CN114732909A - Medical application of TCF7L1 in preventing or treating abdominal aortic aneurysm - Google Patents
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Abstract
The invention belongs to the technical field of medical biology, relates to medical application of TCF7L1 (Transcription factor 7-like 1), and particularly relates to application of intervening TCF7L1 gene expression or inhibiting an active fragment thereof in preparing a medicine for preventing and treating abdominal aortic aneurysm. The invention discloses a biomarker related to abdominal aortic aneurysm and application thereof, wherein the biomarker is TCF7L1, and is a marker with high specificity and good sensitivity; simultaneously discloses a reagent, a kit and a chip for detecting the level of TCF7L1 and application thereof in preparing products for diagnosing abdominal aortic aneurysm; the invention provides a TCF7L1 expression inhibitor, which comprises siRNA and/or shRNA, has good interference effect, can promote the differentiation of vascular smooth muscle cells, can inhibit the migration of abdominal aortic aneurysm, can be used for the treatment of abdominal aortic aneurysm, and is beneficial to the research and development of clinical drugs for abdominal aortic aneurysm; simultaneously discloses the application of the inhibitor containing TCF7L1 in preparing the medicine for treating the abdominal aortic aneurysm, and the TCF7L1 can be used as a new biological target for guiding the treatment of the abdominal aortic aneurysm.
Description
Technical Field
The invention belongs to the technical field of medical biology, relates to medical application of TCF7L1 (Transcription factor 7-like 1), and particularly relates to application of intervening TCF7L1 gene expression or inhibiting an active fragment thereof in preparing a medicine for preventing and treating abdominal aortic aneurysm.
Background
Abdominal Aortic Aneurysm (AAA) is a permanent focal aortic dilatation, caused by a progressive weakening of the Abdominal aortic wall, a common and potentially life-threatening disease in the elderly. Open surgical intervention and endovascular repair are recognized therapeutic approaches for AAA. However, both methods are only applicable to aneurysms or AAA with diameters exceeding 5.5 cm. With the progress of examination, although AAA can be found early, no clinically effective therapeutic agent is currently available to alter the natural course of AAA progression.
Vascular Smooth Muscle Cells (VSMCs) are the main resident cells of the aortic wall. There is increasing evidence that VSMC phenotypic switching plays an important role in the development of AAA. The conversion of VSMC to synthetic phenotype is an early biological change in AAA with a gradual decrease in VSMC contraction phenotype marker expression such as α -SMA. VSMCs of the synthetic phenotype promote the development of aneurysms by promoting the production of inflammatory factors and elastin degrading Matrix Metalloproteinases (MMPs). Targeting improvements in VSMC dysfunction, including inhibition of VSMC phenotypic switching and apoptosis, may be a potential strategy to limit AAA growth during AAA pathogenesis.
Transcription factor 7-like 1 (Transcription factor 7-like 1, TCF7L 1), also known as high mobility group box Transcription factor (TCF 3), is a member of the lymphoenhancer factor/T-cell factor (LEF/TCF) family of Transcription factors, interacts with β catenin (β -catenin), acting as a DNA binding protein. TCF7L1 functions in tumor cell differentiation, proliferation and malignancy by modulating Wnt- β -catenin and c-Myc signaling through direct binding to multiple genes and interaction with proteins. The highly conserved Wnt signaling pathway regulates key processes in embryonic development, including cell fate, and is associated with a number of pathologies. Wnt signaling is thought to modulate VSMC differentiation. However, the role and mechanism of TCF7L1 in AAA is unclear. Therefore, the invention aims to provide the medical application of intervening TCF7L1 expression or inhibiting active fragments thereof in preventing or treating AAA.
The urgent need in the art for the search for diagnostic markers for abdominal aortic aneurysm and the application of marker inhibitors to medicines for abdominal aortic aneurysm is a technical problem that needs to be solved urgently.
Disclosure of Invention
In order to remedy the deficiencies of the prior art, the object of the present invention is to provide a diagnostic effect of TCF7L1 in AAA and to intervene in the medical use of TCF7L1 in the prevention or treatment of AAA.
It is an object of the present invention to provide biomarkers related to the development of an abdominal aortic aneurysm.
It is a further object of the present invention to provide a means and product for diagnosing abdominal aortic aneurysms using the expression levels of biomarkers.
It is a further object of the present invention to provide a means and a medicament for treating abdominal aortic aneurysms using inhibitors of biomarkers.
In order to achieve the purpose, the invention adopts the following technical scheme.
The invention provides application of a TCF7L1 inhibitor in preparing a product for diagnosing abdominal aortic aneurysm.
Furthermore, the product for diagnosing the abdominal aortic aneurysm takes TCF7L1 as a marker, wherein the marker comprises TCF7L1 gene or TCF7L1 protein.
Further, the marker TCF7L1 was up-regulated in abdominal aortic aneurysms.
Furthermore, the TCF7L1 inhibitor is used as a chip, a preparation or a kit in preparing products for diagnosing the abdominal aortic aneurysm.
The invention also provides application of the TCF7L1 inhibitor in preparing a medicament for treating abdominal aortic aneurysm.
