Hypoxic region targeted expression TIA1 gene and application thereof

Abstract

The invention provides an anoxic region targeted expression TIA1 gene and application thereof. The anoxic region targeted expression TIA1 gene comprises nine times of repeated HRE and TIA1 genes. Wherein the nucleotide sequence of 9 times of repeated HRE is as shown in SEQ ID NO: 1, and the TIA1 gene is human NM022037.4. The target gene TIA1 capable of enhancing myocardial cell proliferation in the hypoxia infarction region in a targeted manner is screened from the direction of promoting cardiac function health, so that mass proliferation of myocardium in a non-infarction region can be avoided, and the risk of excessive proliferation in other regions is reduced. The TIA1 gene can promote proliferation of various myocardial cells, and in-vivo and in-vitro myocardial proliferation of the TIA1 gene cannot influence normal myocardial and heart functions, so that other pathological changes of the myocardial cells cannot be caused. The hypoxia region targeted expression TIA1 gene can be used for preparing drugs for promoting myocardial regeneration and repairing damaged heart, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method for targeting the expression of TIA1 gene in hypoxic areas and an application thereof. Background Art

[0002] Cardiovascular diseases accompanied by myocardial damage will eventually develop into heart failure and sudden cardiac death. As an effective treatment, heart transplantation is greatly limited in its application due to the rejection of donated organs and transplantation. Therefore, finding more effective myocardial repair methods has attracted widespread attention.

[0003] Myocardial regeneration studies have found that the original cardiomyocytes in the heart are the main source of supplementary cells after myocardial injury. However, the self-renewal of adult mammalian cardiomyocytes is stagnant, and the repair effect of myocardial regeneration is extremely limited in diseases related to damaged cardiomyocytes, such as myocardial infarction and myocarditis. Therefore, inducing adult cardiomyocytes to re-enter the cell cycle will promote the development of cardiac regeneration-based treatment methods.

[0004] Through the study of developing and exercising hearts, it was found that there is a proliferation trend in the exercising heart that is opposite to that in the developing heart, suggesting that this can be used to explore the mechanism of physiological regulation of myocardial development. The process of myocardial cells exiting the cell cycle as they develop mainly involves the ERBB2, NRG1 and Hippo / YAP signaling pathways, which decay as the myocardial development progresses. Studies have found that in adult myocardial cells, including myeloid ecotropic viral integration site homeobox 1 (MEIS1), retinoblastoma protein (RB), polycomb repressive complex 2 (PRC2) and microRNA, multiple transcriptional regulators and epigenetic modifiers are involved in inhibiting myocardial cells from reentering the cell cycle. Exercise can induce myocardial cells to reenter the cell cycle, which is in stark contrast to the situation in which the proliferation capacity and cell cycle of adult myocardial cells in animals and humans are blocked as the heart develops. However, studies on the development of organs in multiple species have found that organ development is more dependent on post-transcriptional regulation such as coding and non-coding RNA. However, the post-transcriptional regulators of the physiological response of myocardial cell proliferation have not yet been determined.

[0005] In addition, existing myocardial repair technologies show obvious limitations in targeting the infarcted area. Specifically, the current method only targets myocardial cells, but the repair effect is poor, and there is a risk of affecting the myocardium in normal areas, thereby affecting the recovery of cardiac function. Moreover, the traditional method of screening for genes that promote myocardial proliferation is difficult to ensure the long-term effect of the target gene on myocardial cell proliferation. Summary of the invention

[0006] The purpose of the present invention is to provide a method for targeted expression of TIA1 gene in hypoxic areas and its application. The target gene TIA1 is obtained by screening, which can target and enhance the proliferation of myocardial cells in hypoxic infarct areas to promote myocardial regeneration, avoid massive proliferation of myocardial cells in non-infarct areas, and will not cause other pathological changes in myocardial cells.

[0007] The present invention solves the technical problem by adopting the following technical solutions.

[0008] The present invention provides a method for targeted expression of TIA1 gene in hypoxic areas, comprising 9-repeated HRE and TIA1 gene, wherein the nucleotide sequence of the 9-repeated HRE is shown in SEQ ID NO: 1, and the TIA1 gene is human NM_022037.4.

