Fn1 polypeptides, transcripts and uses thereof in the preparation of medicaments for cardiac and cerebral injury and / or cardiovascular diseases

Abstract

The application discloses Fn1 polypeptide, transcript and application thereof in preparation of drugs for heart and brain injury and / or cardiovascular and cerebrovascular diseases. The Fn1 polypeptide provided by the application is shown in sequence 3 of the sequence list. The Fn1-new-112 transcript provided by the application is shown in sequence 1 of the sequence list. The application also protects DNA molecules encoding the polypeptide or the RNA molecule. The application also protects biological materials, which are expression cassettes, recombinant vectors, transgenic microorganisms or transgenic cell lines with the DNA molecules. The polypeptide, the RNA molecule, the DNA molecule or the biological material can be used for preparing drugs; the indications of the drugs are heart injury and / or brain injury and / or cardiovascular diseases and / or cerebrovascular diseases. The application has great application and promotion value for the treatment of heart-related diseases and / or brain-related diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and relates to Fn1 polypeptide, transcript and application thereof in preparation of drugs for heart and brain injury and / or cardiovascular and cerebrovascular diseases. BACKGROUND

[0002] Cardiovascular and cerebrovascular diseases are a general term for diseases of heart blood vessels and brain blood vessels, and refer to ischemic or hemorrhagic diseases of heart, brain and whole body tissues caused by hyperlipidemia, blood viscosity, atherosclerosis, hypertension and the like. Cardiovascular and cerebrovascular diseases are common diseases that seriously threaten human health, especially the health of people over 50 years old, and have the characteristics of high morbidity, high disability rate and high mortality.

[0003] Myocardial infarction (MI) is commonly known as "heart attack", and is defined by pathology as damage or necrosis of myocardial cells due to blood flow obstruction. The most common symptom of MI is angina pectoris, followed by upper abdominal pain, chest tightness, upper limb numbness, dizziness, palpitation, shortness of breath, restlessness and the like.

[0004] Myocardial ischemia, as a pathophysiological state, is caused by coronary artery stenosis, spasm or embolism, etc. The clinical symptoms of myocardial ischemia are manifested as discomfort and pain in the precordial region. Severe myocardial ischemia can be accompanied by arrhythmia and heart failure, and cause various accompanying symptoms. Isopropyl adrenaline (ISO) is a beta-adrenergic receptor agonist, which can directly or indirectly act on myocardial tissue through inflammatory response, oxidative stress, autophagy and apoptosis, etc., thereby causing myocardial ischemic injury, and symptoms such as myocardial hypertrophy and myocardial fibrosis.

[0005] Stroke, commonly known as apoplexy, is divided into two types of ischemic stroke and hemorrhagic stroke, and is a disease caused by various reasons to cause damage to cerebral blood vessels and produce focal or whole brain tissue damage. SUMMARY

[0006] The application aims to provide Fn1 polypeptide, transcript and application thereof in preparation of drugs for heart and brain injury and / or cardiovascular and cerebrovascular diseases.

[0007] The application provides a polypeptide, which is named as Fn1 polypeptide, as shown in sequence 3 of the sequence table.

[0008] The application also protects a polypeptide having 90% or more identity with the Fn1 polypeptide.

[0009] The present application also protects polypeptides modified from Fn1 polypeptides. Specifically, the modification is main chain structure modification and / or side chain group modification. Exemplarily, the modification can be C-terminal modification (amidation, sulfation, etc.), N-terminal modification (acetylation, fatty acidation, etc.), intermediate residue modification (glycosylation modification combined with Ser-, Tyr-, Asn-, Thr-; phosphorylation modification combined with Ser-, Tyr-, Thr-, etc.), cyclization modification, acylation modification (myristoylation, palmitoylation, etc.), polyethylene glycol modification, transmembrane peptide modification, biotinylation modification, fluorescent group modification, etc.

[0010] The present application also provides an RNA molecule, named Fn1-new-112 transcript, as Sequence 1 of the sequence listing Shown. The present application also protects any of the above described polypeptides or any of the above described RNA molecules or any of the above described DNA

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

[0012] The present application also protects DNA molecules encoding any of the above-mentioned polypeptides.

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

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

[0015] The present application 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 application also protects the use of any of the above-mentioned polypeptides or any of the above-mentioned RNA molecules or any of the above-mentioned DNA molecules or any of the above-mentioned biological materials in the preparation of a medicament; the indication of the medicament is heart injury and / or brain injury; the medicament is used for treating and / or preventing the indication.

[0017] The heart injury includes but is not limited to heart injury caused by cardiovascular diseases. Exemplarily, the heart injury can be heart injury caused by myocardial infarction, heart injury caused by myocardial ischemia, heart injury caused by myocardial hypertrophy, etc. Exemplarily, the heart injury can 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 diseases. Exemplarily, the brain injury can be brain injury caused by stroke. Exemplarily, the stroke can be ischemic stroke or hemorrhagic stroke.

[0019] When the heart injury is caused by myocardial infarction, the therapeutic effect of the drug is to improve the cardiac dysfunction after myocardial infarction. When the heart injury is caused by myocardial infarction, the therapeutic effect of the drug is to inhibit the EDD increase caused by myocardial infarction and / or inhibit the ESD increase caused by myocardial infarction and / or inhibit the FS decrease caused by myocardial infarction. When the heart injury is caused by myocardial infarction, the therapeutic effect of the drug is to inhibit the myocardial fibrosis after myocardial infarction. When the heart injury is caused by myocardial infarction, the therapeutic effect of the drug is to inhibit the myocardial cell apoptosis after myocardial infarction. When the heart injury is caused by myocardial infarction, the therapeutic effect of the drug is to promote the cardiac angiogenesis after myocardial infarction.