Further, the application of the TCF7L1 inhibitor in preparing the medicine for treating the abdominal aortic aneurysm, wherein the TCF7L1 inhibitor promotes the differentiation of vascular smooth muscle cells.
Further, the application of the TCF7L1 inhibitor in preparing a medicament for treating the abdominal aortic aneurysm and the application of the TCF7L1 inhibitor in preparing a medicament for treating the abdominal aortic aneurysm as a vascular smooth muscle cell differentiation promoter and/or a vascular smooth muscle cell migration inhibitor.
Further, the inhibitor is in any pharmaceutically therapeutically acceptable dosage form or dosage.
The invention also provides a TCF7L1 inhibitor target sequence shown in SEQ ID NO. 1.
Further, the TCF7L1 inhibitor includes siRNA and/or shRNA of the above target sequence.
Compared with the prior art, the invention has the following beneficial effects.
According to the invention, a large number of animal experiments show that the expression of TCF7L1 is obviously increased in the AAA generation process by establishing an AAA model by adopting an Ang II pump burying method. Constructing an in vivo overexpression or low expression model of a TCF7L1 mouse by adopting a method of tail vein injection of AAV, and finding that the in vivo overexpression of the TCF7L1 in the mouse aggravates the formation of AAA; low expression of TCF7L1 delayed AAA formation. TCF7L1 was significantly up-regulated during mouse VSMC dedifferentiation. After the TCF7L1 is knocked down, the VSMC of a mouse can be promoted to be enhanced in differentiation; conversely, overexpression of TCF7L1 inhibited mouse VSMC differentiation. The results show that TCF7L1 plays an important role in regulation and control during AAA occurrence, and the early intervention on TCF7L1 can be the target point of AAA prevention or treatment.
1. The invention provides a gene marker for diagnosing abdominal aortic aneurysm, which can judge the abdominal aortic aneurysm condition by detecting the expression level of a marker TCF7L1 and provides a molecular marker for detecting and judging abdominal aortic aneurysm with high specificity and high sensitivity.
2. The research of the invention finds that the inhibition agent can promote the differentiation of vascular smooth muscle cells and inhibit the migration of abdominal aortic aneurysm cells by knocking down the expression of the novel TCF7L1, and can be used for treating the abdominal aortic aneurysm.
3. The TCF7L1 expression inhibitor siRNA and/or shRNA provided by the invention has good interference effect when used for expression inhibition of TCF7L1, and has clinical application potential.
4. The invention provides a novel idea and a novel method for screening an abdominal aortic aneurysm medicament by inhibiting expression conditions of novel TCF7L1, and TCF7L1 can be used as a novel biological target for guiding the treatment of the abdominal aortic aneurysm.
Drawings
FIG. 1 shows the expression of TCF7L1 is upregulated in the AAA model established by the Ang II pump-embedded approach.
FIGS. 1A and 1B are statistics of AAA tumor formation rate; FIGS. 1C, 1D are ultrasonically measured maximum abdominal aortic aneurysm diameters; FIGS. 1E and 1F show HE staining and elastin staining; FIGS. 1G, 1H are immunohistochemical staining of blood vessels; FIG. I, J is a Western blot result and a statistical chart thereof; figure 1K is the Real-time PCR assay (n =3,. p <0.001vs. salene panel).
FIG. 2 is a graph showing that overexpression of TCF7L1 in mice exacerbates AAA occurrence.
FIGS. 2A and 2B are statistics of AAA tumor formation rate; FIGS. 2C, 2D are ultrasonically measured maximum abdominal aortic aneurysm diameters; FIGS. 2E, 2F are HE staining, elastin staining and immunohistochemical staining; FIGS. 2G and 2H show the Western blot results and their statistical profiles (n =3, < 0.01, <0.001, # vs. AAV-cmv-GFP-saline group, # p < 0.01vs. AAV-cmv-GFP-AngII group).
FIG. 3 shows that TCF7L1 is underexpressed to delay AAA in mice.
FIGS. 3A and 3B are statistics of AAA tumor formation rate; fig. 3C, 3D are maximum abdominal aortic aneurysm diameters measured ultrasonically; FIGS. 3E and 3F show HE staining, elastin staining and immunohistochemical staining; FIGS. 3G and 3H show the Western blot results and their statistical plots (n =3, p < 0.01, p <0.001, vs. AAV-sh-RNA-salene group, # p < 0.01vs. AAV-sh-RNA-AngII group).
FIG. 4 shows that TCF7L1 is up-regulated during mouse VSMC dedifferentiation.
FIGS. 4A and 4B are the Western blot results and their statistical graphs; FIG. 4C is the Real-time PCR assay; figure 4D is the immunofluorescence staining results (n =3, p <0.001vs. pbs group).
FIG. 5 shows that low expression of TCF7L1 promotes VSMC differentiation.
FIGS. 5A and 5B are Western blot results and statistical graphs thereof; FIG. 5C is the Real-time PCR assay result; FIG. 5D is a transwell migration experiment, and 5E is the number of cells counted in 5D FIG. 3 random fields ([ x ] p <0.001vs. siControl group; # # p < 0.01vs. siControl + AngII group).