[0009] The present invention proposes the use of the hypoxic zone targeted expression of TIA1 gene in the preparation of a drug for promoting myocardial regeneration.

[0010] The present invention proposes the use of the hypoxic zone targeted expression of TIA1 gene in the preparation of a drug for repairing a damaged heart.

[0011] The beneficial effects of the targeted expression of TIA1 gene in hypoxic areas and its application in the embodiments of the present invention are: The present invention screens and obtains the target gene TIA1 that can target and enhance the proliferation of myocardial cells in the hypoxic infarct area from the direction of promoting healthy heart function, thereby avoiding the massive proliferation of myocardium in the non-infarct area and reducing the risk of excessive proliferation in other areas. Moreover, the TIA1 gene can also promote the proliferation of various myocardial cells, and its in vivo and in vitro myocardial proliferation will not affect the normal myocardium and cardiac function, and thus will not cause other pathological changes in myocardial cells. The hypoxic area targeted expression of the TIA1 gene of the present invention can be used to prepare drugs that promote myocardial regeneration and repair damaged hearts, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0013] Figure 1 This is a diagram showing the expression changes of TIA1 during mouse heart development; Figure 2 This is a graph showing the expression changes of TIA1 during mouse embryonic stem cell differentiation; Figure 3 This is the expression of TIA1 in the myocardium of neonatal mice after adenovirus infection; Figure 4 This is a graph showing the change in the proportion of pH3-positive cells after TIA1 overexpression in neonatal rat myocardium; Figure 5 This is a graph showing the change in the proportion of Aurora B-positive cells after TIA1 overexpression in neonatal rat myocardium; Figure 6 This is a graph showing the change in the proportion of Edu-positive cells after TIA1 overexpression in neonatal rat cardiac fibroblasts; Figure 7 This is a graph showing changes in TIA1 expression during human iPS differentiation; Figure 8 This is a graph showing the changes in the proportion of pH3-positive cells after overexpression of TIA1 in human myocardium; Fig. 9 This is a graph showing the change in the proportion of Edu-positive cells after overexpression of TIA1 in human myocardium; Fig.10 Flow chart of the in vivo physiological model for neonatal mice; Fig.11 This is a graph showing the expression of TIA1 in myocardium after AAV9 injection in newborn mice; Fig.12 This is a diagram showing changes in heart morphology and heart-body ratio; Fig.13 This is a graph showing the changes in the proportion of ki67 and Edu-positive cells overexpressing TIA1 in the myocardium of newborn mice; Fig.14 This is a graph showing the changes in the proportion of pH3-positive cells in the myocardium of neonatal mice with overexpression of TIA1; Fig.15 Flow chart of the in vivo physiological model for adult mice; Fig.16 This is the result of Western blot detection of TIA1 expression in heart tissue 3 weeks after adeno-associated virus injection; Fig.17 This is a picture of the mouse heart 4 weeks after adeno-associated virus injection; Fig.18 This is the result of Edu staining of mouse heart sections; Fig.19 Flow chart of adeno-associated virus injection and myocardial infarction experiment in adult mice; Fig. 20 This is the result of ultrasound examination of cardiac function in mice after myocardial infarction; Fig.21 H&E staining of mouse heart sections to examine scar tissue area; Fig. 22 This is the result of Edu staining of mouse heart sections. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0015] The targeted expression of TIA1 gene in hypoxic areas and its application in the embodiments of the present invention are described in detail below.

[0016] Example 1 This embodiment provides a method for targeted expression of TIA1 gene in hypoxic areas, and the specific steps are as follows: C57BL / 6 mice (Jicui Yaokang) were used to establish an exercise mouse model through ramp swimming training. After 4 weeks of training, the mice were euthanized and heart tissues were obtained. RNA and protein were extracted using TRIZOL reagent (Thermo) according to the instructions. RNA was reversed into cDNA using a reverse transcription kit, and the RNA level expression of the TIA1 gene was detected using a qPCR kit and primers. The nucleotide sequence of the primer is: Forward primer 5′-GCCAGTATGTGCCTAACGGTTG-3′ (SEQ ID NO: 2); Reverse primer 5'-CTTGAGGTGGTGGCACACTGTA-3' (SEQ ID NO: 3).

