CN114563330A - Evaluation method for immune regulation correlation between self protein and mesenchymal stem cell Th1 - Google Patents
Evaluation method for immune regulation correlation between self protein and mesenchymal stem cell Th1 Download PDFInfo
- Publication number
- CN114563330A CN114563330A CN202111483872.4A CN202111483872A CN114563330A CN 114563330 A CN114563330 A CN 114563330A CN 202111483872 A CN202111483872 A CN 202111483872A CN 114563330 A CN114563330 A CN 114563330A
- Authority
- CN
- China
- Prior art keywords
- mscs
- transfection
- self
- protein
- mesenchymal stem
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 77
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 55
- 230000003832 immune regulation Effects 0.000 title claims abstract description 29
- 102000004169 proteins and genes Human genes 0.000 title claims description 20
- 238000011156 evaluation Methods 0.000 title abstract description 11
- 238000001890 transfection Methods 0.000 claims abstract description 60
- 210000004027 cell Anatomy 0.000 claims abstract description 36
- 238000012163 sequencing technique Methods 0.000 claims abstract description 20
- 230000014509 gene expression Effects 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 14
- 230000007365 immunoregulation Effects 0.000 claims abstract description 11
- 238000012216 screening Methods 0.000 claims abstract description 11
- 238000004113 cell culture Methods 0.000 claims abstract description 6
- 230000035755 proliferation Effects 0.000 claims description 22
- 210000003954 umbilical cord Anatomy 0.000 claims description 17
- 210000000447 Th1 cell Anatomy 0.000 claims description 16
- 210000003074 dental pulp Anatomy 0.000 claims description 16
- 210000001519 tissue Anatomy 0.000 claims description 11
- 230000001506 immunosuppresive effect Effects 0.000 claims description 10
- 108020004459 Small interfering RNA Proteins 0.000 claims description 9
- 230000003169 placental effect Effects 0.000 claims description 9
- 101000669460 Homo sapiens Toll-like receptor 5 Proteins 0.000 claims description 8
- 102100039357 Toll-like receptor 5 Human genes 0.000 claims description 8
- 230000002519 immonomodulatory effect Effects 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 5
- 230000004957 immunoregulator effect Effects 0.000 claims description 5
- 206010062016 Immunosuppression Diseases 0.000 claims description 4
- 230000033228 biological regulation Effects 0.000 claims description 4
- 210000001185 bone marrow Anatomy 0.000 claims description 4
- 238000005206 flow analysis Methods 0.000 claims description 4
- 230000001900 immune effect Effects 0.000 claims description 4
- 238000011529 RT qPCR Methods 0.000 claims description 3
- 102000002689 Toll-like receptor Human genes 0.000 claims description 3
- 108020000411 Toll-like receptor Proteins 0.000 claims description 3
- 210000001691 amnion Anatomy 0.000 claims description 3
- 210000003785 decidua Anatomy 0.000 claims description 3
- 210000000207 lymphocyte subset Anatomy 0.000 claims description 3
- 238000010200 validation analysis Methods 0.000 claims description 3
- 238000001262 western blot Methods 0.000 claims description 3
- 238000010276 construction Methods 0.000 claims description 2
- 230000002452 interceptive effect Effects 0.000 claims description 2
- 230000002596 correlated effect Effects 0.000 claims 1
- 230000005764 inhibitory process Effects 0.000 abstract description 20
- 201000010099 disease Diseases 0.000 abstract description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 5
- 210000004271 bone marrow stromal cell Anatomy 0.000 abstract 1
- 238000001514 detection method Methods 0.000 abstract 1
- 239000012224 working solution Substances 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 101100153381 Homo sapiens TLR5 gene Proteins 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 101150035068 TLR5 gene Proteins 0.000 description 3
- 239000007640 basal medium Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010201 enrichment analysis Methods 0.000 description 2
- 230000008629 immune suppression Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 201000004569 Blindness Diseases 0.000 description 1
- 208000011231 Crohn disease Diseases 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 101000998181 Homo sapiens Interleukin-17B Proteins 0.000 description 1
- 101000831496 Homo sapiens Toll-like receptor 3 Proteins 0.000 description 1
- 102100033101 Interleukin-17B Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 210000004241 Th2 cell Anatomy 0.000 description 1
