CN114438230A - Cell development map and marker gene of human three-month-old embryo mandible tissue - Google Patents
Cell development map and marker gene of human three-month-old embryo mandible tissue Download PDFInfo
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Abstract
The invention provides a cell development map of a mandible tissue of a human three-month-old embryo, which is characterized by comprising 19 cell groups of endothelial cells, Schwann cells, proliferating cells, satellite cells, muscle cells, erythrocytes, myoblasts, transformed cells 3, transformed cells 1, mesenchymal stem cells, monocytes, osteoclasts, transformed cells 2, neural crest cells, osteoblasts, epithelial cells, chondrocytes, pericytes and tendon cells. The present invention also provides a marker gene for the development of the mandible tissue of a human three-month-old embryo, comprising the 19 cell populations according to claim 1. The invention also provides an application of the cell development marker gene of the human three-month-old embryonic mandibular tissue in judging the developmental stage of the embryonic mandibular tissue and other conditions. The invention provides a cell development map of the mandible tissue of a human three-month-old embryo, and provides new basic data for researching the growth and development of the mandible.
Description
Technical Field
The invention belongs to the technical field of bioscience, relates to a cell development map and a marker gene of a mandible tissue of a human three-month-old embryo, and particularly relates to a cell development map of the mandible tissue of the human three-month-old embryo, a cell development marker gene of the mandible tissue of the human three-month-old embryo, application of the cell development marker gene in judging the development condition of the mandible tissue of the embryo, and a construction method of the cell development map of the mandible tissue of the human three-month-old embryo.
Background
At present, research in developmental biology is mainly based on model organisms. Because of practical challenges, human embryonic development (from zygote to fetal birth) remains a poorly understood "black box". Recently, a combination of studies from multiple national research institutes has described roadmaps constructed from Human Developmental Cell maps and gestational reference maps, with associated results published in the journal of Nature, entitled "A roadmap for the Human development Cell Atlas".
The Human Development Cell Atlas (HDCA) plan is a part of the Human Cell Atlas (HCA) plan, and aims to construct a comprehensive reference Cell Atlas at each stage of Human development (from fertilized egg to fetus birth) and realize the Cell visualization of the temporal and spatial expression of Human embryo development.
The construction of HDCA requires the realization of single cell measurements across three-dimensional space and time to capture dynamic developmental processes including cell proliferation, migration, and regulation, etc., all of which face significant challenges in experimental techniques, computational analysis, and visualization algorithms, etc. In this study, researchers developed concepts and computational frameworks to capture embryonic cells and their morphological changes; integration of single-cell molecular profiles based on RNA, chromatin accessibility, methylation or specific proteins, allowing a more detailed definition of cell type and state; the calculation integration and visualization of multigroup data are realized, so that the practicability of the map is improved. The construction of HDCA not only helps to reveal the pathogenesis of many pediatric diseases, but also will play an important role in cell/tissue engineering, clinical therapy, regenerative medicine, and other fields.
However, the development map of the human embryonic mandibular tissue has not attracted attention and is not reported at present, so that the construction of the cell development map of the human embryonic mandibular tissue plays an important role in the research of the growth and development of the mandibular tissue. The inventor group continuously carries out related research and provides a more accurate development map based on the existing basis.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems or the defects in the prior art, the invention provides a cell development map and a marker gene of a mandible tissue of a human three-month-old embryo.
In order to achieve the above object, an embodiment of the present invention provides a cell development map of mandibular tissue of a human three-month-old embryo, characterized in that: including endothelial cells, Schwann cells, proliferating cells, satellite cells, muscle cells, erythrocytes, myoblasts, transformed cells 3, transformed cells 1, mesenchymal stem cells, monocytes, osteoclasts, transformed cells 2, neural crest cells, osteoblasts, epithelial cells, chondrocytes, pericytes, tendon cells.
