protein levels of p55γ were down-regulated in the aortic tissues of the mouse model of aortic dissection. The immunofluorescence results showed that α-SMA in the aorta was down-regulated after BAPN induction; since α-SMA is a typical contractile protein of vascular smooth muscle cells in the media, the expression of p55γ in vascular smooth muscle cells labeled by α-SMA was detected, and it was found that the expression of p55γ in the medial smooth muscle cells was down-regulated.

[0044] Example 2

[0045] (1) Construction of mice with specific knockdown of p55γ in vascular smooth muscle cells SMKO To explore the function of p55γ in the formation of aortic dissection, mice with specific knockdown of p55γ in vascular smooth muscle cells were constructed. SMKO First, according to the genomic structure of mp55γ and the conserved region of protein function, Exon4 was found. Exon4 is a common exon located in the conserved region of p55γ protein function: SH2_nSH2_p85_like. Moreover, the number of bases in the protein coding region of Exon4 is 181bp, which is not a multiple of 3. After conditional deletion of this exon, the SH2_nSH2_p85_like domain will be disrupted, 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 at both sides of Exon4. mpik3r3-FloxP mice were mated with Cre mice specifically expressing SM22α in vascular smooth muscle cells, so that the Exon4 exon of mpik3r3 was deleted, mpik3r3 would not be translated or would have a frameshift mutation, and the mpik3r3 protein was inactivated, thus achieving the purpose of conditional knockout of the mpik3r3 gene and obtaining mice with specific knockdown of p55γ in vascular smooth muscle cells. SMKO The construction process is as shown in Figure 2As shown in A, wild-type littermate p55γ mice of the same age were used as controls. f / f Mouse tails were used to extract DNA. PCR reaction systems were designed according to the mouse genotypes and primers, and then agarose gel electrophoresis was used to identify the genotypes of the mice. The results are shown in

[0046] B. It can be seen that the p55γ mice had both Flox and cre bands, while the p55γ Figure 2 mice only had Flox bands, indicating that the mice with specific knockdown of p55γ in vascular smooth muscle cells were successfully constructed. SMKO mice only had Flox bands, indicating that the mice with specific knockdown of p55γ in vascular smooth muscle cells were successfully constructed. f / f mice only had Flox bands, indicating that the mice with specific knockdown of p55γ in vascular smooth muscle cells were successfully constructed.

[0047] (2) Experimental grouping: 32 three-week-old p55γ SMKO mice and 36 wild-type littermate p55γ f / f mice of the same age were used as controls. Among them, 18 p55γ SMKO mice and 22 p55γ f / f mice were fed with BAPN (concentration 0.25%) in drinking water for 4 weeks at 3 weeks of age, as shown in Figure 2 C, and were denoted as the p55γ SMKO +BAPN group and the p55γ f / f +BAPN group respectively; 14 p55γ SMKO mice and 14 p55γ f / f mice were fed with sterile water for 4 weeks at 3 weeks of age, and were denoted as the p55γ SMKO +Water group and the p55γ f / f +Water group respectively.

[0048] (3) Experimental measurements: After 4 weeks of feeding, the degree of ascending aortic dilation in the four groups of mice was detected by Doppler ultrasound. The entire aortic tissue was photographed under a stereomicroscope, and the mortality rate was counted. The results are shown in Figure 2 D-F. It can be seen that BAPN induction could increase ascending aortic dilation and mortality rate; under BAPN induction, compared with the control group (p55γ f / f mice), the p55γ SMKO mice showed higher ascending aortic dilation and mortality rate.

[0049] The ascending aortas of the four groups of mice were fixed, sectioned after paraffin embedding, and stained with HE and EVG respectively to compare the differences in the rupture of vascular elastic fibers. The results are shown in Figure 2 G. It can be seen that BAPN induction could significantly increase the rupture of elastic fibers in the media; under BAPN induction, compared with p55γ f / f mice, the p55γ SMKOThe rupture of elastic fibers in the media of mice was significantly increased.

[0050] Subsequently, the aortic tissues of the 4 groups of mice frozen in liquid nitrogen were taken out for detecting the mRNA levels and protein levels of the indicators related to the phenotypic transformation of smooth muscle cells (SM22α, α-SMA, MMP2, MMP9), and the results are as Figure 2 shown in H-I in the figure. 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 the condition of BAPN induction, compared with p55γ f / f mice, p55γ SMKO mice significantly aggravated the down-regulation of the expressions of α-SMA and SM22α and the up-regulation of the expressions of MMP2 and MMP9 induced by BAPN.

