CN113046308A - Novel fibroblast for promoting repair after heart injury and identification method thereof - Google Patents
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Abstract
The invention provides a novel fibroblast for promoting repair after heart injury and an identification method thereof, which explores the difference change in the aspects of epigenetic inheritance and gene regulation in a mouse myocardial infarction disease model by using a single-cell ATAC-seq technology and chromatin patency research of high-throughput sequencing. The invention discovers a novel fibroblast in a myocardial infarction disease model, and the fibroblast has the characteristics of both a myocardial cell and a fibroblast, which has profound significance for researching the effect of the fibroblast on repairing the heart in the pathological process.
Description
Technical Field
The invention belongs to the technical field of cell biology, and relates to novel fibroblasts for promoting repair of damaged heart and a marking method thereof.
Background
Myocardial fibroblasts (CF) play a central role in ventricular remodeling associated with different types of fibrosis. At present, Farbehi, Patrick et al have found various cell subsets in myocardial tissue of myocardial-infarcted mice using the single-cell RNA-seq technique (Farbehi N, Patrick R, Dorison A, Xaymardan M, Janbandhu V, Wystub-Lis K, Ho JW, Nordon RE, Harvey RP.Single-cell expression profiling modified dynamic flux of cardiac molecular, vascular and animal cells in height and in essence.2019; 8: e 43882.). Recent studies have shown that fibroblasts do not respond uniformly to cardiac injury. Since true fibroblast markers are limited, the heterogeneity of fibroblast populations in response to cardiac injury remains poorly characterized.
Disclosure of Invention
In view of the above problems, the present invention aims to provide a novel fibroblast for promoting repair after cardiac injury and a method for identifying the same, which utilizes a single-cell ATAC-seq technique to explore the difference changes in epigenetic and genetic regulation in a mouse myocardial infarction disease model through chromatin patency study of high throughput sequencing.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for identifying a novel class of fibroblasts that promote repair following cardiac injury, comprising:
1) constructing a mouse myocardial infarction disease model;
2) collecting a tissue sample, and carrying out single cell ATAC-seq and subsequent data processing, calibration and cluster analysis to obtain a transcription factor with differential expression;
3) immunofluorescence localizes proteins or novel fibroblasts specifically expressed in two groups of mouse heart tissues.
Preferably, in the step 1), SPF-grade 8-10-week-old C57BL/6J male black mice are selected for constructing a mouse myocardial infarction disease model.
Preferably, step 2) comprises cell clustering based on chromatin opening enrichment peaks or motifs and footprint analysis of Gata 5.
Preferably, step 3) comprises: fixing frozen tissue section with 4% paraformaldehyde for 10min, penetrating with 0.3% Triton X-100 for 10min, sealing with 10% goat serum for 1h, and incubating with primary antibodies from Cola1 and Gata5 at 4 deg.C overnight; the slides were washed 3 times with 0.3% Tween 20 and the corresponding secondary antibodies, Goat Anti-Rabbit IgG H & L and Goat Anti-Mouse IgG H & L, were incubated for 1H at room temperature; after 3 washes with 0.3% Tween 20, the Isl1 primary standard antibody was incubated for 1h at room temperature.
The invention also provides fibroblasts obtained by the above identification method, the subpopulation of which expresses the gene Col1a1 characteristic of fibroblasts and also expresses Gata5 and Isl 1.
The invention has the following beneficial effects:
(1) by constructing a mouse myocardial infarction disease model, the single-cell ATAC-seq results of left ventricular tissues of myocardial infarction groups and sham operation groups show that, except for the detection of Farbehi, most of the fibroblast subgroups discovered by Patrick et al through scRNA-seq, we also discover an unreported fibroblast subgroup; this subgroup expresses genes characteristic of fibroblasts such as Col1a1, while also expressing Gata5 and Isl1, both of which have been shown to be associated with differentiation and identification of cardiac progenitors, which we speculate to be associated with repair following cardiac injury due to myocardial infarction.
(2) Cell clustering based on chromatin opening enrichment peaks or motifs indicates that novel fibroblasts have both cardiomyocytes and fibroblasts characteristics.
(3) Footprinting analysis of Gata5 indicated that Gata5 was upregulated in binding in novel fibroblasts and cardiomyocytes.
The invention discovers a novel fibroblast in a myocardial infarction disease model, and the fibroblast has the characteristics of both a myocardial cell and a fibroblast, which has profound significance for researching the effect of the fibroblast on repairing the heart in the pathological process.
Drawings
FIG. 1 shows the single-cell ATAC-seq results of the left ventricular tissue of the myocardial infarction group and sham operation group according to the present invention.
FIG. 2a Browser track shows the scaTAC-seq signal of Gata5 in different cell populations and FIG. 2b Browser track shows the scaTAC-seq signal of Isl1 in different cell populations.
Figure 3UAMP results show that Gata5+ fibroblast subpopulation simultaneously expressed as fibroblast and cardiomyocyte marker genes.