[0017] Analysis showed that the expression of proliferation genes was upregulated, but there was no difference in the expression of myocardial hypertrophy-related genes, confirming that the physiological myocardial proliferation model initiated by exercise was successfully established. The aforementioned heart samples were subjected to transcriptomic sequencing to analyze the expression changes of RNA binding proteins in the mouse heart after swimming training.

[0018] Using SD rats (Jicui Yaokang), neonatal rat cardiomyocytes and adult rat cardiomyocytes were isolated and collected, and transcriptomic sequencing was performed to analyze the expression differences of RNA binding proteins in the proliferative neonatal rat cardiomyocytes and adult rat mature cardiomyocytes.

[0019] The transcriptomic data of the exercise heart and the developing heart were further compared and analyzed to screen the RNA binding proteins that were upregulated in the transcriptome of the exercise heart and downregulated in the transcriptome of the developing heart, and to analyze the overlapping genes. Using the siRNA library, the aforementioned overlapping RNA binding proteins were downregulated in the cardiomyocytes of neonatal rats and mice, and the proliferation markers ki67 and Edu were used to analyze the effects of each gene on the proliferation ability of neonatal rats and mice cardiomyocytes. In the analysis of the two sets of transcriptomic data, TIA1 was one of the genes with the most significant expression changes. In the siRNA knockdown experiment of the neonatal rats and mice cardiomyocytes, it was further clarified that the downregulation of TIA1 expression had the most significant effect on the proliferation of neonatal rat cardiomyocytes.

[0020] The hypoxic region targeted expression TIA1 gene of the present invention comprises 9-repeated HRE and TIA1 gene, wherein the nucleotide sequence of the 9-repeated HRE is shown in SEQ ID NO: 1, which is 9 HRE sequence fragments connected continuously. The TIA1 gene is human NM_022037.4. Among them, human NM_022037.4 has multiple subtypes, and the nucleic acid sequences range from type 1 to type 6, including 1: NM_003252.4; 2: NM_001033925.2; 3: NM_001323964.2, NM_001323965.2, NM_001323967.2; 4: NM_001323968.2; 5: NM_001323969.2; 6: NM_001323970.2. For example, the nucleotide sequence of NM_003252.4 is shown in SEQ ID NO: 4, and the nucleotide sequence of NM_001033925.2 is shown in SEQ ID NO: 5. The targeted expression of TIA1 gene in the hypoxic area of ​​the present invention can target and enhance myocardial proliferation in the hypoxic infarct area and avoid massive myocardial proliferation in the non-infarct area.

[0021] Example 2 In this example, the TIA1 gene was used to study the effect of promoting mouse cardiomyocyte proliferation. Heart tissues of mice at embryonic day 16.5, day 0, day 7, day 10, and day 60 after birth were collected. Immunoblotting and qPCR were used to detect the expression changes of TIA1 in the hearts of mice at different stages. The specific steps were as follows: C57BL / 6 mice were used. After mating, pregnant female mice (16.5 days) were taken and anesthetized for laparotomy to remove the embryos and isolate the heart tissue. In addition, mice 0, 7, 10, and 60 were taken after euthanasia to isolate the heart tissue. RNA and protein were extracted using TRIZOL reagent (Thermo) according to the instructions. RNA was reversed into cDNA using a reverse transcription kit, and the RNA level expression of the mouse TIA1 gene was detected using a qPCR kit and primers. The nucleotide sequence of the primer is: Forward primer 5′-GCCAGTATGTGCCTAACGGTTG-3′ (SEQ ID NO: 2); Reverse primer 5'-CTTGAGGTGGTGGCACACTGTA-3' (SEQ ID NO: 3).

[0022] After SDS-PAGE electrophoresis and transfer, the protein was blocked with milk for 1 hour, incubated with primary antibodies: TIA1 (Proteintech) and GAPDH (ABclonal) overnight, and incubated with secondary antibodies (ABclonal) at room temperature for 1 hour. The developer was added for development, and the gray value was used to calculate TAI1 / GAPDH of each sample to analyze the expression of TIA1 gene protein level.

