Fn1 polypeptide, transcript and application of Fn1 polypeptide and transcript in preparation of medicine for treating cardio-cerebral injury and / or cardiovascular and cerebrovascular diseases

Abstract

The invention discloses an Fn1 polypeptide, a transcript and application of the Fn1 polypeptide and the transcript in preparation of drugs for treating cardio-cerebral injury and / or cardiovascular and cerebrovascular diseases. The Fn1 polypeptide provided by the invention is shown as a sequence 3 in a sequence table. The Fn1-new-112 transcript provided by the invention is shown as a sequence 1 in a sequence table. The invention also protects a DNA molecule for coding the polypeptide or the RNA molecule. The invention also protects a biological material which is an expression cassette, a recombinant vector, a transgenic microorganism or a transgenic cell line with the DNA molecule. The polypeptide, the RNA molecule, the DNA molecule or the biological material can be used for preparing a medicine; the indications of the medicine are heart injury and / or brain injury and / or cardiovascular diseases and / or cerebrovascular diseases. The invention has great application and popularization value for the treatment of heart-related diseases and / or brain-related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to Fn1 polypeptides, transcripts and applications thereof in the preparation of drugs for treating cardiocerebral injury and / or cardiovascular and cerebrovascular diseases. Background Art

[0002] Cardiovascular and cerebrovascular diseases are a general term for cardiovascular and cerebrovascular diseases, and generally refer to ischemic or hemorrhagic diseases of the heart, brain and systemic tissues caused by hyperlipidemia, blood viscosity, atherosclerosis, hypertension, etc. Cardiovascular and cerebrovascular diseases are common diseases that seriously threaten the health of humans, especially those over 50 years old, and are characterized by high morbidity, high disability and high mortality.

[0003] Myocardial infarction (MI) is commonly known as a "heart attack". Pathologically, it is defined as damage or necrosis of myocardial cells due to obstructed blood flow. The most common symptom of MI is angina pectoris, followed by upper abdominal pain, chest tightness, numbness of the upper limbs, dizziness, palpitations, shortness of breath, irritability, etc.

[0004] Myocardial ischemia, as a pathophysiological state, is an inadequate blood and oxygen supply to the heart caused by coronary artery stenosis, spasm or embolism. The clinical symptoms of myocardial ischemia are discomfort and pain in the precordial area. Severe myocardial ischemia may be accompanied by arrhythmias and heart failure, leading to various accompanying symptoms. Isoproterenol (ISO) is a β-adrenergic receptor agonist that can directly or indirectly act on myocardial tissue through inflammatory response, oxidative stress, autophagy and apoptosis, thereby causing myocardial ischemic damage, myocardial hypertrophy and myocardial fibrosis and other symptoms.

[0005] Stroke, commonly known as stroke, is divided into two types: ischemic stroke and hemorrhagic stroke. It is a disease caused by various reasons that lead to damage to cerebral blood vessels, resulting in focal or overall brain tissue damage. Summary of the invention

[0006] The purpose of the present invention is to provide Fn1 polypeptides, transcripts and their use in preparing drugs for treating cardiocerebral injury and / or cardiovascular and cerebrovascular diseases.

[0007] The present invention provides a polypeptide, named Fn1 polypeptide, as shown in Sequence 3 of the sequence listing.

[0008] The present invention also protects polypeptides having more than 90% identity with the Fn1 polypeptide.

[0009] The present invention also protects the polypeptide after the Fn1 polypeptide is modified. Specifically, the modification is a modification of the main chain structure and / or a modification of the side chain group. Exemplarily, the modification can be a C-terminal modification (amidation, sulfation, etc.), an N-terminal modification (acetylation, fatty acidization, etc.), an intermediate residue modification (glycosylation modification binding to Ser-, Tyr-, Asn-, Thr-; phosphorylation modification binding to Ser-, Tyr-, Thr-, etc.), a cyclization modification, an acylation modification (myristoylation, palmitoylation, etc.), a polyethylene glycol modification, a transmembrane peptide modification, a biotinylation modification, a fluorescent group modification, etc.

[0010] The present invention also provides an RNA molecule named Fn1-new-112 transcript, such as sequence 1 in the sequence listing. Place Show.

[0011] The present invention also protects RNA molecules having more than 90% identity with the Fn1-new-112 transcript.

[0012] The present invention also protects a DNA molecule encoding any of the above polypeptides.

[0013] The present invention also protects a DNA molecule encoding any of the above-mentioned RNA molecules.

[0014] Specifically, the DNA molecule is shown as Sequence 2 in the sequence listing.

[0015] The present invention also protects biological materials, which are expression cassettes, recombinant vectors, transgenic microorganisms or transgenic cell lines having any of the above-mentioned DNA molecules.

[0016] The present invention also protects the use of any of the above polypeptides, any of the above RNA molecules, any of the above DNA molecules, or any of the above biological materials in the preparation of drugs; the indications of the drugs are heart damage and / or brain damage; the drugs are used to treat and / or prevent the indications.

[0017] The heart damage includes but is not limited to heart damage caused by cardiovascular disease. For example, the heart damage may be heart damage caused by myocardial infarction, heart damage caused by myocardial ischemia, heart damage caused by myocardial hypertrophy, etc. For example, the heart damage may be manifested as heart failure, myocardial fibrosis, myocardial cell apoptosis, etc.

[0018] The brain injury includes, but is not limited to, brain injury caused by cerebrovascular disease. Exemplarily, the brain injury may be brain injury caused by stroke. Exemplarily, the stroke may be ischemic stroke or hemorrhagic stroke.

[0019] When the heart damage is caused by myocardial infarction, the therapeutic effect of the drug is reflected in improving heart failure after myocardial infarction. When the heart damage is caused by myocardial infarction, the therapeutic effect of the drug is reflected in inhibiting the increase of EDD caused by myocardial infarction and / or inhibiting the increase of ESD caused by myocardial infarction and / or inhibiting the decrease of FS caused by myocardial infarction. When the heart damage is caused by myocardial infarction, the therapeutic effect of the drug is reflected in inhibiting myocardial fibrosis after myocardial infarction. When the heart damage is caused by myocardial infarction, the therapeutic effect of the drug is reflected in inhibiting myocardial cell apoptosis after myocardial infarction. When the heart damage is caused by myocardial infarction, the therapeutic effect of the drug is reflected in promoting cardiac angiogenesis after myocardial infarction.

[0020] When the heart injury is caused by myocardial ischemia, the therapeutic effect of the drug is manifested in inhibiting myocardial hypertrophy caused by myocardial ischemia. When the heart injury is caused by myocardial ischemia, the therapeutic effect of the drug is manifested in inhibiting the increase of EDD caused by myocardial ischemia and / or inhibiting the increase of ESD caused by myocardial ischemia and / or inhibiting the decrease of FS caused by myocardial ischemia. When the heart injury is caused by myocardial ischemia, the therapeutic effect of the drug is manifested in inhibiting myocardial fibrosis caused by myocardial ischemia. When the heart injury is caused by myocardial ischemia, the therapeutic effect of the drug is manifested in inhibiting myocardial cell apoptosis after myocardial ischemia. When the heart injury is caused by myocardial ischemia, the therapeutic effect of the drug is manifested in promoting cardiac angiogenesis after myocardial ischemia.