FIG. 4 shows GO analysis of the Gata5+ fibroblast subpopulation marker gene.
FIG. 5 shows labeling of fibroblasts in myocardial tissue with Col1a1, while locating cells expressing Gata 5.
FIG. 6 shows labeling of fibroblasts in myocardial tissue with Col1a1, while locating cells expressing Gata5 and Isl 1.
Detailed Description
In order to more clearly illustrate the technical solution of the present invention, the present invention is further described by the following examples so that those skilled in the art can further the present invention, but the following examples do not limit the present invention in any way.
If not specifically stated, the materials adopted in the application are all the existing materials, can be directly purchased from the market, and the adopted method can also be realized by the conventional technical means.
Examples
The implementation process of the embodiment is as follows:
1. establishing mouse myocardial infarction disease model
6 SPF-grade C57BL/6J male black mice with age of 8-10 weeks were selected and randomly divided into myocardial infarction group (n-3) and sham operation group (n-3). The isoflurane is sucked before skin preparation and operation, a 2cm oblique incision parallel to the costal arch is made on the left side of the xiphoid process, the heart is slightly pushed to the right side of a mouse by the thumb of a left hand, the heart is fixed leftwards by the index finger and the middle finger of the left hand, force is slightly applied to the fourth five intercostals by the elbow hemostatic forceps, the heart is pressed leftwards and downwards by the index finger and the middle finger of the left hand while the elbow hemostatic forceps enter a heart chamber, the heart apex part is exposed out of the body, the junction of the continuation line of the left atrial appendage towards the heart apex and 1/3 in the heart, namely the approximate position of the left anterior descending branch, the needle is inserted by 6-0 silk thread, the needle insertion thickness is 2-3. After the chest gas was fully evacuated, the mouse skin incision was sutured and the mouse was placed on a thermostatic table until awakened. The sham group performed the same procedure as the myocardial infarction group, except that ligation was not performed. Mice were euthanized 14 days after left anterior descending ligation, left ventricular infarct area tissue was taken from the myocardial infarction group, and left ventricular tissue was taken from the sham operation group. Tissues were snap frozen in liquid nitrogen and subsequently stored at-80 ℃ until single cell ATAC sequencing.
2. Single cell ATAC-seq and processing, calibration and cluster analysis of data.
In this example, the raw scATAC-seq data was first processed by a 'cellrange-atac' tool downloaded from 10XGenomics that maps sequencing reads to MM10 genomic references and generates fragment files. Downstream analysis was then performed using R packs 'archR' with the fragment file as input. For quality control, less than 4000 cells with unique fragments or a TSS enrichment fraction of less than 8 were removed. The duplets are also deleted using default settings. The cells that pass the filter are then clustered by using the 'search' method implemented in the 'archR' packet with high resolution value (1.5 or 2). The 30 neighborhoods of UMAP are used for dimensionality reduction and visualization. For cell cluster annotation, we first integrated the cell annotation generated from previous scRNA-seq data using the 'addGeneIntegrationmatrix' function of 'archR', and then further collated the cell cluster annotation for a particular marker gene. For peak calls, we used the recommended "MACS 2" and set default settings on the aggregate coverage profile of the identified cell population. For the motif interpretation, footprint analysis, and the rest of the downstream analysis of the trace map, we used the functionality provided by the "archR" software package with the recommended settings.
The important conclusion is as follows
(1) Single cell ATAC-seq results for myocardial infarction and sham operated left ventricular tissue are shown in FIG. 1. in addition to Farbehi being detected, the majority of the fibrous cell subpopulations found by Patrick et al by scRNA-seq, we also found an unreported fibroblast subpopulation (as indicated by the arrow in FIG. 1); the population of fibroblasts at Gata5+ is up-regulated at Gata5 and Isl 1.
Note: in the actual picture, the new fibroblast subpopulation is shown as a dot of a different color from the existing fibroblasts, and since the drawing is a black and white drawing, only a portion of the representative dots are indicated by arrows for illustration.
(2) FIGS. 2a and 2b Browser track show the scaTAC-seq signals of Gata5 and Isl1 in different cell populations. There are no reported subpopulations of fibroblasts expressing fibroblast signature genes, such as Ddr2, Col1a1, Adgrd1, and Twist 1. According to the expression patterns of Myom3, Myo5b, and Actl11, Gata5+ fibroblasts are more similar to myofibroblasts and cardiomyocytes than cardiac fibroblasts.
(3) Cell clustering based on chromatin opening enrichment peaks or motifs showed that the Gata5+ fibroblast subpopulation expressed both fibroblast and cardiomyocyte marker genes as shown by the UAMP results in fig. 3.
(4) This subpopulation expresses genes characteristic of fibroblasts such as Col1a1, while also expressing Gata5 and Isl1, both of which have been shown to be associated with differentiation and identification of cardiac progenitors, which we speculate to be associated with repair of such fibroblasts following cardiac injury caused by myocardial infarction, fig. 4 GO analysis of Gata5+ fibroblast subpopulation marker genes.