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

[0021] When the brain injury is caused by ischemic stroke, the therapeutic effect of the drug is to reduce the infarction area. When the brain injury is caused by ischemic stroke, the therapeutic effect of the drug is to increase the cerebral blood perfusion. When the brain injury is caused by ischemic stroke, the therapeutic effect of the drug is to promote the cerebral angiogenesis after ischemic stroke.

[0022] Figure 1 The molecule or any of the biological materials described above is used in the preparation of a drug; the indication of the drug is cardiovascular disease and / or cerebrovascular disease; the drug is used for treating and / or preventing the indication.

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

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

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

[0026] The present application also protects the use of any one of the above polypeptides or any one of the above RNA molecules or any one of the above DNA molecules or any one of the above biomaterials in the preparation of a medicament; the use of the medicament 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):

[0027] (a) for improving heart dysfunction;

[0028] (b) for inhibiting myocardial fibrosis;

[0029] (c) for inhibiting myocardial cell apoptosis;

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

[0031] (e) for improving the extent of cerebral infarction;

[0032] (f) for increasing cerebral blood perfusion;

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

[0034] (h) for promoting endothelial cell formation of blood vessels;

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

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

[0037] (k) for improving cell hypoxic injury.

[0038] Specifically, the cells can be myoblasts or myocardial cells.

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

[0040] The improvement of heart dysfunction is to improve the heart function of the patient. The inhibition of myocardial fibrosis is to inhibit the myocardial fibrosis of the patient. The inhibition of myocardial cell apoptosis is to inhibit the 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 heart injury patient and / or a cardiovascular disease patient.

[0041] The improvement of the extent of cerebral infarction is to improve the extent of cerebral infarction of the patient. The increase of cerebral blood perfusion is to increase the cerebral blood perfusion of the patient. The improvement of impaired brain function is to improve the brain function of the patient. The patient is a brain injury patient and / or a cerebrovascular disease patient.

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

[0043] The heart injury includes but is not limited to heart injury caused by cardiovascular disease. Exemplarily, the heart injury can be heart injury caused by myocardial infarction, heart injury caused by myocardial ischemia, heart injury caused by myocardial hypertrophy, etc. Exemplarily, the heart injury can 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 can be brain injury caused by stroke. Exemplarily, the stroke can be ischemic stroke or hemorrhagic stroke.

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

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

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

[0048] The present application also protects a medicine comprising any one of the above polypeptides or any one of the above RNA molecules or any one of the above DNA molecules or any one of the above biomaterials.

[0049] The indication of the medicine is as any one of the above.

[0050] The use of the medicine is as any one of the above.

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

[0052] Figure 2 The relevant results of new transcripts significantly up-regulated and down-regulated in the process of myocardial infarction analyzed by three generations of sequencing in Example 1.

[0053] Figure 3Statistical results for relative expression levels of Fn1 -new-112 transcript and Fn1 transcript in Step Two of Example 2.

[0054] Figure 4 Statistical results for Alamar Blue cell viability assay in Step Three of Example 2.

[0055] Figure 5 Photos and statistical results for propidium iodide / calcein detection of cell death in Step Three of Example 2.

[0056] Figure 6 Statistical results for Fn1 polypeptide inhibition of OGD-induced cell damage in Example 4.

[0057] Figure 7 Related results for echocardiography measurements in Example 5.

[0058] Figure 8 Related results for Masson's trichrome staining detection of fibrosis in Example 5.

[0059] Figure 9 Related results for TUNEL apoptosis detection in Example 5.

[0060] Figure 10 Related results for echocardiography measurements in Example 6.

[0061] Figure 11 Related results for Masson's trichrome staining detection of fibrosis in Example 6.

[0062] Figure 12 Results related to the detection of cerebral infarct area by tartrazine staining in Example 7.

[0063] Figure 13 Related results for laser speckle blood flow imaging detection of cerebral perfusion in Example 7.

[0064] Figure 14 Related results for behavioral experiments in Example 7.

[0065] Figure 15 Related results for human coronary artery endothelial cell (HCAEC) vasculogenesis in Example 8.

[0066] Figure 16 Related results for human coronary artery endothelial cell (HCAEC) migration in Example 8.

[0067] Figure 17 Related results for Fn1 polypeptide promotion of post-MI cardiac angiogenesis in Example 8.

[0068] Example 1, discovery of the transcript Fn1 -new-112Results related to the promotion of post-tMCAO cerebral angiogenesis by the Fn1 polypeptide in Example 8. DETAILED DESCRIPTION

[0069] The application will be further described in conjunction with the specific embodiments, and the examples given are only to illustrate the application, but not to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0070] The experimental methods in the following examples are all routine methods, unless otherwise specified, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially. The quantitative tests in the following examples, unless otherwise specified, are all set up in triplicate, and the results are averaged. OGD: oxygen-glucose deprivation. siRNA (small interfering RNA): a double-stranded RNA molecule, 20-25 bp in length.

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

[0072] Real-time PCR analysis of transcript levels. RNA was isolated from heart tissue using Trizol reagent and synthesized into cDNA using Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions.