[0021] When the brain injury is caused by ischemic stroke, the therapeutic effect of the drug is reflected in reducing the infarct area. When the brain injury is caused by ischemic stroke, the therapeutic effect of the drug is reflected in increasing the cerebral blood perfusion. When the brain injury is caused by ischemic stroke, the therapeutic effect of the drug is reflected in promoting cerebral angiogenesis after ischemic stroke.

[0022] The present invention also protects any of the above polypeptides or any of the above RNA molecules or any of the above DNA Use of a molecule or any of the above-mentioned biomaterials in the preparation of a drug; the drug is indicated for cardiovascular disease and / or cerebrovascular disease; the drug is used to treat and / or prevent the indicated disease.

[0023] Exemplarily, the cardiovascular disease may be coronary heart disease, myocardial infarction, myocardial hypertrophy, hypertension, cardiomyopathy, myocarditis, angina pectoris, coronary atherosclerosis, valvular heart disease, ischemic heart disease, congenital heart disease, rheumatic heart disease, heart failure, arrhythmia, endocarditis, deep vein thrombosis and pulmonary embolism, heart function decline caused by aging, etc.

[0024] Exemplarily, the cerebrovascular disease may be stroke.

[0025] Exemplarily, the stroke may be ischemic stroke or hemorrhagic stroke.

[0026] The present invention also protects the use of any of the above polypeptides, or any of the above RNA molecules, or any of the above DNA molecules, or any of the above biological materials in the preparation of drugs; the uses of the drugs are as follows (a) and / or (b) and / or (c) and / or (d) and / or (e) and / or (f) and / or (g) and / or (h) and / or (i) and / or (j) and / or (k):

[0027] (a) Used to improve heart failure;

[0028] (b) for inhibiting myocardial fibrosis;

[0029] (c) for inhibiting cardiomyocyte apoptosis;

[0030] (d) for improving impaired heart function;

[0031] (e) used to improve the extent of cerebral infarction;

[0032] (f) used to increase cerebral blood perfusion;

[0033] (g) for improving impaired brain function;

[0034] (h) for promoting endothelial cells to form blood vessels;

[0035] (i) for promoting endothelial cell migration;

[0036] (j) for promoting angiogenesis in the heart and / or brain;

[0037] (k) Used to improve cell hypoxic damage.

[0038] Specifically, the cells may be myoblasts or cardiomyocytes.

[0039] Specifically, the endothelial cells may be coronary artery endothelial cells.

[0040] The improvement of cardiac insufficiency is to improve the cardiac function of the patient. The inhibition of myocardial fibrosis is to inhibit myocardial fibrosis of the patient. The inhibition of myocardial cell apoptosis is to inhibit myocardial cell apoptosis of the patient. The improvement of impaired cardiac function is to improve the cardiac function of the patient. The patient is a patient with heart damage and / or a patient with cardiovascular disease.

[0041] The improving degree of cerebral infarction is to improve the degree of cerebral infarction of the patient. The increasing cerebral blood perfusion is to increase the cerebral blood perfusion of the patient. The improving brain function impairment is to improve the brain function of the patient. The patient is a patient with brain injury and / or a patient with cerebrovascular disease.

[0042] The promoting of angiogenesis in the heart and / or brain is promoting angiogenesis in the heart and / or brain of a patient. The patient is a patient with heart damage and / or a patient with cardiovascular disease and / or a patient with brain damage and / or a patient with cerebrovascular disease.

[0043] The heart damage includes but is not limited to heart damage caused by cardiovascular disease. For example, the heart damage may be heart damage caused by myocardial infarction, heart damage caused by myocardial ischemia, heart damage caused by myocardial hypertrophy, etc. For example, the heart damage may be manifested as heart failure, myocardial fibrosis, myocardial cell apoptosis, etc.

[0044] The brain injury includes, but is not limited to, brain injury caused by cerebrovascular disease. Exemplarily, the brain injury may be brain injury caused by stroke. Exemplarily, the stroke may be ischemic stroke or hemorrhagic stroke.

[0045] Exemplarily, the cardiovascular disease may be coronary heart disease, myocardial infarction, myocardial hypertrophy, hypertension, cardiomyopathy, myocarditis, angina pectoris, coronary atherosclerosis, valvular heart disease, ischemic heart disease, congenital heart disease, rheumatic heart disease, heart failure, arrhythmia, endocarditis, deep vein thrombosis and pulmonary embolism, heart function decline caused by aging, etc.

[0046] Exemplarily, the cerebrovascular disease may be stroke.

[0047] Exemplarily, the stroke may be ischemic stroke or hemorrhagic stroke.

[0048] The present invention also protects a drug, which comprises any of the above polypeptides, any of the above RNA molecules, any of the above DNA molecules, or any of the above biological materials.

[0049] The indications of the drug are as described above.

[0050] The use of the drug is as described above.

[0051] The present invention can be used to improve the survival rate of patients with heart damage and / or cardiovascular disease and / or brain damage and / or cerebrovascular disease. The present invention has great application and promotion value for the treatment of heart-related diseases and / or brain-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 These are the results of analyzing new transcripts that were significantly upregulated and downregulated during myocardial infarction by third-generation sequencing in Example 1.

[0053] Figure 2This is the statistical result of the relative expression levels of Fn1-new-112 transcript and Fn1 transcript in step 2 of Example 2.

[0054] Figure 3 The statistical results of the Alamar Blue cell viability assay in step 3 of Example 2 are shown.

[0055] Figure 4 These are photos and statistical results of cell death detection using propidium iodide / calcein in step 3 of Example 2.

[0056] Figure 5 The statistical results of Fn1 polypeptide inhibiting OGD-induced cell damage in Example 4 are shown in FIG.

[0057] Figure 6 These are the results related to the echocardiographic measurements in Example 5.

[0058] Figure 7 This is the result of Masson staining to detect fibrosis in Example 5.

[0059] Figure 8 These are the results of TUNEL cell apoptosis detection in Example 5.

[0060] Fig. 9 These are the results related to the echocardiographic measurements in Example 6.

[0061] Fig.10 This is the result of Masson staining to detect fibrosis in Example 6.

[0062] Fig.11 These are the results related to the detection of cerebral infarction area by tar violet staining in Example 7.

[0063] Fig.12 These are the results related to the detection of cerebral blood perfusion by laser speckle blood flow imaging in Example 7.

[0064] Fig.13 These are the results related to the behavioral experiment in Example 7.

[0065] Fig.14 These are the results related to the angiogenesis of human coronary artery endothelial cells (HCAECs) in Example 8.

[0066] Fig.15 These are the results related to the migration of human coronary artery endothelial cells (HCAECs) in Example 8.

[0067] Fig.16 These are the results related to the Fn1 polypeptide promoting cardiac angiogenesis after MI in Example 8.

[0068] Fig.17These are the results related to the promotion of cerebral angiogenesis by Fn1 polypeptide in Example 8 after tMCAO. DETAILED DESCRIPTION

[0069] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0070] The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels. Unless otherwise specified, the quantitative tests in the following examples are set up with three repeated experiments, and the results are averaged. OGD: oxygen-glucose deprivation. siRNA (small interfering RNA): double-stranded RNA molecule, 20-25bp in length.