Fig. 6 shows that TCF7L1 overexpression inhibits VSMC differentiation.
FIGS. 6A and 6B are Western blot results and statistical graphs thereof; FIG. 6C is the Real-time PCR assay; FIG. 6D is a transwell migration experiment, and 6E is the number of cells calculated from the random fields of FIG. 3 in FIG. 6D (. about.. about.p <0.001vs. pcDNA3.1-Flag group;. # #. # p < 0.01vs. pcDNA3.1-Flag + AngII group).
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are commercially available, and are not indicated by manufacturers. The experimental data of the invention are all percentages, the comparison of the two sample rates applies chi-square test, the statistical treatment applies GraphPad prism8.0 software package treatment, and the P <0.05 is taken as the statistical difference.
Example 1: in the AAA model established by the Ang II pump-embedded method, the expression of TCF7L1 was up-regulated.
1. Experimental animals and breeding.
Species, sex, week age and source of experimental animals: ApoE-/-Mouse, male, 8 weeks old. ApoE-/-Mice were purchased from Jiangsu Jiejiaokang Biotech limited.The mice are raised in a Specific Pathogen Free (SPF) animal house at room temperature of (22 +/-2) DEG C and humidity of 45-70% with free access to food and water every 12h of light cycle.
2. And (3) establishing a mouse abdominal aortic aneurysm model.
ApoE-/-Mice were randomly divided into control and experimental groups, with the experimental group mice being subcutaneously embedded with AngII (1000 ng/kg/min) osmotic pumps (Alzet model 1004; AlzaCorp, Mountain View, Calif., USA) for 28 days, and the control group mice being embedded with the same volume of physiological saline for 28 days. AngII was purchased from APExBIO Technology (Houstun, US).
The results show that: the tumor formation rate of the AngII group mice is obviously higher than that of the normal saline group (figure 1A and figure 1B).
3. Ultrasound imaging measures the mouse maximum abdominal aorta diameter.
The incidence of abdominal aortic aneurysms and the maximum abdominal aortic aneurysm diameter were measured using the small animal ultrasound system Vevo 2100 apparatus (Visual sonic, Toronto, Canada), with local expansion of the aorta over 50% of its adjacent intact aortic section defined as aneurysm formation.
The results show that: the maximum abdominal aortic aneurysm diameter was significantly higher in the AngII group mice than in the saline group (fig. 1C, 1D).
4. HE staining detects aortic aneurysm formation in mice.
A mouse abdominal aortic aneurysm model is established by using an Ang II pump-embedded method. Collecting the aorta of the mice with 28 days of pump burying in the normal saline group and the AngII group respectively, and carrying out HE staining, wherein the specific steps are as follows.
1) And preparing paraffin sections.
A. Material taking: the mouse aorta was placed in 4% paraformaldehyde solution overnight.
B. And (3) dehydrating: the dehydration is carried out according to different alcohol concentrations and respectively comprises 2h of 70 percent alcohol, 2h of 80 percent alcohol, 2h of 90 percent alcohol, 4h of 95 percent alcohol, overnight in 95 percent alcohol II, 1.5 h of 100 percent alcohol I and 1.5 h in 100 percent alcohol II.
C. And (3) transparency: and (3) soaking the tissue block in a xylene I solution for 1h, taking out the tissue block, and then soaking the tissue block in a xylene II solution for 1 h.
D. Wax dipping: paraffin I was placed overnight, Paraffin II was placed for 1h, and Paraffin III was placed for 1 h.
E. Embedding: the tissue blocks were embedded in paraffin and left at room temperature.
F. Slicing: the tissue block was sectioned with a paraffin microtome to a thickness of 3 μm and the sections were mounted on glass slides.
G. Baking slices and baking slices: after the glass slide is placed on a 60 ℃ baking machine for 1h, the glass slide is placed in a 65 ℃ baking machine for 48 h.
2) Slicing and dewaxing: the slices are respectively placed in the following reagents according to the steps of 20min in xylene I, 20min in xylene II, 15min in 95% alcohol I, 15min in 95% alcohol II, 10min in 90% alcohol, 5min in 80% alcohol and 5min in 70% alcohol, and finally placed in distilled water for 30 min.
3) And (3) cell nucleus staining:
A. the sections were immersed in hematoxylin solution for 20 min.
B. Paraffin sections were placed in 1% hydrochloric acid for 30s for differentiation and washed with running water.
C. And (3) placing the paraffin sections in ammonia water for 30s, and carrying out cell nucleus bluing and running water washing.
4) Cytoplasmic staining: the paraffin sections are placed in water-soluble eosin solution for 5min, stained and washed by running water.
5) And (3) transparency: the paraffin sections are placed in the following reagents according to the steps of 80% alcohol for 5min, 90% alcohol for 5min, 100% alcohol I for 5min, 100% alcohol II for 5min, xylene I for 5min and xylene II for 5 min.
6) Sealing: the paraffin sections were placed in a fume hood for air drying and mounted with neutral resin.
7) The HE staining results were observed under a microscope and photographed for pictures.