Further, the percentage of 19 of the cell populations was as follows:
the embodiment of the invention also provides a cell development marker gene of the mandibular tissue of a human three-month-old embryo, which is characterized by comprising 19 cell groups of endothelial cells, Schwann cells, proliferating cells, satellite cells, muscle cells, erythrocytes, myoblasts, transformed cells 3, transformed cells 1, mesenchymal stem cells, monocytes, osteoclasts, transformed cells 2, neural crest cells, osteoblasts, epithelial cells, chondrocytes, pericytes and tendon cells.
The embodiment of the invention also provides application of the cell development marker gene of the human three-month-old embryonic mandibular tissue in judging the developmental stage of the embryonic mandibular tissue and other conditions.
Further investigations described: the marker gene comprises 19 cell groups of endothelial cells, Schwann cells, proliferating cells, satellite cells, muscle cells, erythrocytes, myoblasts, transformed cells 3, transformed cells 1, mesenchymal stem cells, monocytes, osteoclasts, transformed cells 2, neural crest cells, osteoblasts, epithelial cells, chondrocytes, pericytes and tendon cells.
The invention also provides a construction method of the cell development map of the mandibular tissue of the human three-month-old embryo, which is characterized by comprising the following steps: single cell sequencing is carried out on the mandible tissue of the human three-month-old embryo, the UMAP dimension reduction and grouping are carried out, and the Marker gene is utilized to annotate the cell group, so that the cell development map of the mandible tissue of the human three-month-old embryo is constructed.
Furthermore, the construction method of the cell development map of the mandible tissue of the human three-month-old embryo specifically comprises the following steps:
s1, single cell suspension preparation: washing the mandible tissue with Hanks equilibrium solution for three times, and cutting into 1-2 mm2The tissue pieces of (4) were digested in 2ml of GEXSCOPET tissue dissociation solution at 37 ℃ for 15 minutes, after which the cells were separated from cell debris and impurities using a 40 μm sterile filter, centrifuged at 1000rpm for 5min at 4 ℃, resuspended in 1ml of PBS, and counted using a TC20 automated cell counter.
S2, single cell library construction and sequencing: PBS was used to adjust the concentration of single cell suspension to 1X 105cells/ml, then adding the single cell suspension into a microfluidic chip, and constructing a single cell RNA-seq library according to the kit instruction; after the single cell suspension flows into the microchip, the single cell enters a single-hole chamber on the chip, a cell bar code bead is added and cleaned, then 100 mu l of single cell lysate is added into the chip, the cell is lysed at room temperature and mRNAs are captured; the cell barcode beads and the captured RNAs together were isolated from the microchip, followed by reverse transcription, cDNA amplification and library construction; after fragment size selection and purification, performing double-end sequencing on the scRNA-seq library on an Illumina HiSeq X10 instrument; and processing and filtering the original data by using an SCOPE-tools software toolkit to generate a gene expression matrix.
S3, single cell sequencing analysis: single cell sequencing analysis was performed using the securat packet in the R language, filtering to remove genes expressed in 3 cells and below; keeping the cells with the gene expression quantity of 200 to 4000 genes and 50 percent less mitochondrial genes for subsequent analysis; normalizing the expression data by using a sctransform algorithm, firstly, selecting a mitochondrial ratio, the number of UMIs of cells and the number of genes of the cells, and performing first normalization on an expression matrix; calculating a Cell cycle score by using a Cell cycle score method in Seurat based on the expression of Cell cycle-associated genes in S phase and G2M phase; selecting a mitochondrial ratio, the number of UMIs of the cells, the gene factors of the cells, the expression score of the S-phase related genes and the expression score of the G2M-phase related genes, and performing second normalization on an expression matrix; finally, 3000 characteristic genes are selected for multi-sample integration; extracting the first 50 main components of the expression matrix, performing dimension reduction on the expression matrix by using a UMAP algorithm, grouping cell groups by using FindNeighbors and FindClusters functions, identifying 19 groups of cells in total, and calculating Marker genes of each group by using FindAllMarkers functions; annotating the types of the mandibular tissue cell populations of human three-month-old embryos based on Marker genes of different cell types reported in literature to obtain the mandibular tissue cell development map.