[0023] like Figure 1 The figure shows the expression changes of TIA1 during mouse heart development. Figure 1 It can be seen that the expression of TIA1 decreases with the age of mice.

[0024] Example 3 In this example, mouse embryonic stem cells were differentiated into cardiomyocytes, embryonic stem cells, mesoderm cells, cardiomyocyte progenitor cells and cardiomyocytes were obtained, and the expression changes of TIA1 at different differentiation stages were detected by immunoblotting and qPCR. The specific steps were as follows: Mouse embryonic stem cells R1 (Pnosai Bio) were grown to a confluency of 80% to 90%. After digestion with 0.05% EDTA trypsin (Gibco), they were resuspended in cardiomyocyte differentiation medium (DMEM / F12 containing 20% ​​FBS, 0.1 mM NEAA, LIF (Gibco), 1 mM sodium pyruvate and β-mercaptoethanol (Sigma)). After adhering to the wall in a 0.1% gelatin-coated culture dish for 20 minutes to remove mouse embryonic fibroblasts, the cells were counted and diluted to 40 cells / µL, and suspended on the cover of the culture dish with 25 µL as a drop. After 4 days, the hanging drops were collected and transferred to a 0.1% gelatin-coated culture dish to allow the embryoid bodies to adhere to the wall, and the medium was changed every day. Then, immunoblotting and qPCR were performed according to the same steps as in Example 2 to detect the expression changes of TIA1 at different differentiation stages.

[0025] like Figure 2 Shown is the expression change of TIA1 during mouse embryonic stem cell differentiation. Figure 2 It is shown in Figure 1 that the expression of TIA1 gradually decreases after mouse embryonic stem cells differentiate into cardiomyocytes. It can be seen from Examples 1 and 2 that TIA1 plays an important role in the development of cardiomyocytes.

[0026] Example 4 In this example, adenovirus expressing TIA1 was transfected into neonatal rat ventricular myocardial cells for 72 hours to study the effect of TIA1 gene on promoting the proliferation of neonatal rat myocardial cells. The specific steps are as follows: Isolation of rat myocardium: Rats were purchased from Jicui Yaokang and bred. One day after the birth of the rats, they were euthanized, the chest cavity was cut open, the heart was carefully removed, and it was quickly placed in a pre-cooled PBS solution and rinsed several times to remove blood and non-cardiac tissue. The heart was cut into small pieces of about 1 mm³, and digestion solution containing trypsin and collagenase (0.1% trypsin and 0.03% collagenase II, Gibco) was added. Digestion was carried out at 37°C for 10-15 minutes and neutralized with DMEM / F12 (Gibco) containing 10% FBS. All digestion solutions were collected and filtered through a 40 µm cell sieve to remove incompletely dissociated tissues. After centrifugation at 1,000 rpm for 5 minutes, the supernatant was discarded and the cell pellet was resuspended in DMEM / F12 medium. The cells were plated on a culture plate coated with 0.2% gelatin for 1 hour and differentially adhered to the wall to remove fibroblasts. Non-adherent cells were collected and counted at 5×10 5 / well was plated in a six-well plate coated with rat tail collagen (Sigma). One day later, after the myocardial beating of the neonatal rats was observed, the neonatal rat myocardium was transfected with adenovirus at an MOI of 50, wherein the construction of adenovirus was entrusted to Weigen Biotech. RNA and protein were extracted using TRIZOL reagent (Thermo) according to the instructions. RNA was reversed into cDNA using a reverse transcription kit, and the RNA level expression of the rat TIA1 gene was detected using a qPCR kit and primers. The nucleotide sequence of the primer is: Forward primer 5′- AAAGATACAAGCAGTAGTACCG-3′ (SEQ ID NO: 6); Reverse primer 5'-CAGTTTATGAGAATCCATGTGC-3' (SEQ ID NO: 7).