The results show that: after 28 days post pump implantation, the maximum abdominal aortic aneurysm diameter was significantly increased in the AngII group mice compared to the saline group (fig. 1E).
5. And (4) dyeing by using elastin.
1) Slicing and dewaxing: staining with HE (steps 1 and 2).
2) Vascular elastin staining was performed according to the kit Verhoef van Gibson Elastic (H Sigma, T25A).
The results show that: after 28 days after pump implantation, the blood vessels of the AngII mice had a significant rupture of elastic fibers compared to the saline group (fig. 1E, 1F).
6. Immunohistochemical staining.
Aortas from mice in the experimental and control groups were harvested and subjected to TCF7L1 immunohistochemical staining as follows.
1) Slicing and dewaxing: staining with HE (steps 1 and 2).
2) Immunohistochemical staining.
A. Antigen retrieval: soaking the slices in the antigen retrieval solution, boiling at 100 ℃ for 40min, and cooling to room temperature.
B. The sections were placed in a wet box with the tissue side facing up, one drop or 50 μ L of peroxidase blocking agent was added to each section, incubated at room temperature for 10min, and washed three times with PBS for 10min each.
C. PBS was removed and one drop or 50. mu.L goat serum was added to each section and incubated for 0.5h at room temperature.
D. Serum was removed and 50. mu.L of TCF7L1 primary antibody (1: 100 PBS dilution) was added to each section overnight at 4 ℃.
E. Rewarming for 30min at room temperature, washing with PBS for 10min three times.
F. After incubation for 10min, three washes with PBS were performed, 10min each.
G. PBS was removed and 50. mu.L of LAB developing solution was added to each section. Diluting DAB color development liquid by 20 times.
H. Hematoxylin solution counterstained nuclei.
I. Paraffin sections were placed in 1% hydrochloric acid for 30s for differentiation and washed with running water.
J. And (3) placing the paraffin sections in ammonia water for 30s, and carrying out cell nucleus blueing and running water washing.
K. Dehydrated, xylene transparent, neutral gum seal.
And L, observing a dyeing result under an upright microscope, photographing and storing pictures.
The results show that: VSMC differentiation indices α -SMA and SM22 α expression were significantly reduced and TCF7L1 expression was significantly upregulated in the AngII group mice compared to the saline group (fig. 1G, H).
7. The expression condition of TCF7L1 in an abdominal aortic aneurysm model is detected by a Western blot method.
In order to determine the effect of TCF7L1 in abdominal aortic aneurysm, a mouse abdominal aortic aneurysm model is established by adopting an AngII embedded pump method, and the expression condition of TCF7L1 in blood vessels is detected by utilizing a western blot method. Respectively collecting blood vessels of experimental group and control group, adding appropriate amount of protein lysate, cracking on ice for 30min, and mixing evenly at intervals of 5 min. Centrifugation was carried out at 12000 g for 20min at 4 ℃ to collect the supernatant as total cell protein. And (3) determining the protein concentration in the lysate by using a BCA colorimetric kit. Using the protein lysate, the final concentration was 3 mg/ml for dilution. Adding 5 Xloading buffer solution in proportion, mixing well, boiling in water bath at 100 deg.C for 5min, centrifuging at room temperature 12000 g for 30 s. 20. mu.g of protein sample was added to each sample well, and power was turned on to start electrophoresis. The operation was performed at the following voltages and times: and (3) switching off the power supply after the bromophenol blue is electrophoresed to the bottom of the glass plate at 100V for 30min and 120V for 60 min. The sample was transferred to the PVDF membrane at a voltage of 90V for a period of 2 h. The PVDF membrane is put into 5% milk sealing solution for 1h, and primary antibody is added to incubate overnight at 4 ℃. The antibodies were raised to 1:1,000 anti-TCF 7L1 (Abcam, usa) antibody, 1: a1000 anti-beta-actin (Cellsignalling, USA) antibody is used as a primary antibody, a horseradish peroxidase-labeled goat anti-mouse (or anti-rabbit) antibody (Cellsignalling, USA) is used as a secondary antibody, Western blot detection is carried out, and ECL kit (Amersham, USA) is used for luminescent development. Protein expression bands of about 78kD and 45kD were detected with TCF7L1 antibody and beta actin antibody, respectively. Grey value measurements of the bands were performed and statistically analyzed using ImageJ 1.51 software.
The results show that: TCF7L1 protein expression was increased in blood vessels of AngII group mice compared to saline group (fig. 1I, 1J).
8. The expression condition of TCF7L1 in a mouse abdominal aortic aneurysm model established by Ang II is detected by a fluorescent Real-time quantitative PCR (Real-time PCR) method.
1) And (4) extracting RNA of the vascular tissue.
A. Vascular tissues of mice were placed in RNase-free EP tubes, 1ml of Trizol was added, three RNase-free milling beads were placed, and milling was performed in a tissue milling homogenizer for 180s at 50 Hz.
B. Standing at room temperature for 5min, adding 1/5 chloroform, mixing by turning upside down, and standing for 15 min.
C.12000rpm, 4 ℃, centrifugal 15 min.