[0071] SYRB Green real-time fluorescence quantitative PCR reagent: Vazyme, catalog number Q712. Trizol: Invitrogen, catalog number 15596018. Jetprime transfection reagent: PolyPlus, catalog number 101000046. Alamar blue solution: Solarbio, catalog number A7631. TUNEL fluorescent staining kit: Vazyme, catalog number A112. Fetal bovine serum (FBS): Vivacell, catalog number C04001500. Penicillin-streptomycin (solution): BIOLOGICAL INDUSTRIES, catalog number 03-033-1B. Phosphate buffered saline (PBS, pH 7.4): VivaCell, catalog number C3580-0500. Matrigel: Corning, catalog number 354230. Y27632 (Rho kinase inhibitor): Sellek, catalog number S1049. CHIR99021: Selleck, catalog number S2924. IWR-1: Selleck, catalog number S7086. RPMI medium: Gibco, catalog number 11875093. Sugar-free RPMI medium: Gibco, catalog number 11879020. B27-ins (B-27supplementminus insulin): Invitrogen, catalog number A1895601. TrypLE Express enzyme: Gibco, catalog number 12605010. DMEM high glucose medium: Gibco, catalog number C11995500BT; the glucose content of DMEM high glucose medium is 4500 mg / L. Low glucose DMEM medium: BIOLOGICAL INDUSTRIES, catalog number 01-051-1ACS; the glucose content of low glucose DMEM medium is 1000 mg / L. Sugar-free DMEM medium: Gibco, item number 11966025; the glucose content of sugar-free DMEM medium is 0. Growth factor-reduced Matrigel: Corning, item number 356230. Calcein-AM: Bio-Tech, item number C2012-0.1ml. 24-well 8.0um TRANSWELL plate: Corning, item number 3422. 0.1% crystal violet stain: Solebol, item number G1063.

[0072] Example 1. Discovery of transcript Fn1-new-112

[0073] 1. Prepare full-length transcriptome sequencing libraries using the SQK-PCB109 kit (Oxford Nanopore sequencing technology, third-generation sequencing technology). Specifically, add 2 μM VN primer (VNP) and 10 mM dNTP to the sample to be sequenced and incubate at 65 ° C for 5 minutes. Then cool the sample quickly and add 5xRT buffer, RNaseOUT and 10 μM chain switching primer (SSP) and incubate at 42 ° C for 2 minutes. Then, add 1 μl Maxima H Minus Reverse Transcriptase to reverse transcribe the full-length transcript into cDNA in vitro for further PCR. Finally, the cDNA of four samples under each condition was combined to obtain three mixed samples, which were labeled with barcodes and sequenced in two technical replicates. Sequencing used PromethION (FLO-PRO002) to generate fast5 files, and the sequencing was set to run for 72 hours, or stop early when there were less than 10 active wells. Reads with a quality score (Q score) <7 were filtered out to ensure sequencing quality. At the same time, these samples were subjected to second-generation short-read sequencing.

[0074] 2. A total of 12 samples (3 mixed samples) from 3 groups (MI-Border, MI-Center and MI-Sham) were sequenced, with four samples in each group. During analysis, Fast5 files were called by Guppy (version 5.0.11) to generate fastq files. Pychopper (version 2.5.0) was used to identify full-length sequences from filtered reads, and NanoFilt (version 2.8.0) was used to remove low-quality (Q < 7) and relatively short sequences (L < 500) to ensure the accuracy of new transcripts. The GENCODE M26 (GRCm39) transcript reference and genome reference (primary assembly) with primary annotations were used for sequence alignment and quantification.

[0075] 3. Identification of new transcripts was achieved by alignment and folding with FLAIR (version 1.5) and SQANTI3 (version 4.2). First, the fastq files from nanopore RNA sequencing were merged and aligned to the genome reference by Minimap2 (version 2.21) with the parameter "-ax splice-uf-k14--secondary=no". Secondly, the alignment results were further processed using the given annotation file to correct the adapter sequences by FLAIR to obtain reliable and high-quality transcript isoforms after removing redundant sequences. Finally, SQANTI3 was used to identify the ORF region, polyA tail and other information of the transcript.

[0076] 4. The position information of the identified new transcripts was added to the transcript annotation file obtained from GENCODE M26 by the AGAT suite (version 0.8.1) to obtain updated transcript annotations for transcript quantification.

[0077] 5. For the second-generation short-read data, Hisat2 (version 2.2.1) was used for genome alignment, and Samtools (version 1.7) was used to convert the Sam file to a bam file. Then featureCounts (version 2.0.1) was used to quantify gene expression (parameter "-g gene_id") and transcript expression (parameter "-g transcript_id"). The distribution of the number of transcripts in each group was obtained by analyzing the expression matrix of the short-read data of the new transcripts.

[0078] Differential expression analysis was performed using DESeq2 (version 1.34.0) with the transcript count matrix as input file. The log2 fold change (log2FC) and adjusted p-value were calculated for each annotated transcript, and an adjusted p-value < 0.05 was considered statistically significant.

[0079] 6. Quantitative RT-PCR: Use SYRB Green real-time fluorescence quantitative PCR reagent to perform real-time PCR and Over real time PCR analysis of transcription levels. RNA was isolated from heart tissue using Trizol reagent and synthesized as cDNA. Quantitative RT-PCR conditions were: 37 cycles of 94°C for 10 sec, 57°C for 15 sec, and 72°C for 5 sec.

[0080] The third-generation sequencing technology was used to analyze and identify new transcripts that were significantly upregulated and downregulated during myocardial infarction, and real-time quantitative PCR was used to verify their changes. The results showed the accuracy of the sequencing analysis process for the quantification of new transcripts. Figure 1 .

[0081] Through the above steps, a new transcript was discovered, which was named Fn1-new-112 transcript. Fn1-new-112 transcript was significantly upregulated during myocardial infarction and was closely involved in the process of myocardial infarction.

[0082] The transcript of Fn1-new-112 is shown in Sequence 1 of the Sequence Listing.

[0083] The coding frame of the Fn1-new-112 gene is shown in Sequence 2 of the Sequence Listing.

[0084] Example 2: Specific knockdown of Fn1-new-112 transcripts aggravates OGD-induced cell damage

[0085] 1. Preparation of siRNA

[0086] siRNA targeting Fn1-new-112 transcript and control siRNA were prepared respectively. The siRNA targeting Fn1-new-112 transcript was named siFn1-new-112. The control siRNA was named siNC.

[0087] siFn1-new-112: 5'-GCTGGTCTGCTGTAACATACA-3'.

[0088] siNC: 5'-GCACTTACGAGTACGATCTGT-3'.

[0089] 2. Verify the effectiveness and specificity of siRNA

[0090] 1. Take a 12-well cell culture plate, inoculate C2C12 cells (mouse myoblasts) and add DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, 2×10 5 cells / well and cultured until the cell density reached 50%.

[0091] 2. After completing step 1, use Jetprime transfection reagent to transfect the test siRNA (the test siRNA is siFn1-new-112 or siNC), 150 ng siRNA / well, and culture for 48 hours.

[0092] 3. After completing step 2, collect cells, extract total RNA and reverse transcribe to obtain cDNA. Using cDNA as a template and 18S gene as an internal reference gene, qPCR is used to detect the relative expression levels of Fn1-new-112 transcripts and Fn1 transcripts (since the primer pair used can theoretically detect all Fn1 transcripts known in the prior art, the detection target of the primer pair is called Fn1 transcript).

[0093] The primers used to detect Fn1-new-112 transcripts are as follows:

[0094] Upstream primer: 5′-caggctggtgttctctgagt-3′;

[0095] Downstream primer: 5′-tacctcccaagtcctcctga-3′.