[0073] 1. Full-length transcriptome sequencing libraries were prepared using the SQK-PCB109 kit (Oxford Nanopore sequencing technology, third generation sequencing technology). Specifically, 2 mM VN primers (VNP) and 10 mM dNTPs were added to the samples to be sequenced and incubated at 65 °C for 5 minutes. The sample was then quickly cooled and 5x RT buffer, RNaseOUT and 10 mM strand switch primers (SSP) were added and incubated at 42 °C for 2 minutes. Then, 1 pl of Maxima H Minus Reverse Transcriptase was added to reverse the full-length transcripts into cDNA in vitro for further PCR. Finally, the cDNA of four samples under each condition were combined together to get three mixed samples and labeled with barcodes, while two technical replicates were sequenced. Sequencing was performed using PromethION (FLO-PRO002) to generate fast5 files, with sequencing settings running for 72 hours or stopping early when the number of active pores was less than 10. Reads with a quality score (Q-score) <7 were filtered out to ensure sequencing quality. At the same time, these samples were also 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 4 samples per group. When analyzing, the 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. GENCODE M26 (GRCm39) transcript reference and genome reference (primary assembly) were used for sequence alignment and quantification with main annotations.

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

[0076] 4. Add the position information of the identified novel transcripts to the transcript annotation file obtained from GENCODE M26 for the AGAT suite (version 0.8.1) to obtain updated transcript annotation for transcript quantification.

[0077] 5. For second-generation short read data, genome alignment was performed using Hisat2 (version 2.2.1), 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 novel transcripts.

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

[0079] 6. Quantitative RT-PCR: Real-time PCR was performed using SYRB Green real-time fluorescent quantitative PCR reagents, and the expression of the novel transcript was verified by real-time quantitative PCR. Figure 1 Upstream primer: 5'-GCTTAATTTGACTCAACACGGGA-3'; cDNA. The quantitative RT-PCR conditions were: 37 cycles, 94°C for 10 seconds, 57°C for 15 seconds, and 72°C for 5 seconds.

[0080] By analyzing and identifying novel transcripts significantly up-regulated and down-regulated during myocardial infarction through third-generation sequencing technology and verifying their changes using real-time quantitative PCR, the results showed that the sequencing analysis process was accurate for the quantification of novel transcripts. The relevant results are shown in Figure 2 .

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

[0082] Fn1-new-112 transcript is shown in SEQ ID NO: 1 of the sequence listing.

[0083] The coding frame of Fn1-new-112 gene is shown in SEQ ID NO: 2 of the sequence listing.

[0084] Example 2, Specific knockdown of Fn1-new-112 transcript aggravates OGD-induced cell injury

[0085] I. Preparation of siRNA

[0086] siRNAs targeting Fn1 -new-112 transcript and control siRNAs were prepared, respectively. The siRNAs targeting Fn1 -new-112 transcript were named as siFn1 -new-112. The control siRNAs were named as siNC.

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

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

[0089] II. Verification of effectiveness and specificity of siRNAs

[0090] 1. A 12-well cell culture plate was taken, C2C12 cells (mouse myoblast cells) were inoculated and DMEM high-sugar culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was added, 2 x 10 5 cells / well, and cultured until the cell density reached 50%.

[0091] 2. After step 1 was completed, the test siRNAs (the test siRNAs were siFn1 -new-112 or siNC) were transfected using Jetprime transfection reagent, 150 ng siRNA / well, and cultured for 48 hours.

[0092] 3. After step 2 was completed, the cells were collected, total RNA was extracted and cDNA was obtained by reverse transcription. The cDNA was used as a template, 18S gene was used as an internal reference gene, and the relative expression levels of Fn1 -new-112 transcript and Fn1 transcript (since the primer pair used can theoretically detect all known Fn1 transcripts in the prior art, the detection target of the primer pair is referred to as Fn1 transcript) were detected by qPCR, respectively.

[0093] The primers for detecting Fn1 -new-112 transcript are as follows:

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

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

[0096] The primers for detecting Fn1 transcript are as follows:

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

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

[0099] Primers for detecting 18S gene are as follows:

[0100] Figure 3

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

[0102] Three replicates were set and the results were averaged ± standard deviation.

[0103] Statistical results of relative expression levels of Fn1-new-112 transcript and Fn1 transcript are shown in Table 1. Figure 4 .

[0104] III. Specific knockdown of Fn1-new-112 transcript aggravates OGD-induced cell injury

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

[0106] 2. After completing step 1, transfect the test siRNA (test siRNA is siFn1-new-112 or siNC) using Jetprime transfection reagent, 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 medium containing 1% penicillin-streptomycin per well, and place it in a culture incubator (the gas composition in the culture incubator is: 0.1% O2, 5% CO2, 94.9% N2; volume ratio) at 37°C for 8h.

[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 per well, incubate at 37°C for 2h, then measure the fluorescence change using the Cytation 5 cell imaging microplate detection system (excitation / emission wavelength is set to 545 / 590nm), and output the result as the fluorescence intensity value.

[0110] Subtract the fluorescence intensity value of the background well (i.e. the well with only PBS added) from the fluorescence intensity value of the test well, and the result is the fluorescence intensity correction value of the test well.

[0111] Take the fluorescence intensity correction value of the siNC group as reference 1, calculate the relative value of the siFn1-new-112 group, which is the relative cell viability.

[0112] Three replicates were set and the results were averaged ± standard deviation.

[0113] Results are shown in Fn1 polypeptide is shown in SEQ ID NO: 3 of the sequence listing. Compared with the siNC group, the number of dead cells in the siFn1-new-112 group increased and the number of live cells decreased. The results showed that under OGD conditions, knocking down the Fn1-new-112 transcript reduced cell viability and cell survival ability.