S4, statistics of proportion distribution of each cell population: based on the grouping obtained in step S3 and the information on the number of cells in each group, the proportion of each cell group was counted using a mapping tool provided in the R language.
The technical scheme of the invention has the following beneficial effects: the method collects the mandible tissues of the human three-month-old embryos and combines a single cell sequencing technology to successfully construct the cell development map of the mandible tissues of the human three-month-old embryos; the invention can fill the blank of the construction of the cell development map of the mandible tissue of the human three-month-old embryo, and open up a new visual angle for the subsequent research of the growth and development of the mandible tissue; in the invention, the mandible tissue of a human three-month-old embryo is divided into 19 cell groups, wherein part of the cell groups are reported in a literature and are new cell groups of the mandible tissue identified for the first time; the invention provides a complete and comprehensive cell development map of a mandible tissue of a three-month-old embryo; the Marker genes used for group annotation in the invention are reported in documents and verified in experiments, and the reality and accuracy of the cell map are further enhanced by adopting the Marker genes to annotate the cell group.
Drawings
FIG. 1 is a UMAP cell population map of the mandibular tissue of a human three-month-old embryo of the present invention.
FIG. 2 is a thermal map of the Marker gene of the mandibular tissue cell population of a human three month old embryo of the invention.
FIG. 3 is a drawing showing a ratio of the mandibular tissue cell population of a human three-month-old embryo according to the present invention.
FIG. 4 is a GO analysis (cell function) diagram of the human triply-aged embryos mandibular tissue cell population GO.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is given with reference to specific embodiments.
The method carries out single cell sequencing on the mandible tissue of a human three-month-old embryo, reduces Dimension and divides groups through UMAP (Uniform Manifold Approximation and Projection algorithm), and annotates cell groups by using Marker genes (cell group characteristic expression genes) to construct a cell development map of the mandible tissue of the human embryo. The method comprises the following specific steps:
1. single cell suspension preparation
The mandible tissue was washed three times with Hanks equilibration solution, cut into 1-2 mm tissue pieces, digested in 2ml GEXSCOPETM (Singleron Biotechnologies) tissue dissociation solution at 37 ℃ for 15 minutes, after which the cells were separated from cell debris and impurities with a 40 μm sterile filter (Corning), centrifuged at 1000rpm at 4 ℃ for 5min, resuspended in 1ml PBS (HyClone), and counted using a TC20 automated cell counter (Bio-Rad).
2. Single cell library construction and sequencing
PBS was used to adjust the concentration of single cell suspension to 1X 105cells/ml, and then adding the Single Cell suspension to a microfluidic chip (Single Cell RNA-seq Kit, Single Electron Biotechnologies), according to the Kit instructionsThe instructions construct a single-cell RNA-seq library.
After the single cell suspension flowed into the microchip, the single cells were placed in the single well chamber on the chip, the cell barcode beads were added and washed, then 100. mu.l of single cell lysate was added to the chip, the cells were lysed at room temperature and the mRNAs were captured. The cell barcode beads and the captured RNAs were isolated together from the microchip, followed by reverse transcription, cDNA amplification and library construction. After fragment size selection and purification, the scRNA-seq library was double-ended sequenced on the Illumina HiSeq X10 instrument. The raw data was processed and filtered using the SCOPE-tools (https:// https:// githu. com/singleron Bio/SCOPE-tools) software toolkit to generate a gene expression matrix. The concrete process is briefly described as follows: poor quality reads and linker sequences are removed from the original fastq file to obtain clean reads. The obtained clean reads were mapped onto the human genome (GRCh38, annotated version 92). The mapping results are integrated with the cell barcodes and Unique Molecular Identifiers (UMIs) extracted from read 1 and converted into an expression matrix.