[0096] Primers used to detect Fn1 transcripts are as follows:

[0097] Upstream primer: 5′-cccctcctgatagtgtggtg-3′;

[0098] Downstream primer: 5′-ggacagcccagtgatttcag-3′.

[0099] The primers used to detect the 18S gene are as follows:

[0100] Upstream primer: 5′-GCTTAATTTGACTCAACACGGGA-3′;

[0101] Downstream primer: 5′-AGCTATCAATCTGTCAATCCTGTC-3′.

[0102] Three repeated treatments were set up, and the results were expressed as mean ± standard deviation.

[0103] The statistical results of the relative expression levels of Fn1-new-112 transcripts and Fn1 transcripts are shown in Figure 2 .

[0104] Specific knockdown of Fn1-new-112 transcripts aggravates OGD-induced cell damage

[0105] 1. Take a 96-well cell culture plate, inoculate C2C12 cells and add DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, 2×10 4 cells / well and cultured until the cell density reached 50%.

[0106] 2. After completing step 1, use Jetprime transfection reagent to transfect the test siRNA (the test siRNA is siFn1-new-112 or siNC), 15 ng siRNA / well, and culture for 48 hours.

[0107] 3. After completing step 2, discard the culture supernatant, wash the cells with PBS, then add 100 μl of sugar-free DMEM culture medium containing 1% penicillin-streptomycin to each well, and place in an incubator (the gas composition in the incubator is: 0.1% O2, 5% CO2, 94.9% N2; volume ratio) for culturing at 37°C for 8 hours.

[0108] 4. Alamar Blue Cell Viability Assay

[0109] After completing step 3, discard the culture supernatant, wash the cells with PBS, then add 100 μl of 10% Alamar blue solution to each well, incubate at 37°C for 2 h, and then use the Cytation 5 cell imaging microplate detection system to measure fluorescence changes (excitation / emission wavelengths set to 545 / 590 nm), and the output result is the fluorescence intensity value.

[0110] The fluorescence intensity value of the test well minus the fluorescence intensity value of the background well (i.e., the well with only PBS added) is the fluorescence intensity correction value of the test well.

[0111] The fluorescence intensity correction value of the siNC group was used as reference 1, and the relative value of the siFn1-new-112 group was calculated, which was the relative cell viability.

[0112] Three repeated treatments were set up, and the results were expressed as mean ± standard deviation.

[0113] Results Figure 3 Compared with the siNC group, the cell viability in the siFn1-new-112 group decreased.

[0114] 5. Propidium iodide / calcein detection of cell death

[0115] After completing step 3, discard the culture supernatant, wash the cells with PBS, and then add 100 μl of staining solution (the staining solution is a solution containing 1 μg / μl propidium iodide and 2 μg / μl calcein) to each well, incubate at 37°C for 30 minutes, and then take pictures using the Cytation 5 cell imaging microplate detection system (propidium iodide stains dead cells, the detection channel is 561 / 600nm; calcein stains live cells, the detection channel is 488 / 525nm).

[0116] Five repeated treatments were set up, and the results were expressed as the mean ± standard deviation.

[0117] Results Figure 4 Compared with the siNC group, the number of dead cells in the siFn1-new-112 group increased, while the number of live cells decreased. The results showed that knocking down Fn1-new-112 transcripts reduced cell viability and cell survival under OGD conditions.

[0118] Example 3: Preparation of Fn1 polypeptide

[0119] The Fn1 polypeptide is shown in Sequence 3 of the Sequence Listing.

[0120] Sequence 3: MDRLVFSEYFLLVCCNIHSFASVGSTVRGLCPG.

[0121] The Fn1 peptide was artificially synthesized by Shanghai Nuoyou Biotechnology Co., Ltd.

[0122] Fn1 polypeptide solution: Fn1 polypeptide was dissolved in PBS to make the concentration of Fn1 polypeptide be 5 μM.

[0123] The Fn1 polypeptide solution was used in Example 4, Example 5, Example 6, Example 7 and Example 8.

[0124] Example 4: Fn1 polypeptide inhibits OGD-induced cell damage

[0125] 1. Fn1 peptide inhibits OGD-induced C2C12 cell injury

[0126] 1. Take a 96-well cell culture plate, inoculate C2C12 cells and add DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, 2×104 cells / well and then cultured until the cell density reached 80%-90%.

[0127] 2. Group processing

[0128] MIB-PBS group: After completing step 1, the culture supernatant was discarded, and 100 μl of low-glucose DMEM medium containing 4% fetal bovine serum and 1% penicillin-streptomycin and 10 μl of PBS were added to each well, and the cells were placed in an incubator (the gas composition in the incubator was: 4% O2, 5% CO2, 91% N2; volume ratio) and cultured at 37°C for 24 h.

[0129] MIB-Fn1 polypeptide group: After completing step 1, the culture supernatant was discarded, 100 μl of low-glucose DMEM medium containing 4% fetal bovine serum and 1% penicillin-streptomycin and 10 μl of Fn1 polypeptide solution were added to each well, and the cells were placed in an incubator (the gas composition in the incubator was: 4% O2, 5% CO2, 91% N2; volume ratio) and cultured at 37°C for 24 h.

[0130] MIC-PBS group: After completing step 1, the culture supernatant was discarded, 100 μl of sugar-free DMEM medium containing 1% penicillin-streptomycin and 10 μl of PBS were added to each well, and the cells were cultured at 37°C in an incubator (the gas composition in the incubator was: 0.1% O2, 5% CO2, 94.9% N2; volume ratio) for 8 h.

[0131] MIC-Fn1 polypeptide group: After completing step 1, the culture supernatant was discarded, 100 μl of sugar-free DMEM medium containing 1% penicillin-streptomycin and 10 μl of Fn1 polypeptide solution were added to each well, and the cells were cultured at 37°C in an incubator (the gas composition in the incubator was: 0.1% O2, 5% CO2, 94.9% N2; volume ratio) for 8 h.

[0132] Normoxia-PBS group: After completing step 1, the culture supernatant was discarded, and 100 μl of DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin and 10 μl of PBS were added to each well, and the cells were placed in an incubator (the gas composition in the incubator was: 5% CO2, 95% air; volume ratio) and cultured at 37°C for 24 h.

[0133] Normoxia-Fn1 polypeptide group: After completing step 1, discard the culture supernatant, add 100 μl of DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin and 10 μl of Fn1 polypeptide solution to each well, and place in an incubator (the gas composition in the incubator is: 5% CO2, 95% air; volume ratio) at 37°C for 24 hours.

[0134] Three replicate treatments were set up for each group.

[0135] 3. Cell Viability Assay

[0136] The method is the same as step 4 of step 3 in Example 2.

[0137] 30-50 replicates were set up and the results were expressed as mean ± standard deviation.

[0138] Results Figure 5 The left figure (PBS represents the PBS group, and F peptide represents the Fn1 peptide group). Fn1 peptide can inhibit OGD-induced mouse myoblast injury and has a protective effect on cell hypoxia injury.