3. Immunofluorescence mapping of specifically expressed proteins or novel fibroblasts in two groups of mouse heart tissues to construct a mouse myocardial infarction disease model as described above, euthanization was performed 14 days after surgery. After perfusion with PBS, left ventricular tissue was removed, fixed in 4% paraformaldehyde overnight, dehydrated with 30% sucrose, embedded in optimal cutting temperature compound, and refrigerated at-80 ℃. The myocardial tissue was cut into 5 μm thick sections at-20 ℃ and stored in a cryostat at-20 ℃ for staining. Frozen tissue sections were fixed with 4% paraformaldehyde for 10min, infiltrated with 0.3% Triton X-100 for 10min, blocked with 10% goat serum for 1h, and primary antibodies from two non-identical species, Cola1(Abcam, ab260043) and Gata5(Santa Cruz, sc-373684) were incubated overnight at 4 ℃. The slides were washed 3 times with 0.3% Tween 20, the corresponding secondary antibody, Goat Anti-Rabbit IgG H&L(Alexa
647) (Abcam, ab150083) and Goat Anti-Mouse IgG H&L(Alexa
488) (Abcam, ab150117) incubated at room temperature for 1 h. By usingAfter 3 washes with 0.3% Tween 20, Isl1 direct primary antibody (Abcam, ab203406) was incubated for 1h at room temperature. Sections were washed 3 times with 0.3% Tween 20 before mounting with DAPI-containing anti-quench mounting agent. After standing at room temperature for 5min, the slide was placed in a refrigerator at 4 ℃ until observed under a confocal laser microscope. The immunofluorescence results are shown in FIG. 5 and FIG. 6.
In FIG. 5, the Col1a1 is used to mark the fibroblasts in the myocardial tissue, and the cells expressing Gata5 are located to be the novel fibroblasts. In FIG. 6, the Col1a1 is used to mark the fibroblasts in the myocardial tissue, and the cells expressing both Gata5 and Isl1 are located as novel fibroblasts.
Claims (5)
1. A method for identifying a novel class of fibroblasts that promote repair following cardiac injury, comprising:
1) constructing a mouse myocardial infarction disease model;
2) collecting a tissue sample to carry out single cell ATAC-seq and subsequent data processing, calibration and cluster analysis to obtain a transcription factor with differential expression;
3) immunofluorescence localizes proteins or novel fibroblasts specifically expressed in two groups of mouse heart tissues.
2. The method for identifying a novel fibroblast for promoting repair after heart injury according to claim 1, wherein in the step 1), SPF-grade C57BL/6J male black mice with age of 8-10 weeks are selected for constructing a mouse myocardial infarction disease model.
3. The method of claim 1, wherein step 2) comprises a cell clustering analysis based on chromatin opening enrichment peaks or motifs and a footprint analysis of Gata 5.
4. The method of claim 1, wherein step 3) comprises: fixing frozen tissue section with 4% paraformaldehyde for 10min, penetrating with 0.3% Triton X-100 for 10min, sealing with 10% goat serum for 1h, and incubating with primary antibodies from Cola1 and Gata5 at 4 deg.C overnight; the slides were washed 3 times with 0.3% Tween 20 and the corresponding secondary antibodies, Goat Anti-Rabbit IgG H & L and Goat Anti-Mouse IgG H & L, were incubated for 1H at room temperature; after 3 washes with 0.3% Tween 20, the Isl1 primary standard antibody was incubated for 1h at room temperature.
5. Fibroblast cells obtained by the method for identifying a novel class of fibroblasts promoting repair after cardiac injury according to any one of claims 1 to 4, a subgroup of which expresses the gene characteristic of fibroblast cells, Col1a1, and at the same time also expresses Gata5 and Isl 1.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130216503A1 (en) * | 2010-04-28 | 2013-08-22 | The J. David Gladstone Institutes | Methods for Generating Cardiomyocytes |
| US20190062696A1 (en) * | 2017-08-23 | 2019-02-28 | Procella Therapeutics Ab | Use of neuropilin-1 (nrp1) as a cell surface marker for isolating human cardiac ventricular progenitor cells |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130216503A1 (en) * | 2010-04-28 | 2013-08-22 | The J. David Gladstone Institutes | Methods for Generating Cardiomyocytes |
| US20190062696A1 (en) * | 2017-08-23 | 2019-02-28 | Procella Therapeutics Ab | Use of neuropilin-1 (nrp1) as a cell surface marker for isolating human cardiac ventricular progenitor cells |
Non-Patent Citations (1)
| Title |
|---|
| ADRIAN RUIZ-VILLALBA等: "Single-cell RNA sequencing analysis reveals a crucial role for CTHRC1 (collagen triple helix repeat containing 1) cardiac fibrolasts after myocardial infaction", 《CIRCULATION》 * |
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Application publication date: 20210629 |