D. Sucking the supernatant, adding equal volume of isopropanol, inverting, mixing, and standing at room temperature for 10 min.
E.12000rpm, 4 ℃, centrifugal 15min, abandoning the supernatant.
F. Adding 1mL of 75% ethanol, gently shaking the centrifugal tube, and suspending and precipitating.
G.12000rpm, 4 ℃, centrifuging for 15min, and discarding the supernatant.
H. Drying at room temperature, and making it transparent.
I. Add 25. mu.L of enzyme-free water to dissolve the RNA.
2) Reverse transcription of RNA into cDNA
The RNA extracted in step 1 was reverse transcribed to synthesize cDNA using Takara reverse transcription kit, as follows:
A. removal of genomic DNA
B. Reverse transcription reaction
Reaction conditions are as follows: 15min at 37 ℃; 5s at 85 ℃; storing at 4 ℃.
3) A primer sequence.
4) And (3) fluorescent quantitative PCR.
According to the volume of the table, a 20 μ L qPCR reaction system was prepared, and each group of samples was provided with 3 duplicate wells, respectively. Reaction conditions are as follows: 95 ℃ for 5min, (95 ℃ for 5s, 60 ℃ for 30s, 72 ℃ for 30 s) x 40 cycles, 72 ℃ for 5s, 95 ℃ for 15 s. By using 2－△△CtMethod, expression of each group of samples was analyzed.
The results show that: TCF7L1 transcript levels were increased in the blood vessels of AngII mice compared to the saline group (fig. 1K).
The above results all show that TCF7L1 is up-regulated in abdominal aortic aneurysm, suggesting that TCF7L1 may be involved in the development of abdominal aortic aneurysm in mice.
Example 2 overexpression of TCF7L1 in mice exacerbates the development of AAA.
1. ApoE-/-The mice were purchased from Jiangsu Jiejiegaokang Biotech Co., Ltd under the same feeding conditions as in example 1.
2. Mice were injected intravenously with adeno-associated virus.
To verify the effect of TCF7L1 on mouse abdominal aortic aneurysm formation in vivo, AAV-cmv-TCF7L1, and its negative control AAV-cmv-GFP were constructed in Shanghai and Yuan Biotechnology Ltd. TCF7L1 was overexpressed in vivo by intravenous injection of mouse tail for 3 weeks.
3. And (5) establishing a mouse abdominal aortic aneurysm model.
AAV-cmv-TCF7L1 was injected into tail vein, and 4 groups of mice with negative control were used to construct abdominal aortic aneurysm model by AngII pump-embedded method, as in example 1.
The results show that: compared with the normal saline group, the AngII group mice have obviously increased tumor formation rate. The tumor formation rates of 4 groups of mice are shown in FIGS. 2A and 2B.
4. Ultrasound imaging measures the maximum abdominal aorta diameter as in example 1.
The results show that: the maximum abdominal aortic aneurysm diameter was significantly increased in the AngII group mice compared to the saline group; compared with AAV-cmv-GFP + AngII group, the diameter of maximum abdominal aortic aneurysm of AAV-cmv-TCF7L1+ AngII group mice is increased more obviously. Ultrasound images of 4 groups of mice and the maximum abdominal aortic aneurysm diameter are shown in fig. 2C and 2D.
5. HE staining detects aortic aneurysm formation.
AAV-cmv-GFP + salene, AAV-cmv-GFP + AngII, AAV-cmv-TCF7L1+ salene, and AAV-cmv-TCF7L1+ AngII group mice were harvested for HE staining of aorta 28 days after pump embedding.
The results show that: compared with the normal saline group, the blood vessels of the mice in the AngII group are obviously widened; compared with AAV-cmv-GFP + AngII group, mice in AAV-cmv-TCF7L1+ AngII group broadened more significantly (FIGS. 2E, 2F).
6. And (4) dyeing by using elastin.
And collecting the aorta tissues 28 days after AAV-cmv-GFP + saline, AAV-cmv-GFP + AngII, AAV-cmv-TCF7L1+ saline and AAV-cmv-TCF7L1+ AngII mice are buried for elastin staining.
The results show that: compared with the normal saline group, the elastic fibers of the blood vessels of the mice in the AngII group are obviously broken; the AAV-cmv-TCF7L1+ AngII group mice fragmented more significantly than the AAV-cmv-GFP + AngII group (FIGS. 2E, 2F).
7. Immunohistochemical staining.
And (3) respectively collecting the aortic tissues 28 days after AAV-cmv-GFP + salene, AAV-cmv-TCF7L1+ salene and AAV-cmv-TCF7L1+ AngII mice are buried in a pump, and performing immunohistochemical staining of alpha-SMA, SM22 alpha, MMP2 and TCF7L 1.
The results show that: compared with the normal saline group, the expression of alpha-SMA and SM22 alpha in the blood vessel of the mice in the AngII group is obviously reduced, and the expression of MMP2 is obviously increased; compared with AAV-cmv-GFP + AngII group, the expression of mouse alpha-SMA and SM22 alpha of AAV-cmv-TCF7L1+ AngII group was more reduced, and the expression of MMP2 was increased (FIGS. 2E and 2F).