[0139] 2. Fn1 peptide inhibits OGD-induced NRVM cell injury

[0140] Method for obtaining NRVM cells (primary rat cardiomyocytes): ① Take the heart of a newborn SD rat, wash it with HBSS solution, cut the heart open and drain the blood in the visceral cavity, then cut it into pieces and add it to a centrifuge tube containing digestion solution, stir it in a 37°C water bath for 5 minutes, and discard the supernatant blood cells; ② Add the digestion solution to the precipitate in step ① again, stir it in a 37°C water bath for 5 minutes, collect the supernatant, add the supernatant to a centrifuge tube containing DMEM high-glucose culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, centrifuge it twice, discard the supernatant, and then add Resuspend the precipitate with new DMEM culture medium to obtain the cell suspension; ③ Repeat step ② for digestion. When no cell precipitate is observed in the centrifuge tube and the digestion solution is visibly clear, the digestion is terminated; ④ Filter the cell suspension obtained above with a sieve, rinse the cells with DMEM culture medium, and then adjust the volume of the cells to a culture dish, and place it in a 37°C incubator for differential adhesion for 1 hour; ⑤ After the cells adhere, aspirate the supernatant into a volumetric flask, repeatedly blow with DMEM culture medium, add 5-bromopropene to inhibit fibroblast differentiation, and finally inoculate the cells in a culture dish for constant temperature culture at 37°C.

[0141] NRVM cells were used instead of C2C12 cells, and the rest was the same as step 1.

[0142] 10-20 replicates were set up and the results were expressed as mean ± standard deviation.

[0143] Results Figure 5 The middle figure (PBS represents the PBS group, and F peptide represents the Fn1 peptide group). Fn1 peptide can inhibit OGD-induced rat cardiomyocyte injury and has a protective effect on cell hypoxia injury.

[0144] 3. Fn1 peptide inhibits OGD-induced hiPSC-CMs cell injury

[0145] Method for obtaining hiPSC-CMs cells (cardiomyocytes differentiated from human induced pluripotent stem cells): ① hiPSC (human induced pluripotent stem cells, obtained from the National Stem Cell Resource Bank, sample number Q-ips-6P2220230206002) was inoculated in a Matrigel-coated 6-well plate and cultured until the cell density reached 75%-95%; ② After completing step ①, the culture supernatant was aspirated and 3 ml of RPMI medium containing 4 μM CHIR99021 and 1x B27-ins was added to each well, and cultured for 2 days; ③ After completing step ②, the supernatant was aspirated and 3 ml of RPMI medium containing 1x B27-ins was added to each well, and cultured for 1 day; ④ After completing step ③, the supernatant was aspirated and 3 ml of RPMI medium containing 5 μM IWR-1 and 1xB27-inss was added to each well, and cultured for 2 days; ⑤ After completing step ④, the supernatant was aspirated and 3 ml of 1x B27-ins were cultured in RPMI medium for 8 days (new medium was replaced every 48 h); ⑥ After completing step ⑤, the cells were collected, digested with TrypLE Express enzyme, and resuspended in RPMI medium containing 1x B27-ins and the cell concentration was adjusted to 1×10 6 / ml, add Y27632 and make it into a concentration of 10μM, then inoculate into a 24-well plate, 750000 cells / well, and culture for 24 hours; ⑦ After completing step ⑥, replace with sugar-free RPMI medium containing 1x B27-ins and culture for 48 hours for glucose starvation purification; ⑧ After completing step ⑦, replace with RPMI medium containing 1x B27-ins and continue to culture for 4 days, changing the medium every 48 hours to obtain hiPSC-CMs cells.

[0146] 1. Take a 96-well cell culture plate, inoculate hiPSC-CMs cells and add DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin to make the cell content 2.5×10 4 / well and then cultured for 48 hours.

[0147] 2. Same as step 1, step 2.

[0148] 3. Same as step 1, step 3.

[0149] 6-10 replicates were set up and the results were expressed as mean ± standard deviation.

[0150] Results Figure 5 The right figure (PBS represents the PBS group, and F peptide represents the Fn1 peptide group). Fn1 peptide can inhibit OGD-induced human cardiomyocyte injury and has a protective effect on cell hypoxic injury.

[0151] Example 5: Fn1 polypeptide improves cardiac function after myocardial infarction

[0152] Experimental animals: Male C57BL / 6J mice aged 8-10 weeks.

[0153] 1. Construction of mouse acute myocardial infarction model (MI model)

[0154] Modeling method of MI model: The MI model was constructed by permanently blocking the left anterior descending coronary artery (LAD). The specific method of constructing the MI model: the experimental animals were anesthetized by inhaling 2% isoflurane, and a 1.2 cm incision was made on the left chest to expose the fourth intercostal space; a small hole was made in the fourth intercostal space, the heart was squeezed out, a silk thread was placed under the course of the left coronary artery, and the left anterior descending coronary artery (LAD) was ligated; when the anterior wall of the left ventricle turned white, it indicated that the ligation was successful, and the heart was immediately returned to the chest cavity, the air was evacuated, and the muscles and skin were sutured.

[0155] Treatment method of the sham operation group: LAD was not blocked, and other modeling methods were the same as those for constructing the MI model.

[0156] 2. Grouped medication

[0157] Fn1 peptide group: The experimental animals were subjected to MI model establishment according to the method in step 1. Five minutes after MI surgery, each mouse was injected with Fn1 peptide solution through the tail vein. The injection dose of Fn1 peptide was 2.5 mg / (kg·bw);

[0158] PBS control group (PBS): The experimental animals were subjected to MI model establishment according to the method in step 1. 5 minutes after MI surgery, each mouse was injected with PBS via the tail vein. The volume of PBS injected was the same as that of the Fn1 polypeptide solution injected in the Fn1 polypeptide group.

[0159] Sham operation group (Sham): The experimental animals were subjected to sham operation according to the method in step 1. 5 minutes after the sham operation, PBS was injected into the tail vein of each mouse. The volume of PBS injected was the same as the volume of Fn1 polypeptide solution injected into the Fn1 polypeptide group.

[0160] 3. Index detection

[0161] 1. Echocardiography

[0162] Echocardiographic measurements were performed on day 7 after MI surgery or on day 7 after sham surgery to evaluate cardiac function. Each mouse was measured at least three times each time, and the mice were anesthetized with 1.5% isoflurane.

[0163] Exemplary echocardiograms are shown in Figure 6 Left figure. The statistical results of ventricular end-diastolic diameter (EDD), ventricular end-systolic diameter (ESD) and left ventricular shortening fraction (FS) are shown in Figure 6The right figure shows that 5-10 mice were counted in each group. The results showed that compared with the PBS control group, the ventricular end-diastolic diameter (EDD) and ventricular end-systolic diameter (ESD) of the Fn1 polypeptide test group mice were significantly reduced, and the left ventricular shortening fraction (FS) was increased. The results showed that Fn1 peptide can improve heart function after myocardial infarction. Dirty function.

[0164] 2. Masson staining to detect fibrosis

[0165] On the 7th day after MI surgery or sham surgery, mice were killed, hearts were dissected and obtained, hearts were fixed with 4% paraformaldehyde and embedded in paraffin, cross-sectioned and stained with Masson, and then observed and recorded the degree of fibrosis under an optical microscope.

[0166] Exemplary photographs of Masson staining are shown in Figure 7 The statistical results are shown in the left figure of Figure 7 The right figure (4 mice in each group). Compared with the PBS control group, the myocardial fibrosis area of ​​mice in the Fn1 polypeptide group was significantly reduced. The results showed that Fn1 polypeptide can reduce the degree of myocardial fibrosis after myocardial infarction and improve heart damage after myocardial infarction.