[0027] like Figure 3 Shown is the expression of TIA1 in neonatal mouse myocardium after adenovirus infection. Figure 4 This is a graph showing the changes in the proportion of pH3-positive cells after overexpression of TIA1 in the myocardium of neonatal rats. Figure 5 This is a graph showing the changes in the proportion of Aurora B-positive cells after overexpression of TIA1 in the myocardium of neonatal rats. Figure 6 The figure shows the change in the proportion of Edu-positive cells after TIA1 overexpression in neonatal rat cardiac fibroblasts. Figures 3 to 6 It can be seen that after detecting the increase in mRNA and protein levels of TIA1 ( Figure 3 ), it was found that the proportion of phospho-histone H3 (PH3) and Aurora B kinase-positive cardiomyocytes increased ( Figure 4 and Figure 5 ). Six days after transfection, the number of cardiomyocytes was also increased in neonatal rat ventricular myocytes, while the expression of hypertrophic genes did not change. In addition, overexpression of TIA1 in cardiac fibroblasts did not affect fibroblast proliferation, highlighting the selectivity of TIA1 on cardiomyocyte proliferation ( Figure 6 ).

[0028] Example 5 This example uses human iPS differentiated cardiomyocytes to further explore whether the activity of TIA1 in promoting cardiomyocyte proliferation is species-specific. Cells were collected on day 0, day 3, day 7, day 10, day 14, and day 30 of iPS differentiation into cardiomyocytes to detect the expression changes of TIA1 during human cardiomyocyte development. The specific steps are as follows: iPSC-B1 (Saibei Bio) was cultured to a density of 80%-90% using E8 medium (Stemcell), and the differentiation medium was replaced: RPMI1640 (Gibco) containing 2% insulin-free B27 (Thermo), and the Wnt pathway activator 6 µM CHIR99021 was added for 48 hours, and the differentiation medium was replaced with a new one and the Wnt pathway inhibitor 5 µM IWP-2 was added for 48 hours. The differentiation medium was changed every other day until the 7th day, and the medium was switched to the cardiomyocyte medium RPMI1640 (Gibco) containing 2% insulin-containing B27 (Thermo) (RPMI1640 / B27) for 2 days. Next, the cells were cultured in glucose-free RPMI1640 supplemented with 5 mM sodium L-lactate (Sigma) containing 2% insulin-containing B27 for 5 days to obtain highly pure iPSC-derived cardiomyocytes (iPSC-CMs). On day 15, iPSC-CMs were digested with TrypLE 10× (Thermo) and seeded into RPMI1640 / B27 / 10% KOSR (Gibco) / Y-27632 (10 µM). Finally, iPSC-CMs were maintained in RPMI / B27 and reseeded every two weeks with fresh matrix gel. RNA and protein were extracted using TRIZOL reagent (Thermo) according to the instructions on days 0, 3, 7, 10, 14, and 30 of differentiation. RNA was reversed into cDNA using a reverse transcription kit, and the expression of the RNA level of the human TIA1 gene was detected using a qPCR kit and primers. The nucleotide sequences of the primers are: Forward primer 5′- GCCTAATGGTTGGCAAGTTCCTG-3′ (SEQ ID NO: 8); Reverse primer 5'-CCATTTTGCCCTTGAGGCGGTT-3' (SEQ ID NO: 9).

[0029] After SDS-PAGE electrophoresis and transfer, the protein was blocked with milk for 1 hour, incubated with primary antibodies: TIA1 (Proteintech) and GAPDH (ABclonal) overnight, and incubated with secondary antibodies (ABclonal) at room temperature for 1 hour. The developer was added for development, and the gray value was used to calculate TAI1 / GAPDH of each sample to analyze the expression of TIA1 gene protein level.

[0030] like Figure 7 The figure shows the changes in TIA1 expression during human iPS differentiation. Figure 7 It can be seen that qPCR and immunoblotting detection showed that TIA1 expression gradually decreased during human myocardial differentiation, increased when myocardial progenitor cells differentiated to day 14, and decreased when differentiated to myocardial cells ( Figure 7 ), which is consistent with the expression changes during mouse myocardial development, suggesting that TIA1 plays an important role in human myocardial development.