And 8, detecting the expression conditions of AAV-cmv-GFP + salene, AAV-cmv-GFP + AngII, AAV-cmv-TCF7L1+ salene, AAV-cmv-TCF7L1+ AngII group mouse VSMC differentiation indexes alpha-SMA, SM22 alpha and metallomatrix protease MMP2 by using a Western blot method.
The results show that: compared with the normal saline group, the expression of alpha-SMA and SM22 alpha in the blood vessel of the mice in the AngII group is obviously reduced, and the expression of MMP2 is obviously increased; compared with AAV-cmv-GFP + AngII group, the AAV-cmv-TCF7L1+ AngII group mice have more reduced expression of alpha-SMA and SM22 alpha, and increased expression of MMP2 (FIGS. 2G, 2H).
Example 3: the low expression of TCF7L1 in mice delayed the onset of AAA.
1.ApoE-/-Mice were purchased from Jiangsu Jiejiaokang Biotech limited. The feeding conditions were the same as in example 1.
2. Mice were injected intravenously with adeno-associated virus.
Knockdown of TCF7L1 by shRNA interference technique, shRNA interference of target sequence: GAGAAGAACAGGCCAAATA, shRNA shRNA used in the interference experiments was purchased from Shanghai and Yuan Biotechnology (Shanghai) GmbH, as follows.
To verify the effect of TCF7L1 on abdominal aortic aneurysm formation in vivo, AAV-shTCF7L1 and its negative control AAV-sh-RNA were constructed in shanghai and yuans biotechnology limited. TCF7L1 was knocked down in vivo by intravenous injection of mouse tail for 3 weeks.
3. And (5) establishing a mouse abdominal aortic aneurysm model.
4 groups of mice injected with AAV-shTCF7L1 and negative control in tail vein were respectively used for constructing an abdominal aortic aneurysm model by the method of AngII pump burying, as in example 1.
The results show that: compared with the normal saline group, the AngII group mice have obviously increased tumor formation rate. The tumor formation rates of the 4 groups of mice are shown in FIGS. 3A and 3B.
4. Ultrasound imaging measures the maximum abdominal aorta diameter as in example 1.
The results show that: the maximum abdominal aortic aneurysm diameter was significantly increased in the AngII group mice compared to the saline group; compared with the AAV-sh-RNA + AngII group, the AAV-shTCF7L1+ AngII group mice have reduced maximum abdominal aortic aneurysm diameter. The results of the ultrasound images and the maximum abdominal aortic aneurysm diameter of the 4 groups of mice are shown in fig. 3C and 3D.
5. HE staining detects aortic aneurysm formation.
And respectively collecting the aorta tissues 28 days after AAV-sh-RNA + saponin, AAV-sh-RNA + AngII, AAV-shTCF7L1+ saponin and AAV-shTCF7L1+ AngII mice are buried in the pump, and carrying out HE staining.
The results show that: the maximum abdominal aortic aneurysm diameter was significantly broadened in the AngII group mice compared to the saline group; compared with the AAV-sh-RNA + AngII group, the maximum abdominal aortic aneurysm diameter of the mice in the AV-shTCF7L1+ AngII group is reduced. The results of the ultrasound images and the maximum abdominal aortic aneurysm diameter of the 4 groups of mice are shown in fig. 3C and 3D.
6. And (4) dyeing with elastin.
And respectively collecting the aorta tissues 28 days after AAV-sh-RNA + saponin, AAV-sh-RNA + AngII, AAV-shTCF7L1+ saponin and AAV-shTCF7L1+ AngII mice are buried in the pump, and performing elastin staining.
The results show that: compared with the normal saline group, the elastic fibers of the blood vessels of the mice in the AngII group are obviously broken; compared with AAV-sh-RNA + AngII group, the breakage of elastic fibers of AAV-shTCF7L1+ AngII group mice was alleviated (FIGS. 3E and 3F).
7. Immunohistochemical staining.
And respectively collecting the aorta tissues 28 days after AAV-sh-RNA + saponin, AAV-sh-RNA + AngII, AAV-shTCF7L1+ saponin and AAV-shTCF7L1+ AngII group mice are buried in a pump, and performing immunohistochemical staining of alpha-SMA, SM22 alpha, MMP2 and TCF7L 1.
The results show that: compared with the normal saline group, the expression of alpha-SMA and SM22 alpha in the blood vessel of the mice in the AngII group is obviously reduced, and the expression of MMP2 is obviously increased; compared with AAV-sh-RNA + AngII group, the mouse alpha-SMA and SM22 alpha expression of AAV-shTCF7L1+ AngII group was increased, and MMP2 expression was decreased (FIGS. 3E and 3F).
8. The expression conditions of AAV-sh-RNA + salene, AAV-sh-RNA + AngII, AAV-shTCF7L1+ salene, and AAV-shTCF7L1+ AngII group mice vascular smooth muscle differentiation indexes alpha-SMA, SM22 alpha and metal matrix protease MMP2 are detected by a Western blot method.