[0167] 3. TUNEL cell apoptosis detection

[0168] On the third day after MI surgery or sham surgery, mice were killed, dissected and hearts were obtained. The hearts were fixed with 4% paraformaldehyde and embedded in paraffin, cross-sectioned and stained with TUNEL fluorescent staining kit, and then placed under Dragonfly high-speed confocal microscopy imaging system to observe TUNEL fluorescence at 505 nm.

[0169] See the sample photos Figure 8 The statistical results of the percentage of TUNEL-positive cells (%) are shown in the left figure (IZ represents ischemic zone, BZ represents border zone). Figure 8 The right figure (4 mice in each group). The results showed that compared with the PBS group, the number of TUNEL-positive cardiomyocytes around the border zone in the Fn1 polypeptide group was significantly reduced, and the apoptosis of cells in the lesion site was reduced after Fn1 polypeptide treatment. This shows that Fn1 polypeptide can reduce cardiomyocyte apoptosis after myocardial infarction.

[0170] Example 6: Fn1 polypeptide improves ISO-induced myocardial hypertrophy

[0171] Experimental animals: 8-10 week old male C57BL / 6J mice.

[0172] Isoproterenol (ISO) was used to induce myocardial ischemia symptoms (including myocardial hypertrophy and myocardial fibrosis) in mice, and an ISO-induced myocardial hypertrophy model in mice was established.

[0173] 1. Group processing

[0174] Fn1 peptide group (expressed as Fn1 peptide or ISO+Fn1): From the 1st day to the 9th day of the experiment, the mice were injected with Fn1 peptide solution through the tail vein every day, and the single injection dose of Fn1 peptide was 2.5 mg / (kg·bw); from the 3rd day to the 9th day of the experiment, the mice were injected with ISO subcutaneously behind the neck every day, and the single injection dose of ISO was 5 mg / (kg·bw).

[0175] PBS control group (expressed as PBS or ISO+PBS): From the 1st day to the 9th day of the experiment, the mice were injected with PBS through the tail vein every day, and the volume of the injected PBS was the same as the volume of the Fn1 polypeptide solution injected into the Fn1 polypeptide group; from the 3rd day to the 9th day of the experiment, the mice were injected with ISO subcutaneously behind the neck every day, and the single injection dose of ISO was 5 mg / (kg·bw).

[0176] Ctrl control group (denoted by Control): From the 1st day to the 9th day of the experiment, the mice were injected with PBS via the tail vein every day. The volume of the injected PBS was the same as the volume of the Fn1 polypeptide solution injected into the Fn1 polypeptide group.

[0177] 2. Index detection

[0178] 1. Echocardiography

[0179] On day 16 of the experiment, echocardiography was performed to evaluate cardiac function. Each mouse was measured at least three times each time. During this period, mice were maintained under anesthesia using 1.5% isoflurane.

[0180] Echocardiography Fig. 9 The statistical results of ventricular end-diastolic diameter (EDD), ventricular end-systolic diameter (ESD) and left ventricular fractional shortening (FS) are shown in Fig. 9 The right figure shows that there are about 8-10 mice in each group. The results show that compared with the PBS control group, the ventricular end-diastolic diameter (EDD) and ventricular end-systolic diameter (ESD) of the mice in the Fn1 polypeptide group were significantly reduced, and the left ventricular shortening fraction (FS) was increased. The results show that Fn1 polypeptide can improve the impaired cardiac function of mice induced by isoproterenol (ISO).

[0181] 2. Masson staining to detect fibrosis

[0182] On the 16th day of the experiment, the mice were killed, and the hearts were obtained by dissection. The hearts were fixed with 4% paraformaldehyde and embedded in paraffin, and then cross-sectioned and stained with Masson staining, and then observed and recorded the degree of fibrosis under an optical microscope.

[0183] Masson staining results are shown in Fig.10The statistical results are shown in the left figure of Fig.10 The right figure (7 mice in each group). The results showed that compared with the PBS control group, the area of ​​myocardial fibrosis in the Fn1 polypeptide group was significantly reduced, indicating that the Fn1 polypeptide can significantly reduce the degree of ISO-induced myocardial fibrosis and improve the heart damage caused by myocardial hypertrophy.

[0184] Example 7: Fn1 polypeptide improves brain function after stroke

[0185] Experimental animals: Male C57BL / 6J mice aged 8-10 weeks.

[0186] 1. Construction of transient middle cerebral artery occlusion model (tMCAO model) in mice

[0187] Modeling method of tMCAO model: ① Use 3% sodium pentobarbital to anesthetize the test animals, prepare the neck skin, disinfect, insert a rectal temperature probe, and maintain the body temperature at 37±0.5℃. ② Make a midline incision in the neck to expose the right common carotid artery, internal carotid artery and external carotid artery. ③ Use 7-0 silk thread to ligate the distal end of the external carotid artery 2mm away from the bifurcation of the common carotid artery, insert another 7-0 silk thread into the external carotid artery, and tie a slipknot near the bifurcation of the common carotid artery. ④ Use an artery clamp to clamp the common carotid artery, and cut a small incision on the external carotid artery 1.5mm away from the bifurcation of the common carotid artery. A 0.18mm diameter nylon with a processed head The thread is inserted from a small opening into the internal carotid artery and then into the middle cerebral artery. The insertion depth of the nylon thread is The bifurcation is about 9±1mm ⑤ Remove the suture plug, ligate the proximal end of the external artery with 7-0 silk thread, suture the neck wound with 5-0 silk thread, disinfect the wound with iodine, place the mouse on a heating pad, and place it in a constant temperature incubator after it wakes up.

[0188] Sham operation method: do not perform the underlined steps, and the rest are the same as the modeling method of tMCAO model.

[0189] 2. Grouped medication

[0190] Fn1 polypeptide group (IS-FN1P): tMCAO model was established according to the method in step 1; 6 hours after surgery, Fn1 polypeptide solution was injected into the tail vein, and the injection dose of Fn1 polypeptide was 2.5 mg / (kg·bw); Fn1 polypeptide solution was injected into the tail vein on the 3rd, 6th, 9th and 12th days after surgery, and the single injection dose of Fn1 polypeptide was 2.5 mg / (kg·bw);

[0191] PBS control group (IS-PBS): The tMCAO model was established according to the method in step 1; 6 hours after the operation, PBS was injected into the tail vein, and the volume of PBS injected was the same as that of Fn1 polypeptide solution injected into the Fn1 polypeptide group; On the 3rd day after surgery, On days 6, 9, and 12, PBS was injected into the tail vein. The volume of PBS injected wasThe volume of Fn1 polypeptide solution injected was the same as that of the Fn1 polypeptide group;

[0192] Sham operation group (Sham): Sham operation was performed according to the method in step 1; 6 hours after the sham operation, PBS was injected into the tail vein of each mouse; 6 hours after the operation, PBS was injected into the tail vein, and the volume of PBS injected was the same as the volume of Fn1 polypeptide solution injected into the Fn1 polypeptide group; on the 3rd day, 6th day, 9th day and 12th day after the operation, PBS was injected into the tail vein, and the volume of PBS injected was the same as the volume of Fn1 polypeptide solution injected into the Fn1 polypeptide group.

[0193] 3. Index detection

[0194] 1. Tar violet staining to detect cerebral infarction area

[0195] 14 days after tMCAO surgery or 14 days after sham surgery, the mice were killed, dissected and the brains were obtained. The brains were fixed with 4% paraformaldehyde, routinely dehydrated and paraffin-embedded, cross-sectioned and stained with tar violet, and then the degree of cerebral infarction in the mice was observed under a light microscope.