3. Single cell sequencing analysis
Single cell sequencing analysis was performed using the saurat package in the R language (4.0.3) (Stuart et al, 2019), filtering to remove genes expressed in 3 cells and below. The cells with gene expression level between 200 and 4000 genes and less than 50% of mitochondrial genes were retained for subsequent analysis. Expression data were normalized using the sctransform (sct) algorithm, first selecting the mitochondrial ratio (percent of mitochondria), the number of UMIs in the cells (nCount _ RNA), and the cell basis factor (nFeature _ RNA), and first normalizing the expression matrix. Cell cycle scores were calculated using the Cell cycle score method in Seurat based on the expression of Cell cycle associated genes in S phase and G2M phase. The mitochondrial ratio (percentage of mitochondria), the number of UMIs of the cells (nCount _ RNA), the gene factor of the cells (nfear _ RNA), the expression score of the S-phase-related gene (s.score), and the expression score of the G2M-phase-related gene (G2m.score) were selected and normalized for the expression matrix a second time. Finally, 3000 signature genes were selected for multi-sample integration. The first 50 main components of the expression matrix are extracted, the dimension reduction of the expression matrix is carried out by using a UMAP algorithm, the cell groups are grouped by using FindNeighbors and FindClusters (the resolution is 0.25), as shown in figure 1, 19 groups of cells are identified in total, the Marker genes of each group are calculated by using FindAllMarkers functions (min. pct. 0.25 and logfc. threshold. 0.25), and the Marker gene heat map of the cell group of the mandible of the human three-month-old embryo is shown in figure 2. The method is characterized in that the types of the mandible tissue cell groups of the human three-month-old embryos are annotated based on Marker genes of different cell types reported in literature, the Marker genes are used for annotating the mandible tissue development maps of the human three-month-old embryos, and the mandible tissue cell development maps are obtained as shown in the table 1.
TABLE 1 Marker Gene summary Table for annotating human 3-month-old embryo mandibular tissue development profiles
4. Statistics of proportion distribution of individual cell populations
Counting the proportion of each cell group and the proportion of the cell groups of the mandible tissue of the human three-month-old embryo by utilizing a drawing tool carried by the R language based on the grouping obtained in the step 3 and the cell quantity information of each group, as shown in a figure 3; the statistical results of the statistics of the ratios of the cells of the mandibular tissue of human three-month-old embryos are shown in Table 2. GO analysis was performed on each cell population, and the results of GO analysis (cell function) on the cell population of the mandible tissue of a human three-month-old embryo are shown in fig. 4.
TABLE 2 statistical table of ratios of cells in mandibular tissues of human three-month-old embryos
The invention can fill the blank of constructing the cell development map of the mandible tissue of the human three-month-old embryo and open up a new visual angle for the follow-up research of the growth and development of the mandible tissue. In the invention, the mandible tissue of the human three-month-old embryo is divided into 19 cell groups, wherein part of the cell groups are reported in the literature, and the part of the cell groups are new cell groups of the mandible tissue which is firstly identified. The invention provides a complete and comprehensive cell development map of mandibular tissues of three-month-old embryos. The Marker genes for group annotation in the invention are reported in literature and verified in experiments. The fact and accuracy of the cell map are further enhanced by annotating the cell population with Marker genes.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (8)
1. A cell development map of a mandibular tissue of a human three-month-old embryo, comprising 19 cell populations of endothelial cells, Schwann cells, proliferating cells, satellite cells, muscle cells, erythrocytes, myoblasts, transformed cells 3, transformed cells 1, mesenchymal stem cells, monocytes, osteoclasts, transformed cells 2, neural crest cells, osteoblasts, epithelial cells, chondrocytes, pericytes, and tendon cells.
2. The map of claim 1, wherein the percentage of 19 of said cell populations is as follows:
3. a marker gene for the development of cells in the mandibular tissue of a human three-month-old embryo, comprising the 19 cell populations of claim 1.