The results show that: compared with the normal saline group, the expression of alpha-SMA and SM22 alpha in the blood vessel of the mice in the AngII group is obviously reduced, and the expression of MMP2 is obviously increased; compared with AAV-sh-RNA + AngII group, the mouse alpha-SMA and SM22 alpha expression of AAV-shTCF7L1+ AngII group was increased, and MMP2 expression was decreased (FIGS. 3G, 3H).
Example 4: TCF7L1 was up-regulated during mouse VSMC dedifferentiation.
1. Isolation and culture of mouse VSMC.
5-6C 57BL/6J male mice (purchased from Jiejiaokang Biotech, Inc., Jiangsu, China) aged 8 weeks were carefully dissected from the aorta and stored in ice sterile PBS containing 1 Xantibiotics, and the adventitia was carefully removed. The strips of tissue were cut into pieces about 2mm long with an ophthalmic scissors. The aortic media tissue mass was resuspended in 3mL of 0.15% collagenase II digest and transferred to a six-well plate. Digestion was terminated after 2-3h by placing the six-well plate in a 37 ℃ 5% CO2 incubator. The cell suspension was seeded in 1 6-well plate, 2 mL per well. The fluid is changed for the first time at 3d to remove non-adherent cells or tissue blocks. And then changing the solution for 1 time every 3-5 days according to the growth condition of the cells. Culturing the cells in a DMEM medium (growth medium) containing 20% fetal calf serum in a 100 mm culture dish, and carrying out cell passage when the cell growth density reaches 80% -90% fusion. Passage 6-10 cells were used for the experiments.
2. Induction of mouse VSMC synthetic phenotype.
Mouse VSMC is cultured in a growth medium containing 20% fetal calf serum in a 100 mm culture dish, and when the cell density reaches 80% -90%, AngII with the concentration of 1 mu M is added for 24 h.
3. And detecting the expression condition of TCF7L1 in the mouse VSMC dedifferentiation process by using a Western blot method.
Total proteins in VSMCs of AngII group and PBS group were extracted, and the expression change of TCF7L1 during the dedifferentiation of VSMCs was compared, as in example 1.
The results show that: the expression of TCF7L1 protein in AngII group was significantly higher than that in PBS group (fig. 4A, 4B).
4. The expression condition of TCF7L1 in the mouse VSMC dedifferentiation process is detected by a fluorescent Real-time quantitative PCR (Real-time PCR) method.
The total RNA in VSMCs of AngII group and PBS group was extracted, and the expression change of TCF7L1 in the transcriptional level during the dedifferentiation of VSMCs was compared, as described in example 1.
The results show that: the transcriptional level expression of TCF7L1 was significantly higher in the AngII group than in the PBS group (fig. 4C).
5. The expression of TCF7L1 in mouse VSMC dedifferentiation process was detected by immunofluorescence staining method.
1) Cell slide was prepared by placing sterile coverslips into 24-well plates and adding 1mL of cell suspension per well.
2) When the cell density reaches 80-90%, adding AngII with the concentration of 1 μ M for 24 h.
3) After 24h, 3 times with PBS, 10min with 4% paraformaldehyde, and 3 times with PBS.
4)0.1% Triton X-100 was permeabilized for 5min and washed 3 times with PBS.
5) Sealing with 5% goat serum for 30 min.
6) Mouse anti- α -SMA and sm22 α primary antibody (1: 100 dilution) were added to the cell-slide separately and incubated overnight at 4 ℃.
7) PBS was washed 3 times, and goat anti-mouse fluorescent secondary antibody (1: 100) adding onto cell slide, and incubating at room temperature in dark for 30 min.
8) The cells were washed 3 times with PBS and stained with 0.5 μ g/mL DAPI for 1 min.
9) The plate was washed 3 times with PBS and sealed with an anti-quenching fluorescent blocking agent.
10) Staining was observed under an upright fluorescence microscope (zeiss, germany) and photographed.
The results show that: TCF7L1 expression was significantly higher in AngII group than in PBS group (fig. 4D).
Example 5: low expression of TCF7L1 promoted VSMC differentiation.
1. And (3) establishing a TCF7L1 low-expression mouse VSMC model.
Knockdown of TCF7L1 by siRNA interference technique, siRNA interference with target sequence: GAGAAGAACAGGCCAAATA, siRNA siRNA used in interference experiments were purchased from Cantonella, Guangzhou, Inc. as follows.
To prepare TCF7L 1-low expressing mouse VSMCs, cells were plated in six-well plates and siRNA transfection was performed when cells grew to 70% -80%. 0.5mL of Opti-MEM medium was added to each well and siControl and siTCF7L1 (interfering target sequence: GAGAAGAACAGGCCAAATA) were transfected into mouse VSMCs using Lipofectamine RNAImax transfection reagent. Culturing at 37 deg.C in 5% CO2 cell culture box for 4-6h, then changing 6-well plate into DMEM medium containing 20% fetal calf serum and 1% antibiotics, and culturing for 24h for experiment.
2.Western blot detection of the influence of low expression of TCF7L1 on the expression of mouse VSMC differentiation markers alpha-SMA and SM22 alpha and metallomatrix protease MMP2, the specific method is the same as in example 1.