[0196] The results of tar violet staining are shown in Fig.11 The statistical results are shown in the left figure of Fig.11 In the right figure, 5 mice were counted in each group. Compared with the PBS control group, the cerebral infarction area of ​​the mice in the Fn1 polypeptide group was significantly reduced, and the results showed that Fn1 polypeptide can reduce the degree of cerebral infarction after middle cerebral artery embolism.

[0197] 2. Laser speckle blood flow imaging

[0198] Laser speckle images were taken during and 14 days after tMCAO or during and 14 days after sham surgery.

[0199] Laser speckle imaging Fig.12 Figure 1 (baseline represents speckle detection before modeling; tMCAO represents the step ④ of the modeling method in which the plug is inserted and speckle detection is performed 10 minutes after ischemia; reperfusion represents the step ⑤ of the modeling method in which the plug is removed and speckle detection is performed 10 minutes later; 14d represents the speckle detection performed 14 days after surgery). 14 days after surgery, the cerebral blood flow (CBF) of the sham operation group was taken as 100%, and the relative values ​​of each group were calculated. The statistical results are shown in Fig.12 In the figure below, 6-12 mice were counted in each group. Compared with the PBS control group, the cerebral blood perfusion in the Fn1 polypeptide group increased significantly, and the results showed that the Fn1 polypeptide has a protective effect on cerebral blood flow function after stroke.

[0200] 3. Behavioral Experiment

[0201] Behavioral testing was performed on the day of tMCAO or sham operation, 1 day, 3 days, 7 days or 14 days after surgery.

[0202] (1) Modified Neurological Severity Score (mNSS) was performed. The scoring criteria are shown in Table 1.

[0203] Table 1 Nerve damage severity score table

[0204]

[0205]

[0206] The results of the neurological damage severity score are shown in Fig.13 The first row shows that the scores of mice in the IS-FN1P group were lower than those in the IS-PBS group after surgery, indicating that the degree of neurological damage in the IS-FN1P group was lower than that in the IS-PBS group, indicating that FN1P improved the severity of neurological damage in mice after stroke to a certain extent.

[0207] (2) Conduct a suspended rotation test.

[0208] The Elevated Body Swing Test is used to evaluate asymmetric motor behavior. Lift the mouse's tail to about 10 cm above the table. Each time the mouse turns its head away from the vertical axis to any side (≥10°), it is counted as a deflection. If there is no deflection behavior after hanging in the air for more than 5 seconds, gently pinch the tail to induce rotational behavior. Each mouse is performed 10 times in total, with an interval of more than 1 minute, and repeated once, and the number of deviations to the left and right is recorded. The results are statistically calculated as the number of deviations to the right / the number of deviations to the left.

[0209] The results of the suspended rotation experiment are shown in Fig.13 The second row shows that the IS-FN1P group of mice had reduced deflection compared to the IS-PBS group.

[0210] (3) Perform the rotarod test.

[0211] The Rotarod Test is used to evaluate motor coordination and balance. The test requires the animal to maintain balance on a uniformly rotating rod, and the time spent on the rod and the speed of the rod when it falls are recorded. The training was conducted for 3 consecutive days before modeling, 3 times a day. The mouse was placed on the rod to adapt for 1 minute, and then the rod was started, rotating at a constant speed of 20 rpm, and the training was conducted for 5 minutes. If the mouse falls, the rod is put back until the exercise time reaches 5 minutes. On the third day of training, a baseline test was performed after 2 training sessions. The rod accelerated from 0 until it rotated at a constant speed after 40 rpm, and the time when the animal fell was recorded. If the mouse hugged the rod for 2 circles without moving, it was also considered to have fallen. The animal test after modeling was the same as the baseline test. After training or testing, the instrument was cleaned with alcohol and wiped dry. In addition: the holding circle can also be counted as 1s, and the number of seconds for all holding circles can be subtracted from the total number of seconds.

[0212] The results of the rotarod test are shown in Fig.13 The third row shows that the mice in the IS-FN1P group maintained movement on the rotarod longer than those in the IS-PBS group, indicating that FN1P improved some of the neuromotor functions of mice after stroke to a certain extent.

[0213] Example 8: Fn1 polypeptide promotes cardiovascular and cerebrovascular angiogenesis

[0214] 1. Fn1 enhances the angiogenesis ability of human coronary artery endothelial cells (HCAECs)

[0215] 1. HCAECs (Guangzhou Shunping Biotechnology Co., Ltd., catalog number BFN60805939-2) were inoculated in a 10 cm culture dish and cultured in DMEM medium containing 10% FBS until the density reached about 80%. Then, the supernatant was discarded and serum-free DMEM medium was used for 6 hours.

[0216] 2. After completing step 1, collect the cells, digest them with trypsin, add DMEM medium containing 10% FBS, centrifuge at room temperature and 1200 rpm for 3 minutes, collect the cell pellet, resuspend it in serum-free DMEM medium, and count the cells.

[0217] 3. Take a 96-well plate, add Matrigel (50 μl / well), shake the plate to spread the matrix gel evenly, and then place it horizontally in the incubator to polymerize for 1 hour.

[0218] 4. Take the 96-well plate that has completed step 3, and add the cell suspension prepared in step 2 to the test wells (denoted by FN1P) (1×10 4 -2×10 4cells), Calcein-AM, Fn1 polypeptide solution and serum-free DMEM medium (the total volume of the liquid phase system in each well is 100 μl, wherein the concentration of Calcein-AM is 1 μM, and the concentration of Fn1 polypeptide is 5 nM). The control well (expressed by PBS) uses an equal volume of PBS instead of Fn1 polypeptide solution, and the other wells are the same as the test wells, and cultured for 6 hours.

[0219] 5. After completing step 4, discard the supernatant, then fix with 4% paraformaldehyde solution, observe and take pictures with an optical microscope, and use Image J software to calculate the network parameters.

[0220] Microscope photos see Fig.14 The statistical results are shown in the left figure of Fig.14 Compared with the PBS group, the HCAECs in the Fn1 polypeptide group formed more nodes in the tubular network structure, longer branches, and a larger lumen area, indicating that the Fn1 polypeptide promoted the ability of endothelial cells to form blood vessels.

[0221] 2. Fn1 enhances the migration ability of human coronary artery endothelial cells (HCAECs)

[0222] 1. HCAECs were seeded in a 10 cm culture dish and cultured in DMEM medium containing 10% FBS until the density reached about 80%.

[0223] 2. After completing step 1, collect the cells, digest them with trypsin, and then resuspend the cells in DMEM medium containing 10% FBS to a cell concentration of 5×10 8 The cell suspension is 1000 cells / L.

[0224] 3. Perform Transwell assay.

[0225] Test group (expressed as FN1P): Take the cell suspension obtained in step 2, the Fn1 polypeptide solution and make the concentration of the polypeptide in the system 5nM, and then add it to the upper chamber (0.1ml / chamber). Control group (expressed as PBS): Replace the Fn1 polypeptide solution with an equal volume of PBS, and the rest is the same as the test group. After 18 hours of culture, remove the filter membrane, fix it with 4% paraformaldehyde for 10 minutes, rinse with PBS, wipe the residual cells on the surface of the upper chamber with a cotton swab, and stain with crystal violet. Photograph the entire well field of view under a microscope, calculate the number of cells that penetrate the membrane and take the average value.