[0051] The above results indicate that specific knockdown of p55γ in smooth muscle cells promotes the formation of aortic dissection.

[0052] Example 3

[0053] (1) Construction of p55γ overexpression mice: To explore the function of p55γ in the formation of aortic dissection, transgenic mice p55γ TG of p55γ were constructed. The specific construction process is as follows: The cDNA of p55γ was cloned into an expression vector to obtain a transgenic p55γ construct. Then the p55γ construct was injected into single-cell embryos of C57BL / 6 mice and further hybridized with C57BL / 6 mice to obtain p55γ transgenic (p55γ TG ) mice. The p55γ TG mice used in this study and their age-matched wild-type (WT) littermates were used as controls.

[0054] (2) Experimental grouping: Thirty-three 3-week-old p55γ TG mice and 38 littermate wild-type mice WT were selected as controls; among them, 21 p55γ TG mice and 26 WT mice were fed with BAPN (concentration of 0.25%) in drinking water for 4 weeks at 3 weeks of age, and were respectively denoted 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 respectively denoted as p55γ TG +Water group and WT+Water group.

[0055] (3) Experimental determination: After feeding the mice for 4 weeks, the degree of dilation of the ascending aorta was detected by Doppler ultrasound. The whole aortic tissue was photographed under a stereomicroscope, and the mortality rate of each group was counted. The results are as Figure 3 shown in A - C. It can be seen that BAPN induction can increase the dilation of the ascending aorta and the mortality rate; under the condition of BAPN induction, compared with the control group (WT) mice, p55γ TG mice had a decrease in the dilation of the ascending aorta and the mortality rate.

[0056] The ascending aortas of 4 groups of mice were fixed, sectioned after paraffin embedding, and stained with HE and EVG respectively to compare the differences in the rupture of vascular elastic fibers. The results are as Figure 3 shown in D. It can be seen that BAPN induction can significantly increase the rupture of elastic fibers in the media; under the condition of BAPN induction, compared with WT mice, p55γ TG mice had a significantly lower number of ruptured layers of elastic fibers in the media.

[0057] Subsequently, the aortic tissues of 4 groups of mice frozen in liquid nitrogen were taken for detecting the mRNA and protein levels of smooth muscle cell phenotypic conversion-related indicators (SM22α, α-SMA, MMP2, MMP9). The results are as Figure 3 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 the condition of BAPN induction, compared with WT mice, p55γ TG mice significantly alleviated the down-regulation of the expression of α-SMA and SM22α and the up-regulation of the expression of MMP2 and MMP9 induced by BAPN.

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

[0059] Example 4

[0060] Previous studies found that p55γ is involved in the proliferation of smooth muscle cells induced by PDGF-BB, and the deletion and phenotypic conversion of smooth muscle cells (HASMC) in aortic dissection have been widely proven. To verify whether p55γ is involved in the phenotypic conversion of HASMC, aortic smooth muscle cells overexpressing p55γ were constructed.

[0061] (1) Construction of HASMC overexpressing p55γ: Ad-p55γ was transfected into HASMC for 48 h to obtain HASMC overexpressing p55γ (denoted as the Ad-p55γ group); Ad-Lac was transfected into HASMC 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 HASMCs after transfection were detected, and the results are shown in Figure 4 Figures A and B below. It can be seen that the mRNA and protein of p55γ were overexpressed in HASMCs transfected with Ad-p55γ, indicating successful construction.

[0063] After successful construction, TGF-β was added to the culture media of Ad-Lac and Ad-p55γ cells respectively to make the concentration of TGF-β in the culture media 10 ng / mL, and the cells were treated for 48 h to induce phenotypic transformation of smooth muscle cells. Ad-Lac without TGF-β addition was used as a control. The mRNA and protein levels of α-SMA and SM22α in the three groups of cells were detected, and the results are shown in Figure 4 Figures C and D below. It can be seen that the addition of TGF-β up-regulated the mRNA and protein expression levels of HASMC contractile proteins (SM22α, α-SMA); the overexpression of p55γ could further promote the up-regulation of the expression levels of HASMC contractile proteins induced by TGF-β.