[0114] 5. Cell death was detected by iodinated propidium / calcium chlorophyll

[0115] After step 3 was completed, the culture supernatant was aspirated and the cells were washed with PBS, and then 100 μl of staining solution (staining solution was a solution containing 1 μg / μl iodinated propidium and 2 μg / μl calcium chlorophyll) was added to each well, and incubated at 37°C for 30 minutes, and then photographed using the Cytation 5 cell imaging microplate detection system (iodinated propidium stained dead cells, detection channel was 561 / 600 nm; calcium chlorophyll stained live cells, detection channel was 488 / 525 nm).

[0116] Five replicates were set and the results were averaged ± standard deviation.

[0117] Results are shown in Figure 5 Compared with the siNC group, the number of dead cells in the siFn1-new-112 group increased and the number of live cells decreased. The results showed that under OGD conditions, knocking down the Fn1-new-112 transcript reduced cell viability and cell survival ability.

[0118] Example 3, Preparation of Fn1 polypeptide

[0119] Figure 5

[0120] Sequence 3: MDRLVFSEYFLLVCCNIHSFASVGSTVRGLCPG.

[0121] Fn1 polypeptide was artificially synthesized by Shanghai Nuoyou Biological Technology Co., Ltd.

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

[0123] 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] I. Fn1 polypeptide inhibits OGD-induced C2C12 cell damage

[0126] 1. Take a 96-well cell culture plate, inoculate C2C12 cells and add DMEM high-sugar culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, 2 x 104 Cells were cultured to a density of 80-90% confluence.

[0127] 2. Group processing

[0128] MIB-PBS group: After step 1 was completed, the culture supernatant was aspirated, 100 μl of low-sugar 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 incubated in an incubator (the gas composition in the incubator was 4% O2, 5% CO2, and 91% N2 by volume) at 37°C for 24 h.

[0129] MIB-Fn1 polypeptide group: After step 1 was completed, the culture supernatant was aspirated, 100 μl of low-sugar 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 incubated in an incubator (the gas composition in the incubator was 4% O2, 5% CO2, and 91% N2 by volume) at 37°C for 24 h.

[0130] MIC-PBS group: After step 1 was completed, the culture supernatant was aspirated, 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 incubated in an incubator (the gas composition in the incubator was 0.1% O2, 5% CO2, and 94.9% N2 by volume) at 37°C for 8 h.

[0131] MIC-Fn1 polypeptide group: After step 1 was completed, the culture supernatant was aspirated, 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 incubated in an incubator (the gas composition in the incubator was 0.1% O2, 5% CO2, and 94.9% N2 by volume) at 37°C for 8 h.

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

[0133] Normoxia-Fn1 polypeptide group: After step 1 was completed, the culture supernatant was aspirated, 100 μl of DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin-streptomycin and 10 μl of Fn1 polypeptide solution were added to each well, and the cells were incubated in an incubator (the gas composition in the incubator was 5% CO2 and 95% air by volume) at 37°C for 24 h.

[0134] Three replicates were set for each group.

[0135] 3. Cell viability assay

[0136] Method same as step 3 of example 2.

[0137] Set 30-50 repeated treatments, and take the average value ± standard deviation.

[0138] Results are shown in 2, step 1 of 2. The left graph (PBS represents the PBS group, and F peptide represents the Fn1 polypeptide group). Fn1 polypeptide can inhibit OGD-induced damage to mouse myoblast cells and has a protective effect on cell hypoxic injury.

[0139] II. Fn1 polypeptide inhibits OGD-induced damage to NRVM cells

[0140] Method for obtaining NRVM cells (rat primary myocardial cells): ① Take the heart of a newborn SD rat, wash it with HBSS solution, cut it open and remove the blood in the visceral cavity, then cut it into pieces and add it to a centrifuge tube containing a digestive solution, stir at 37°C for 5 min, and discard the supernatant blood cells; ② Add digestive solution to the precipitate in step ① again, stir at 37°C for 5 min, collect the supernatant, and add it to a centrifuge tube containing DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, centrifuge twice to discard the supernatant, and then add new DMEM medium to resuspend the precipitate, which is the cell suspension; ③ Repeat step ② for digestion, and observe that there is no cell precipitate in the centrifuge tube, and the digestive solution is clear to the naked eye; stop digestion; ④ Filter the cell suspension obtained above with a sieve, rinse the cells with DMEM medium, then place the cells in a culture dish, and place it in a 37°C incubator for differential adhesion for 1 h; ⑤ After the cells adhere, aspirate the supernatant into a volumetric flask, repeatedly blow it with DMEM medium, add 5-bromodeoxyuridine, and finally inoculate it in a culture dish at 37°C.

[0141] Use NRVM cells instead of C2C12 cells, and other steps are the same.

[0142] Set 10-20 repeated treatments, and take the average value ± standard deviation.

[0143] Results are shown in Figure 5 The middle graph (PBS represents the PBS group, and F peptide represents the Fn1 polypeptide group). Fn1 polypeptide can inhibit OGD-induced damage to rat myocardial cells and has a protective effect on cell hypoxic injury.