[0226] Microscope images Fig.15 The statistical results are shown in the left figure of Fig.15 The right figure shows that compared with the PBS group, the number of cells penetrating the membrane in the Fn1 polypeptide group increased significantly, indicating that the Fn1 polypeptide promoted the migration of endothelial cells.

[0227] 3. Fn1 peptide promotes cardiac angiogenesis after MI

[0228] Experimental animals: Male C57BL / 6J mice aged 8-10 weeks.

[0229] Fn1 polypeptide group (MI+FN1P): The experimental animals were subjected to MI model establishment according to the method in step 1 of Example 5. Five minutes after the MI operation, each mouse was injected with Fn1 polypeptide solution through the tail vein. The injection dose of Fn1 polypeptide was 2.5 mg / (kg·bw);

[0230] PBS control group (MI+PBS): The experimental animals were subjected to MI model establishment according to the method in step 1 of Example 5. 5 minutes after the MI operation, PBS was injected into the tail vein of each mouse. The volume of PBS injected was the same as the volume of Fn1 polypeptide solution injected into the Fn1 polypeptide group.

[0231] Sham operation group (Sham): The experimental animals were subjected to sham operation according to the method in step 1 of Example 5. 5 minutes after the sham operation, PBS was injected into the tail vein of each mouse. The volume of PBS injected was the same as the volume of Fn1 polypeptide solution injected into the Fn1 polypeptide group.

[0232] CD31 is a marker of vascular endothelial cells. On the third day after MI surgery or sham surgery, mice were killed, dissected and the hearts were obtained. The hearts were fixed with 4% paraformaldehyde and embedded in paraffin, cross-sectioned, immunofluorescently stained with CD31 antibody (Proteintech, 11265-1-AP), and then observed under a laser confocal microscope.

[0233] Microscope images Fig.16 The statistical results of the average CD31 positive area in each field of view are shown in Fig.16 In the right figure, about 5 mice were counted in each group. The number of blood vessels in the Fn1 polypeptide group was significantly greater than that in the PBS control group, indicating that the Fn1 polypeptide promoted angiogenesis in the heart after MI.

[0234] 4. Fn1 peptide promotes cerebral angiogenesis after tMCAO

[0235] Experimental animals: Male C57BL / 6J mice aged 8-10 weeks.

[0236] Fn1 polypeptide group (IS-FN1P): tMCAO model was established according to the method of step 1 of Example 7; 6 hours after surgery, Fn1 polypeptide solution was injected into the tail vein, and the injection dose of Fn1 polypeptide was 2.5 mg / (kg·bw); Fn1 polypeptide solution was injected into the tail vein on the 3rd day, 6th day, 9th day and 12th day after surgery, and the single injection dose of Fn1 polypeptide was 2.5 mg / (kg·bw);

[0237] PBS control group (IS-PBS): tMCAO model was established according to the method of step 1 of Example 7; PBS was injected into the tail vein 6 hours after surgery, and the volume of PBS injected was the same as the volume of Fn1 polypeptide solution injected into the Fn1 polypeptide group; PBS was injected into the tail vein on the 3rd, 6th, 9th and 12th days after surgery, and the volume of PBS injected was the same as the volume of Fn1 polypeptide solution injected into the Fn1 polypeptide group;

[0238] Sham operation group (Sham): Sham operation was performed according to the method of step 1 of Example 7; 6 hours after the sham operation, PBS was injected into the tail vein of each mouse; 6 hours after the operation, PBS was injected into the tail vein, and the volume of the injected PBS was the same as the volume of the Fn1 polypeptide solution injected into the Fn1 polypeptide group; on the 3rd day, 6th day, 9th day and 12th day after the operation, PBS was injected into the tail vein, and the volume of the injected PBS was the same as the volume of the Fn1 polypeptide solution injected into the Fn1 polypeptide group.

[0239] CD31 is a marker of endothelial cells. Ki67 is a marker of proliferating cells. 14 days after tMCAO or 14 days after sham surgery, mice were killed, dissected and the brains were obtained. The brains were fixed with 4% paraformaldehyde and embedded in paraffin, cross-sectioned, and immunofluorescence stained with CD31 and Ki67 antibodies, and then imaged under laser confocal microscope. Observe under a microscope.

[0240] Microscope images Fig.17 The number of CD31+ and Ki67+ cells per unit area in each field of view is shown in the left figure. Fig.17 In the right figure, there are about 8-10 mice in each group. Compared with the PBS group, the number of blood vessels in the Fn1 polypeptide group increased significantly, indicating that the Fn1 polypeptide promoted angiogenesis in the brain after middle cerebral artery occlusion.

[0241] In summary, Fn1 peptide can not only promote the angiogenesis ability of endothelial cells in vitro, but also promote the angiogenesis process in the heart and brain after ischemia.

[0242] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A polypeptide, named Fn1 polypeptide, as shown in Sequence 3 of the Sequence Listing.

2. A polypeptide having 90% or more identity with the polypeptide according to claim 1 and / or a polypeptide obtained by modifying the polypeptide according to claim 1.

3. An RNA molecule, named Fn1-new-112 transcript, as shown in sequence 1 of the sequence listing.

4. An RNA molecule having 90% or more identity with the RNA according to claim 3.

5. A DNA molecule encoding the polypeptide according to claim 1 or 2 or encoding the RNA molecule according to claim 3 or 4.

6. A biological material, which is an expression cassette, a recombinant vector, a transgenic microorganism or a transgenic cell line having the DNA molecule of claim 5.

7. Use of the polypeptide according to claim 1 or the polypeptide according to claim 2 or the RNA molecule according to claim 3 or the RNA molecule according to claim 4 or the DNA molecule according to claim 5 or the biomaterial according to claim 6 in the preparation of a drug; the drug is indicated for heart damage and / or brain damage; the drug is used to treat and / or prevent the indicated disease.

8. Use of the polypeptide according to claim 1, the polypeptide according to claim 2, the RNA molecule according to claim 3, the RNA molecule according to claim 4, the DNA molecule according to claim 5, or the biomaterial according to claim 6 in the preparation of a drug; the drug is indicated for cardiovascular disease and / or cerebrovascular disease; the drug is used to treat and / or prevent the indicated disease.

9. Use of the polypeptide according to claim 1 or the polypeptide according to claim 2 or the RNA molecule according to claim 3 or the RNA molecule according to claim 4 or the DNA molecule according to claim 5 or the biomaterial according to claim 6 in the preparation of a drug; the use of the drug is as follows (a) and / or (b) and / or (c) and / or (d) and / or (e) and / or (f) and / or (g) and / or (h) and / or (i) and / or (j) and / or (k): (a) Used to improve heart failure; (b) for inhibiting myocardial fibrosis; (c) for inhibiting cardiomyocyte apoptosis; (d) for improving impaired heart function; (e) used to improve the extent of cerebral infarction; (f) used to increase cerebral blood perfusion; (g) for improving impaired brain function; (h) for promoting endothelial cells to form blood vessels; (i) for promoting endothelial cell migration; (j) for promoting angiogenesis in the heart and / or brain; (k) Used to improve cell hypoxic damage.

10. A drug comprising the polypeptide according to claim 1 or the polypeptide according to claim 2 or the RNA molecule according to claim 3 or the RNA molecule according to claim 4 or the DNA molecule according to claim 5 or the biomaterial according to claim 6.

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