The results show that: compared with the siControl group, the expression of alpha-SMA and SM22 alpha in the siTCF7L1 group is obviously increased, and the expression of MMP2 is obviously reduced (FIGS. 5A and 5B).
3, Real-time PCR detection of the effect of TCF7L1 low expression on mouse VSMC differentiation markers alpha-SMA and SM22 alpha expression, the specific method is the same as example 1.
The results show that: compared with the siControl group, transcript levels of α -SMA and SM22 α were significantly increased in the siTCF7L1 group and MMP2 was significantly decreased (fig. 5C).
4. The Transwell method detects the effect of low-expression TCF7L1 on mouse VSMC migration.
1) The siTCF7L1 and siControl mouse VSMC transfections were seeded into the upper chamber of a Transwell cell (approximately 2.5X 10)4One), the upper chamber medium was changed to serum-free.
2) The lower chamber was filled with 500. mu.L of 10% serum medium.
3) After 24h incubation, the upper chamber cells were washed and fixed with 4% paraformaldehyde.
4) The upper chamber cells were stained with 0.1% crystal violet solution (solibao, beijing, china) for 20min at room temperature.
5) The number of migrated cells was quantified by taking pictures under a microscope at 40 times magnification and counting the number of cells in 10 random fields.
The results show that: compared to the siControl group, siTCF7L1 inhibited migration of mouse VSMCs (fig. 5D, 5E).
Example 6: TCF7L1 overexpression inhibited VSMC differentiation.
1. Establishment of TCF7L1 overexpression mouse VSMC model.
To prepare mouse VSMC overexpressing TCF7L1, cells were plated in a six-well plate and when the cells grew to 70% -80%, a plasmid containing the TCF7L1 overexpression vector (pcDNA3.1-TCF 7L 1) was added to the mouse VSMC for infection, and a Flag-tagged plasmid (pcDNA3.1-Flag) was used as a control for 24h infection with Lipofectamine 2000 (Invitrogen, USA) and then used for experiments. Plasmids were constructed in Shanghai and Yuan biologies.
Western blot to detect the influence of over-expression of TCF7L1 on the expression of mouse VSMC differentiation markers alpha-SMA and SM22 alpha and MMP2, the specific method is the same as that in example 1.
The results show that: compared with pcDNA3.1-Flag, the expression of the alpha-SMA and SM22 alpha in the pcDNA3.1-TCF7L1 group is obviously reduced, and the MMP2 is obviously increased (FIGS. 6A and 6B).
3, Real-time PCR detection of the effect of TCF7L1 overexpression on the expression of mouse VSMC differentiation markers alpha-SMA and SM22 alpha, the specific method is the same as example 1.
The results show that: compared with the pcDNA3.1-Flag group, the expression of alpha-SMA and SM22 alpha of the pcDNA3.1-TCF7L group was significantly reduced, and MMP2 was significantly increased (FIG. 6C).
The Transwell method is the same as example 5 in the specific method for detecting the influence of over-expression of TCF7L1 on the migration of mouse VSMC.
The results show that: compared with the pcDNA3.1-Flag group, pcDNA3.1-TCF7L1 promoted migration of mouse VSMC (FIGS. 6D, 6E).
SEQUENCE LISTING
<110> general hospital in north war zone of China's liberation army
<120> medical use of TCF7L1 for preventing or treating abdominal aortic aneurysm
<130> 20220318
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213> Artificial sequence
<400> 1
gagaagaaca ggccaaata 19
Claims (10)
- Use of an inhibitor of TCF7L1 in the manufacture of a product for the diagnosis of abdominal aortic aneurysm.
- 2. The use of claim 1, wherein the product for diagnosing abdominal aortic aneurysm contains TCF7L1 as a marker, and the marker comprises TCF7L1 gene or TCF7L1 protein.
- 3. A marker according to claim 2, wherein TCF7L1 is up-regulated in abdominal aortic aneurysms.
- 4. The use according to claim 1, wherein the product is a chip, a preparation or a kit.
- Use of an inhibitor of TCF7L1 in the manufacture of a medicament for the treatment of an abdominal aortic aneurysm.
- 6. The use of claim 5, wherein the TCF7L1 inhibitor promotes vascular smooth muscle cell differentiation.
- 7. The use of claim 5, wherein the TCF7L1 inhibitor is used as a vascular smooth muscle cell differentiation promoter and/or a vascular smooth muscle cell migration inhibitor in the preparation of a medicament for the treatment of an abdominal aortic aneurysm.
- 8. The use according to claim 5, wherein the inhibitor is in any pharmaceutically acceptable dosage form or dosage.
- 9. The use of claim 5, wherein the TCF7L1 inhibitor target sequence is set forth in SEQ ID No. 1.
- 10. The use of claim 5, wherein the TCF7L1 inhibitor comprises an siRNA and/or shRNA of the target sequence of claim 9.
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| Title |
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| JING WANG等: "Tcf7l1 Accelerates Smooth Muscle Cell Phenotypic Switching Suppressing Srf Transcription and Aggravates Abdominal AorticAneurysms" * |
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