[0144] III. Fn1 polypeptide inhibits OGD-induced damage to hiPSC-CMs cells

[0145] Method for obtaining hiPSC-CMs cells (human induced pluripotent stem cell differentiated cardiomyocytes): ① hiPSC (human induced pluripotent stem cells, obtained from the National Stem Cell Resource Library, 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 step ①, the culture supernatant was aspirated, 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 step ②, the supernatant was aspirated, 3 ml of RPMI medium containing 1x B27-ins was added to each well, and cultured for 1 day; ④ After step ③, the supernatant was aspirated, 3 ml of RPMI medium containing 5 μM IWR-1 and 1x B27-ins was added to each well, and cultured for 2 days; ⑤ After step ④, the supernatant was aspirated, 3 ml of RPMI medium containing 1x B27-ins was added to each well and cultured for 8 days (new medium was replaced every 48 h); ⑥ After step ⑤, the cells were collected, TrypLE Express enzyme digestion, resuspended with RPMI medium containing 1x B27-ins and adjusted the cell concentration to 1x10 6 6 / ml, add Y27632 to make the concentration 10 μM, then inoculate in a 24-well plate, 750000 cells / well, culture for 24 hours; ⑦ After step ⑥, replace with 1x B27-ins RPMI medium without sugar for 48 hours for glucose starvation purification; ⑧ After step ⑦, replace with 1x B27-ins RPMI medium and continue to culture for 4 days, replace the medium every 48 hours, and 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, so that the cell content is 2.5x10 4 6 / well, then culture for 48 hours.

[0147] Figure 6

[0148] 3. Synchronize step 1 3.

[0149] Set 6-10 repeated treatments, and take the average value ± standard deviation.

[0150] The results are shown in the right graph of Figure 6 (PBS represents the PBS group, and F peptide represents the Fn1 polypeptide group). Fn1 polypeptide can inhibit OGD-induced human myocardial cell damage and has a protective effect on cell hypoxic injury.

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

[0152] Test animals: 8-10 week old male C57BL / 6J mice.

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

[0154] Modeling method of MI model: permanent blockage of left anterior descending branch of coronary artery (LAD) was used to construct the MI model. The specific method of constructing the MI model: the test animals were anesthetized by inhaling 2% isoflurane, 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, and a silk thread was placed under the left coronary artery to ligate the left anterior descending branch of coronary artery (LAD); when the left ventricular anterior wall turned white, it indicated that the ligation was successful, and the heart was immediately put back into the chest cavity, and the air was emptied and the muscle and skin were sutured.

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

[0156] II. Dosing

[0157] Fn1 polypeptide group (Fn1 peptide): the test animals were modeled according to the method in step one, and 5 minutes after the MI operation, Fn1 polypeptide solution was injected into the tail vein of each mouse, and the injection dose of Fn1 polypeptide was 2.5 mg / (kg·bw);

[0158] PBS control group (PBS): the test animals were modeled according to the method in step one, and 5 minutes after the MI operation, PBS was injected into the tail vein of each mouse, and the volume of PBS injection was the same as the volume of Fn1 polypeptide solution injected in the Fn1 polypeptide group;

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

[0160] III. Index detection

[0161] 1. Echocardiography

[0162] Echocardiography was performed 7 days after the MI operation or 7 days after the sham operation to evaluate cardiac function. Each mouse was measured at least three times each time, and the mouse was maintained under anesthesia with 1.5% isoflurane during the measurement.

[0163] An exemplary photograph of echocardiography is shown in Figure 7 Left. The statistical results of left ventricular end-diastolic diameter (EDD), left ventricular end-systolic diameter (ESD), and left ventricular fractional shortening (FS) are shown in Figure 7The 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 peptide test group were significantly reduced, and the left ventricular fractional shortening (FS) was increased. Figure 8 Figure 8

[0164] 2. Masson staining to detect fibrosis

[0165] Seven days after MI surgery or sham surgery, mice were sacrificed and their hearts were dissected and obtained. The hearts were fixed with 4% paraformaldehyde, embedded in paraffin, and cross-sectioned and stained with Masson's staining. The degree of fibrosis was then observed under a light microscope.

[0166] Exemplary photographs of Masson staining are shown in During the experiment, the mice were maintained under anesthesia using 1.5% isoflurane. The statistical results are shown in the left figure of Figure 9 The right figure (4 mice per group) shows that compared with the PBS control group, the area of ​​myocardial fibrosis in the Fn1 peptide group was significantly reduced. This result indicates that Fn1 peptide 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 day 3 after MI surgery or sham surgery, mice were sacrificed, and hearts were dissected and harvested. Hearts were fixed with 4% paraformaldehyde, embedded in paraffin, and then cross-sectioned and stained using a TUNEL fluorescent staining kit. TUNEL fluorescence was then observed at 505 nm using a Dragonfly high-speed confocal microscopy system.

[0169] See the exemplary photos Figure 9 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 10 The right panel (4 mice per group) shows that compared with the PBS group, the number of TUNEL-positive cardiomyocytes in the border zone of the Fn1 peptide group was significantly reduced, and Fn1 peptide treatment reduced cell apoptosis in the lesion site. This suggests that Fn1 peptide 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 was established in mice.

[0173] I. Grouping

[0174] Fn1 polypeptide group (denoted as Fn1 peptide or ISO+Fn1): from day 1 to day 9 of the experiment, the mice were injected with Fn1 polypeptide solution via tail vein every day, and the single injection dose of Fn1 polypeptide was 2.5 mg / (kg·bw); from day 3 to day 9 of the experiment, the mice were injected with ISO subcutaneously at the back of the neck every day, and the single injection dose of ISO was 5 mg / (kg·bw).

[0175] PBS control group (denoted as PBS or ISO+PBS): from day 1 to day 9 of the experiment, the mice were injected with PBS via tail vein every day, and the volume of PBS injection was the same as the volume of Fn1 polypeptide solution injection in the Fn1 polypeptide group; from day 3 to day 9 of the experiment, the mice were injected with ISO subcutaneously at the back of the neck every day, and the single injection dose of ISO was 5 mg / (kg·bw).