[0031] After iPS differentiated cardiomyocytes were purified, TIA1 was overexpressed in human cardiomyocytes by adenovirus infection, and the cells positive for pH3 and Edu among the cells positive for the cardiomyocyte-specific marker α-actinin were analyzed by immunofluorescence staining. The specific steps were as follows: iPSC-CMs were differentiated and cultured for 25 days, and adenovirus (ordered from Weizhen Bio) was added at an MOI of 50 for 12 hours. After replacing the myocardial culture medium, the cells were cultured for 72 hours. The cells were fixed by incubation with 4% paraformaldehyde for 10 minutes, permeabilized by incubation with 0.5% Triton for 15 minutes, blocked by incubation with 5% donkey serum for 1 hour, incubated overnight with primary antibodies: mouse monoclonal antibody a-actinin (Abcam) and rabbit monoclonal antibody pH3 (Abcam), incubated with secondary antibodies: donkey anti-mouse AF-647 (Thermo) and donkey anti-rabbit AF-555 (Thermo) for 1 hour, and sealed after incubation with DAPI for 10 minutes. The cells were observed and photographed using a Zeiss confocal microscope 980. Edu analysis After incubation with adenovirus for 48 hours, Edu was added to the culture medium and cultured for another 24 hours. After fixing the cells with 4% paraformaldehyde, staining was performed according to the instructions of the Edu kit (Raybo), and observation and photography were performed using a Zeiss confocal microscope 980.

[0032] like Figure 8 Shown is the change in the proportion of pH3-positive cells after overexpression of TIA1 in human myocardium. Fig. 9 The figure shows the change in the proportion of Edu-positive cells after overexpression of TIA1 in human myocardium. Figure 8 and Fig. 9It can be seen that confocal analysis showed that TIA1 overexpression promoted the increase in the proportion of pH3 and Edu positive cells, among which the increase in the proportion of Edu positive cells was higher than that of pH3. The above data preliminarily suggested that TIA1 can promote the proliferation of cardiomyocytes in multiple genera.

[0033] Example 6 In vitro preliminary experiments have shown that TIA1 can promote cardiomyocyte proliferation. This example further uses newborn mice to explore the effect of TAI1 on cardiomyocyte proliferation in vivo. Using AAV9 containing the cardiomyocyte-specific promoter Tnnt2, 1×10 11 AAV9-Tnnt2-TIA1, 14 days later, the mice were euthanized, and the mouse heart tissues were collected to detect the changes in the size and weight of the mouse heart, and the expression of TIA1 protein was analyzed by immunoblotting according to the same steps as in Example 2.

[0034] like Fig.10 Shown is a flow chart of the in vivo physiological model for neonatal mice. Fig.11 This is a graph showing the expression of myocardial TIA1 in newborn mice after injection of AAV9. Fig.12 Figure 2 is a graph showing changes in heart morphology and heart-body ratio. Figure 10~Figure 12 It can be seen that TIA1 protein was overexpressed in the heart as determined by qPCR and immunoblotting, and there was no significant increase in cardiac morphology and cardiac body ratio.

[0035] This example also detects the proportion of proliferation markers pH3, ki67 and Edu positive cells in cardiomyocytes by immunofluorescence staining, and the specific steps are as follows: After euthanasia, the heart was dissected and collected. After embedding in OCT, 5-8 µm heart tissue sections were cut by freezing microtome. The sections were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.5% Triton for 15 minutes, blocked with 5% donkey serum for 1 hour, incubated overnight with primary antibody: mouse monoclonal antibody a-actinin (Abcam) and rabbit polyclonal antibody pH3 (Abcam) or rabbit monoclonal antibody ki67 (CST), incubated with secondary antibody: donkey anti-mouse AF-647 (Thermo) and donkey anti-rabbit AF-555 (Thermo) for 1 hour, and sealed with DAPI for 10 minutes. The sections were observed and photographed using Zeiss confocal microscope 980. Edu analysis After the newborn mice were injected with adeno-associated virus, Edu was injected for 5 consecutive days on the second day. After the heart tissue was sliced, it was stained according to the instructions of the Edu kit (Raybo) and observed and photographed using Zeiss confocal microscope 980.