[0064] (2) Construction of HASMCs with specifically knocked-down p55γ: HASMCs were transfected with siRNAs (hp55γsi1, hp55γsi2) respectively to knock down p55γ, and HASMCs with specifically knocked-down p55γ were obtained (denoted as the p55γsi1 group and the p55γsi2 group respectively); HASMCs transfected with Scrambled were used as a control (denoted as the Scrambled group); the sense strand sequence of Scrambled was: UUCUCCGAACGUGUCACGUTT (SEQ ID NO: 1), and the antisense strand sequence was ACGUGACACGUUCGGAGAATT (SEQ ID NO: 2); the sense strand sequence of hp55γsi1 was: GAAGGACAGUUCUGUUUCUTT (SEQ ID NO: 3), and the antisense strand sequence was: AGAAACAGAACUGUCCUUCTT (SEQ ID NO: 4); the sense strand sequence of hp55γsi2 was: GAGAUUCAUGAUAGCAAAATT (SEQ ID NO: 5), and the antisense strand sequence was: UUUUGCUAUCAUGAAUCUCTT (SEQ ID NO: 6).

[0065] The mRNA and protein levels of p55γ in the three groups of HASMCs after transfection were detected, and the results are shown in Figure 4 Figures E and F below. It can be seen that the mRNA and protein expression levels of p55γ in HASMCs with specifically knocked-down p55γ (p55γsi1, p55γsi2) decreased, indicating successful construction.

[0066] After successful construction, TGF-β was added to the culture media of HASMCs with specific knockdown of p55γ (p55γsi1, p55γsi2) and HASMCs without p55γ knockdown (Scrambled) respectively, so that the concentration of TGF-β in the culture media was 10 ng / mL, and the cells were treated for 48 h to induce phenotypic transformation of smooth muscle cells. Scrambled without TGF-β addition was used as a control. The mRNA and protein expression levels of α-SMA and SM22α in the four groups of cells were detected, and the results are as shown in Figure 4 G and H in the figure. 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 the expression levels of smooth muscle cell contractile proteins induced by TGF-β.

[0067] From this, we drew the conclusion that p55γ maintained the contractile phenotype of vascular smooth muscle. Overexpression of p55γ promoted the maintenance of the contractile phenotype of smooth muscle cells, and knockdown of p55γ promoted the transformation of smooth muscle cells from the contractile phenotype to the synthetic phenotype.

[0068] Example 5

[0069] (1) To study the mechanism by which p55γ regulates the formation of aortic dissection, p55γ and WT mice after 4 weeks of BAPN feeding in Example 3 were taken, the aortic tissues of the mice were dissected, RNA was extracted, and high-throughput sequencing was performed to search for downstream target genes of p55γ. The results are as shown in TG A - C in the figure. The results of differential gene pathway enrichment showed that the extracellular matrix interaction and focal 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 classical pathway for smooth muscle cells to maintain the contractile phenotype, we detected the change in the expression content of Smad2 by p55γ and found that p55γ could upregulate the expression level of Smad2. Figure 5 A - C in the figure. The results of differential gene pathway enrichment showed that the extracellular matrix interaction and focal 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 classical pathway for smooth muscle cells to maintain the contractile phenotype, we detected the change in the expression content of Smad2 by p55γ and found that p55γ could upregulate the expression level of Smad2.

[0070] (2) In Example 4, Ad-Lac and Ad-p55γ induced by TGF-β, and Ad-Lac without TGF-β addition were taken, and the protein expression level of Smad2 in the three groups of cells was detected. The results are as shown in Figure 5 D in the figure. It can be seen that the protein expression level of Smad2 in HASMCs increased after TGF-β induction, and overexpression of p55γ could further promote the increase in the expression level of Smad2 induced by TGF-β.

[0071] (3) In Example 4, TGF-β-induced p55γ si1, p55γ si2, and Scrambled, as well as Scrambled without TGF-β addition, were used to detect the protein expression level of smad2 in the four groups of cells. The results are as shown in Figure 5 Figure E. It can be seen that after TGF-β induction, the protein expression level of Smad2 in HASMC increased, and the increase in Smad2 expression level induced by TGF-β could be inhibited by knockdown of p55γ.

[0072] The above results indicate that p55γ plays a role in maintaining the contractile phenotype of smooth muscle cells by upregulating the expression of Smad2.

[0073] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of a p55γ activator in the preparation of a medicament for preventing and / or treating aortic dissection, characterized in that, The p55γ activator is a construct overexpressing p55γ.

2. Use of the p55γ protein in the preparation of a medicament for preventing and / or treating aortic dissection.

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