[0176] Ctrl control group (denoted as Control): from day 1 to day 9 of the experiment, the mice were injected with PBS via tail vein every day, and the volume of PBS injection was the same as the volume of Fn1 polypeptide solution injection in the Fn1 polypeptide group.

[0177] II. 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, Figure 10

[0180] The echocardiography is shown in the left panel of A head-processed 0.18 mm diameter nylon thread was inserted from the small mouth into the internal carotid artery and inserted into the middle cerebral artery, the insertion depth of the nylon thread was about 9±1 mm from the carotid artery bifurcation. . The statistical results of left ventricular end-diastolic diameter (EDD), left ventricular end-systolic diameter (ESD), and left ventricular fractional shortening (FS) are shown in the right panel of On the 3rd, 6th, 9th and 12th day after surgery, PBS was injected through the tail vein, the volume of PBS injected . The results show that compared with the PBS control group, the left ventricular end-diastolic diameter (EDD) and the left ventricular end-systolic diameter (ESD) of the Fn1 polypeptide group mice are significantly reduced, and the left ventricular fractional shortening (FS) is increased. The results show that Fn1 polypeptide can improve the impaired cardiac function of isoproterenol (ISO)-induced mice.

[0181] 2. Masson staining for fibrosis

[0182] On day 16 of the experiment, the mice were sacrificed, dissected, and the hearts were obtained. The hearts were sequentially fixed with 4% paraformaldehyde and paraffin-embedded, and then subjected to Masson staining after being cut transversely, and then the degree of fibrosis was observed and recorded under an optical microscope.

[0183] The results of Masson staining are shown in Figure 11The statistical results are shown in the left figure of Figure 11 The right figure (7 mice per group) showed that compared with the PBS control group, the area of ​​myocardial fibrosis in the Fn1 peptide group was significantly reduced, indicating that Fn1 peptide can significantly reduce the degree of ISO-induced myocardial fibrosis and improve heart damage caused by myocardial hypertrophy.

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

[0185] Experimental animals: 8-10 week old male C57BL / 6J mice.

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

[0187] The modeling method of tMCAO model: ① Use 3% sodium pentobarbital to anesthetize the test animals, prepare the neck skin, disinfect it, 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. Figure 12 Figure 12 Figure 13 ⑤ Remove the suture plug, ligate the proximal end of the external artery with 7-0 silk suture, suture the neck wound with 5-0 silk suture, 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: The underlined steps were not performed, and the other modeling methods were the same as those of the tMCAO model.

[0189] 2. Grouped Dosing

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

[0191] PBS control group (IS-PBS): The tMCAO model was established according to the method of step 1; 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; Figure 13 Figure 13The volume of Fn1 polypeptide solution injected was the same as that in the Fn1 polypeptide group;

[0192] Sham group (Sham): Sham surgery was performed according to the method in step 1; 6 hours after the sham surgery, each mouse was injected with PBS through the tail vein; 6 hours after the surgery, 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, 6th, 9th and 12th day after the surgery, 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. Detection of cerebral infarction area by tar violet staining

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

[0196] The results of tar violet staining are shown in Figure 14 The statistical results are shown in the left figure of Figure 14 In the right figure, five mice were included in each group. Compared with the PBS control group, the cerebral infarction area of ​​mice in the Fn1 peptide group was significantly reduced, indicating that Fn1 peptide can reduce the severity of cerebral infarction after middle cerebral artery occlusion.

[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 Figure 15 Figure 1 (baseline represents speckle detection before modeling; tMCAO represents the modeling method step ④ in which the suture plug is inserted and speckle detection is performed 10 minutes after ischemia; reperfusion represents the modeling method step ⑤ in which the suture 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-operated group was set as 100%, and the relative values ​​of each group were calculated. The statistical results are shown in Figure 1. Figure 15 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 peptide group was significantly increased, indicating that Fn1 peptide has a protective effect on cerebral blood flow function after stroke.

[0200] 3. Behavioral experiments

[0201] Behavioral tests were performed on the day of tMCAO surgery or sham surgery, 1 day after surgery, 3 days after surgery, 7 days after surgery, or 14 days after surgery, respectively.

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

[0203] Table 1. The modified Neurological Severity Score (mNSS) table

[0204]

[0205]

[0206] The results of the modified Neurological Severity Score (mNSS) are shown in Table 2. Test animals: 8-10 week old male C57BL / 6J mice. The first row of Table 2 shows that the score of the IS-FN1P group is lower than that of the IS-PBS group, indicating that the degree of neurological impairment of the IS-FN1P group is lower than that of the IS-PBS group, indicating that FN1P to some extent improves the severity of neurological impairment in mice after stroke.

[0207] (2) The Elevated Body Swing Test was performed.

[0208] The Elevated Body Swing Test was used to assess asymmetric motor behavior. The mouse tail was lifted to about 10 cm from the tabletop. Each time the mouse turned its head to deviate from the vertical axis to either side (≥ 10°) was counted as a deflection. If there was no deflection behavior for more than 5 s, the tail was gently pinched to induce rotation behavior. Each mouse was performed a total of 10 times, with an interval of more than 1 min, and then repeated once, and the number of left and right deviations was recorded. The results were statistically analyzed by the number of right deviations / the number of left deviations.

[0209] The results of the Elevated Body Swing Test are shown in Table 3. Figure 16 The second row of Table 3 shows that the IS-FN1P group has less deflection than the IS-PBS group.