4. Use of the marker gene for the development of the mandibular tissue of the human three-month-old embryo according to claim 3 for the determination of the developmental status of the mandibular tissue of the embryo.
5. The use of claim 4, wherein said marker gene comprises 19 cell populations of endothelial cells, Schwann cells, proliferating cells, satellite cells, muscle cells, erythrocytes, myoblasts, transformed cell 3, transformed cell 1, mesenchymal stem cells, monocytes, osteoclasts, transformed cell 2, neural crest cells, osteoblasts, epithelial cells, chondrocytes, pericytes, tendon cells.
6. The use according to claim 4, wherein the percentage of 19 of said cell populations is as claimed in claim 2.
7. A method for constructing a cellular development map of the mandibular tissue of a human three month old embryo according to claim 1, comprising the steps of: single cell sequencing is carried out on the mandible tissue of the human three-month-old embryo, the UMAP dimension reduction and grouping are carried out, and the Marker gene is utilized to annotate the cell group, so that the cell development map of the mandible tissue of the human three-month-old embryo is constructed.
8. The construction method according to claim 7, comprising the steps of:
s1, single cell suspension preparation: washing the mandible tissue with Hanks equilibrium solution for three times, and cutting into 1-2 mm2The tissue pieces of (4) were digested in 2ml of GEXSCOPETM tissue dissociation fluid at 37 ℃ for 15 minutes, after which the cells were separated from cell debris and impurities using a 40 μm sterile filter, centrifuged at 1000rpm for 5min at 4 ℃, resuspended in 1ml of PBS, and counted using a TC20 automated cell counter;
s2, single cell library construction and sequencing: PBS was used to adjust the concentration of single cell suspension to 1X 105cells/ml, then monomersAdding the cell suspension into a microfluidic chip, and constructing a single-cell RNA-seq library according to the kit instruction; after the single cell suspension flows into the microchip, the single cell enters a single-hole chamber on the chip, a cell bar code bead is added and cleaned, then 100 mu l of single cell lysate is added into the chip, the cell is lysed at room temperature and mRNAs are captured; the cell barcode beads and the captured RNAs together were isolated from the microchip, followed by reverse transcription, cDNA amplification and library construction; after fragment size selection and purification, performing double-end sequencing on the scRNA-seq library on an Illumina HiSeq X10 instrument; processing and filtering original data by using an SCOPE-tools software toolkit to generate a gene expression matrix;
s3, single cell sequencing analysis: single cell sequencing analysis was performed using the securat packet in the R language, filtering to remove genes expressed in 3 cells and below; keeping the cells with the gene expression quantity of 200 to 4000 genes and less than 50 percent of mitochondrial genes for subsequent analysis; normalizing the expression data by using a sctransform algorithm, firstly, selecting a mitochondrial ratio, the number of UMIs of cells and the number of genes of the cells, and performing first normalization on an expression matrix; calculating a Cell cycle score using the Cell cycle score method in Seurat based on the expression of Cell cycle associated genes in S phase and G2M phase; selecting a mitochondrial ratio, the number of UMIs of the cells, the gene factors of the cells, the expression score of the S-phase related genes and the expression score of the G2M-phase related genes, and performing second normalization on an expression matrix; finally, 3000 characteristic genes are selected for integration of multiple samples; extracting the first 50 main components of the expression matrix, performing dimension reduction on the expression matrix by using a UMAP algorithm, grouping cell groups by using FindNeighbors and FindClusters functions, identifying 19 groups of cells in total, and calculating Marker genes of each group by using FindAllMarkers functions; annotating the types of the mandibular tissue cell populations of human three-month-old embryos based on Marker genes of different cell types reported in literature to obtain a mandibular tissue cell development map;
s4, statistics of proportion distribution of each cell population: based on the grouping obtained in step S3 and the information on the number of cells in each group, the proportion of each cell group was counted using a mapping tool provided in the R language.
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