[0036] like Fig.13 Shown is the change in the proportion of ki67 and Edu positive cells overexpressing TIA1 in the myocardium of newborn mice. Fig.14The figure shows the change in the proportion of pH3-positive cells in the myocardium of neonatal mice with overexpression of TIA1. Fig.13 and Fig.14 It can be seen that the proportion of Edu-positive cells in the heart increased significantly, and the proportion of pH3-positive cells also increased significantly. At the same time, some myocardial sarcomeres were observed to be disordered, which is a characteristic of myocardial cell division and proliferation. The above results suggest that TIA1 can promote myocardial cell proliferation in vivo.

[0037] Example 7 Experiments on newborn mice have shown that TIA1 can promote cardiomyocyte proliferation. This example further uses adult mice to explore the effect of TAI1 on cardiomyocyte proliferation in vivo. The adeno-associated virus (AAV9-Tnnt2-TIA1) packaging was commissioned to be synthesized by Weigen Biotech. The specific steps are as follows: Using AAV9 containing the myocardium-specific promoter Tnnt2, 3×10 13 AAV9-Tnnt2-TIA1, 28 days later, the mice were euthanized, and the mouse heart tissues were collected to detect the changes in the size and weight of the mouse heart, and the expression of TIA1 protein was analyzed by immunoblotting according to the same steps as in Example 2.

[0038] like Fig.15 Flowchart of the in vivo physiological model for adult mice. Fig.16 This is the result of Western blotting to detect TIA1 expression in heart tissue 3 weeks after adeno-associated virus injection. Fig.17 This is a picture of a mouse heart 4 weeks after adeno-associated virus injection. Fig.18 This is the result of Edu staining of mouse heart slices. Figure 15~Figure 18 It can be seen that TIA1 protein was overexpressed in the heart as determined by qPCR and immunoblotting, and there was no significant increase in cardiac morphology and cardiac body ratio. Immunofluorescence staining was used to detect the proportion of cells positive for proliferation markers pH3 and Edu in cardiomyocytes ( Fig.18 The results showed that the proportion of Edu-positive cells in the heart increased significantly, and the proportion of pH3-positive cells also increased significantly. At the same time, some myocardial sarcomeres were observed to be disordered, which is a sign of myocardial cell division and proliferation ( Fig.18 ). The above results suggest that TIA1 can promote cardiomyocyte proliferation in vivo.

[0039] Example 8 Experiments on newborn mice and adult mice have shown that TIA1 can promote cardiomyocyte proliferation in normal physiological mice. This example uses a mouse model of myocardial infarction to explore whether TAI1 can promote cardiomyocyte proliferation to repair damaged hearts. Using AAV9 containing the cardiomyocyte-specific promoter Tnnt2, 6-8 week-old C57BL / 6 mice were injected with 3×10 13 AAV9-Tnnt2-TIA1, 7 days later, myocardial infarction surgery was performed. 28 days after the surgery, the heart function of the mice was examined by cardiac ultrasound, and then the mice were euthanized and the heart tissues were collected.

[0040] like Fig.19 Shown is the flow chart of adeno-associated virus injection and myocardial infarction experiment in adult mice. Fig. 20 This is the result of ultrasound examination of cardiac function in mice after myocardial infarction. Fig.21 H&E staining of mouse heart sections to examine scar tissue area. Fig. 22 This is the result of Edu staining of mouse heart slices. The results of ultrasound detection of mouse heart function showed that overexpression of myocardial TIA1 can promote the recovery of mouse heart function after myocardial infarction ( Fig. 20 H&E staining showed that the scar area at the myocardial infarction site was significantly reduced in mice with TIA1 overexpression ( Fig.21 ). By immunofluorescence staining, the proportion of Edu-positive cells in cardiomyocytes was detected, confirming that cardiomyocytes overexpressing TIA1 proliferated during the development of myocardial infarction. ( Fig. 22 ).

[0041] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A method for targeted expression of TIA1 gene in hypoxic areas, characterized in that: It comprises 9-times repeated HRE and TIA1 gene, wherein the nucleotide sequence of the 9-times repeated HRE is shown in SEQ ID NO: 1, and the TIA1 gene is human NM_022037.

4.

2. Use of the hypoxic zone targeted expression of TIA1 gene according to claim 1 in the preparation of a drug for promoting myocardial regeneration.

3. Use of the hypoxic zone targeted expression of TIA1 gene according to claim 1 in the preparation of a drug for repairing a damaged heart.

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