[0210] (3) The Rotarod Test was performed.

[0211] Rotarod Test was used to assess motor coordination and balance. The test required animals to balance on a rotating rod at a constant speed and the time spent on the rotarod and the speed of the rotarod when the animal fell were recorded. Animals were trained for 3 consecutive days before modeling, 3 times a day. Mice were placed on the rotarod for 1 minute of adaptation, then the rotarod was started, rotating at a constant speed of 20 revolutions per minute, and trained for 5 minutes. If the mouse fell, it was placed back on the rotarod until the movement time reached 5 minutes. On the third day of training, a baseline test was performed after 2 training sessions, the rotarod was accelerated from 0 to a constant speed of 40 revolutions per minute, and the time when the animal fell was recorded. If the mouse held the rotarod for 2 revolutions without moving, it was also considered to have fallen. The animals were tested after modeling as in the baseline test. After training or testing, the instruments were cleaned with alcohol and dried. Alternatively: holding a circle can also be counted as 1s, and the total number of seconds is reduced by the number of seconds of all holding circles.

[0212] The results of the rotarod test experiment are shown in Table 3. Figure 16 The third row of Table 3 shows that the IS-FN1P group of mice maintained movement on the rotarod for a longer time than the IS-PBS group, indicating that FN1P improved the motor function of mice to some extent after stroke.

[0213] Example 8, Fn1 polypeptide promotes cardiovascular neovascularization

[0214] I. Fn1 enhances the ability of human coronary artery endothelial cells (HCAECs) to form blood vessels

[0215] 1. HCAECs (Guangzhou Shunping Biotechnology Co., Ltd., Item No. BFN60805939-2) were inoculated in 10 cm culture dishes and cultured in DMEM medium containing 10% FBS until the density reached about 80%. Then, the supernatant was discarded and the cells were cultured in serum-free DMEM medium for 6 hours.

[0216] 2. After completing step 1, the cells were collected, trypsinized, and then added with DMEM medium containing 10% FBS, centrifuged at 1200 rpm for 3 min at room temperature, and the cell precipitate was collected and resuspended in serum-free DMEM medium for cell counting.

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

[0218] 4. Take the 96-well plate completed in step 3, add the cell suspension prepared in step 2 to the test wells (indicated by FN1P) (1 x 10 4 -2 x 10 4Cell suspension, Calcein-AM, Fn1 polypeptide solution and serum-free DMEM medium, (the total volume of each liquid system was 100 μl, the concentration of Calcein-AM was 1 μM, and the concentration of Fn1 polypeptide was 5 nM), the control group (represented by PBS) was replaced with an equal volume of PBS instead of Fn1 polypeptide solution, and the rest was the same as the experimental group. Incubate for 6 hours.

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

[0220] The photos under the microscope are shown in the left graph of Observed under a microscope. , and the statistical results are shown in the right graph of Figure 17 . Compared with the PBS group, the HCAECs in the Fn1 polypeptide group have more nodes, longer branches, and larger statistical lumen area, indicating that Fn1 polypeptide promotes the ability of endothelial cells to form blood vessels.

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

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

[0223] 2. After step 1, collect the cells, perform trypsin digestion, then resuspend the cells in DMEM medium containing 10% FBS to make the cell concentration 5 × 10 8 cells / L, which is the cell suspension.

[0224] 3. Perform the Transwell experiment.

[0225] Experimental group (represented by FN1P): Take the cell suspension obtained in step 2, Fn1 polypeptide solution, and make the polypeptide concentration in the system 5 nM, then add to the upper chamber (0.1 ml / chamber). Control group (represented by PBS): Replace Fn1 polypeptide solution with an equal volume of PBS, and the rest is the same as the experimental group. After 18 h of culture, remove the filter membrane, fix with 4% paraformaldehyde for 10 min, rinse with PBS, and use a cotton swab to wipe off the residual cells on the surface of the upper chamber. Crystal violet staining. Take pictures of the entire field of view under a microscope, and calculate the average number of cells that have crossed the membrane.

[0226] The images under the microscope are shown in the left graph of Figure 17 , and the statistical results are shown in the right graph of ​ . Compared with the PBS group, the number of cells that have crossed the membrane in the Fn1 polypeptide group increased significantly, indicating that Fn1 polypeptide promotes the migration of endothelial cells.

[0227] III. Fn1 polypeptide promotes angiogenesis after MI

[0228] ​

[0229] Fn1 polypeptide group (MI + FN1P): the test animals were modeled according to the method in step one of Example 5, and 5 minutes after the MI operation, Fn1 polypeptide solution was injected into the tail vein of each mouse, and the injection dose of Fn1 polypeptide was 2.5 mg / (kg·bw);

[0230] PBS control group (MI + PBS): the test animals were modeled according to the method in step one of Example 5, and 5 minutes after the MI operation, PBS was injected into the tail vein of each mouse, and the volume of PBS injection was the same as that of Fn1 polypeptide solution injection in the Fn1 polypeptide group;

[0231] Sham operation group (Sham): the test animals were sham operated according to the method in step one of Example 5, and 5 minutes after the sham operation, PBS was injected into the tail vein of each mouse, and the volume of PBS injection was the same as that of Fn1 polypeptide solution injection in the Fn1 polypeptide group.

[0232] CD31 is a marker of vascular endothelial cells. Three days after the MI operation or sham operation, the mice were sacrificed, dissected and the heart was obtained. The heart was sequentially fixed with 4% paraformaldehyde and paraffin-embedded, and after being cut, immunofluorescence staining was performed with CD31 antibody (Proteintech, 11265-1-AP), and then observed under a laser confocal microscope.

[0233] The microscopic image is shown in the left image of ​ , and the statistical results of the average CD31 positive area in each field of view are shown in the right image of ​ , and about 5 mice were counted in each group. The number of blood vessels in the Fn1 polypeptide group was significantly more than that in the PBS control group, indicating that Fn1 polypeptide promoted angiogenesis in the heart after MI.

[0234] Four, Fn1 polypeptide promotes cerebral angiogenesis after tMCAO

[0235] Test animals: 8-10 week old male C57BL / 6J mice.

[0236] Fn1 polypeptide group (IS-FN1P): the tMCAO model was modeled according to the method in step one of Example 7; 6 hours after the operation, 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 at 3 days, 6 days, 9 days and 12 days after the operation, and the single dose of Fn1 polypeptide injection was 2.5 mg / (kg·bw);

[0237] PBS control group (IS-PBS): the modeling of tMCAO model was performed according to the method of Step one of Example 7; 6h after the operation, PBS was injected into the tail vein, and the volume of PBS injection was the same as the volume of Fn1 polypeptide solution injection in the Fn1 polypeptide group; 3 days, 6 days, 9 days and 12 days after the operation, PBS was injected into the tail vein, and the volume of PBS injection was the same as the volume of Fn1 polypeptide solution injection in the Fn1 polypeptide group;

[0238] Sham operation group (Sham): sham operation was performed according to the method of Step one of Example 7; 6h after the sham operation, PBS was injected into the tail vein of each mouse; 6h after the operation, PBS was injected into the tail vein, and the volume of PBS injection was the same as the volume of Fn1 polypeptide solution injection in the Fn1 polypeptide group; 3 days, 6 days, 9 days and 12 days after the operation, PBS was injected into the tail vein, and the volume of PBS injection was the same as the volume of Fn1 polypeptide solution injection in the Fn1 polypeptide group.

[0239] CD31 is a marker of vascular endothelial cells. Ki67 is a marker of proliferating cells. 14 days after tMCAO operation or 14 days after sham operation, the mice were sacrificed, dissected and the brain was obtained. The brain was sequentially fixed with 4% paraformaldehyde and paraffin-embedded, and after being cut, immunofluorescence staining was performed with CD31 antibody and Ki67 antibody, and then laser confocal imaging was performed ​

[0240] The microscopic images are shown in the left panel of FIG. 6. The number of CD31+ and Ki67+ cells per unit area in each field is shown in the right panel of FIG. 6, and 8-10 mice were counted in each group. Compared with the PBS group, the number of blood vessels in the Fn1 polypeptide group was significantly increased, indicating that the Fn1 polypeptide promoted the angiogenesis in the brain after middle cerebral artery occlusion. ​ ​ In summary, the Fn1 polypeptide not only promotes the vascular formation ability of endothelial cells in vitro, but also promotes the angiogenesis in the heart and brain after ischemia.

[0241] In summary, the Fn1 polypeptide not only promotes the vascular formation ability of endothelial cells in vitro, but also promotes the angiogenesis in the heart and brain after ischemia.

[0242] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wide range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, the present application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the present application. Some basic features can be applied within the scope of the following appended claims.​

Claims

1. A polypeptide, named Fn1 polypeptide, the sequence of which is shown in SEQ ID NO:

3.

2. An RNA molecule, named Fn1-new-112 transcript, the sequence of which is shown in SEQ ID NO:

1.

3. A DNA molecule encoding the polypeptide of claim 1 or encoding the RNA molecule of claim 2.

4. 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 3.

5. Use of the polypeptide of claim 1 or the RNA molecule of claim 2 or the DNA molecule of claim 3 or the biological material of claim 4 in the preparation of a medicament; the indication of the medicament is cardiac injury caused by myocardial infarction and / or brain injury caused by stroke; the medicament is used for treating and / or preventing the indication.

6. The use of claim 5, wherein: the use of the medicament is (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) for improving cardiac dysfunction; (b) for inhibiting myocardial fibrosis; (c) for inhibiting cardiomyocyte apoptosis; (d) for improving impaired cardiac function; (e) for improving the extent of cerebral infarction; (f) for increasing cerebral blood perfusion; (g) for improving impaired brain function; (h) for promoting endothelial cell formation of blood vessels; (i) for promoting endothelial cell migration; (j) for promoting angiogenesis in the heart and / or brain; (k) for improving hypoxic injury of myoblasts or cardiomyocytes.

7. Use of the polypeptide of claim 1 or the RNA molecule of claim 2 or the DNA molecule of claim 3 or the biological material of claim 4 in the preparation of a medicament; the indication of the medicament is myocardial infarction and / or stroke; the medicament is used for treating and / or preventing the indication.

8. The use of claim 7, wherein: the use of the medicament is (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) for improving cardiac dysfunction; (b) for inhibiting myocardial fibrosis; (c) for inhibiting cardiomyocyte apoptosis; (d) for improving impaired cardiac function; (e) for improving the extent of cerebral infarction; (f) for increasing cerebral blood perfusion; (g) for improving impaired brain function; (h) for promoting endothelial cell formation of blood vessels; (i) for promoting endothelial cell migration; (j) for promoting angiogenesis in the heart and / or brain; (k) for improving hypoxic injury of myoblasts or cardiomyocytes.

9. A medicament comprising the polypeptide of claim 1 or the RNA molecule of claim 2 or the DNA molecule of claim 3. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

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