- 102100024324 Toll-like receptor 3 Human genes 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000001612 chondrocyte Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000000513 principal component analysis Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 208000037922 refractory disease Diseases 0.000 description 1
- 210000003289 regulatory T cell Anatomy 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 102000037983 regulatory factors Human genes 0.000 description 1
- 108091008025 regulatory factors Proteins 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000009168 stem cell therapy Methods 0.000 description 1
- 238000009580 stem-cell therapy Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N2015/1486—Counting the particles
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Pathology (AREA)
- Dispersion Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an evaluation method of self-protein and mesenchymal stem cell Th1 immunoregulation correlation, comprising the following steps: cell culture, gene sequencing, differential gene screening, differential gene assignment, cell transfection, Th1 immune regulation and control ability detection and evaluation of Th1 immune regulation and control ability. According to the method, heterogeneity exists in the aspect of Th1 immune regulation and inhibition of MSCs from different sources, the correlation between the self-protein and the Th1 immune regulation capacity of the mesenchymal stem cells is further explored and evaluated, and a basis is provided for detecting the cell quality by using the self-protein expressed by the mesenchymal stem cells; when the immune regulation is involved in treating diseases, a standard is provided for evaluating the immune regulation capacity of the mesenchymal stem cells by detecting the expression quantity of self-protein.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an evaluation method for immune regulation correlation between self-protein and mesenchymal stem cells Th 1.
Background
Mesenchymal Stromal Cells (MSCs), also commonly referred to as Mesenchymal stem cells, have multipotentiality and immunoregulatory ability, and have been widely used for treating various refractory diseases such as type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, crohn's disease, and the like. In recent years, the research on MSCs has led to encouraging results, some of which have entered the clinical phase from the preclinical phase, and some MSCs-based therapeutic products have received marketing approval from various national regulatory bodies.
MSCs may be derived from a range of tissues including umbilical cord, placenta, adipose tissue, bone marrow, gums, dental pulp, and the like. MSCs of different tissue origin have basic biological properties: such as proliferative capacity; multidirectional differentiation potential, which has the capability of differentiating into various cells such as osteoblasts, adipocytes and chondrocytes under a suitable in vivo or in vitro environment; the immune regulation function, which inhibits the proliferation of T cells and the differentiation of subgroups through the interaction among cells and the secretion of regulatory factors, thereby playing the role of immune reconstruction; some specific surface markers are expressed, and allograft rejection is light. MSCs derived from different tissues have different biological properties and exhibit significant tissue heterogeneity. One of the obvious functional characteristics of MSCs is their ability to secrete immune mediators or to interact directly with immune cells, exerting immunomodulatory effects. MSCs can obviously inhibit the proliferation of CD4+ T cells in vitro, obviously inhibit the differentiation of the T cells to Th1 and Th17 subgroups and obviously promote the differentiation of Tregs subgroups in PBMCs induced by IL-2.
Patent CN202110285278.8 discloses a quantitative standard for screening high-quality human umbilical cord mesenchymal stem cells with immunoregulatory ability, and during stem cell therapy, stem cells meeting conditions are selected according to the immunoregulatory ability requirement of stem cells transplanted into a body so as to improve the therapeutic effect. However, when the quantitative standard relates to the treatment of related diseases and clinical transformed cell selection of MSCs through Th1 immunoregulation function, the method has certain blindness, and cannot further evaluate the correlation between the immunoregulation capability of mesenchymal stem cells from different sources and self proteins, and cannot identify the self proteins capable of assisting in screening high-quality cells.
Disclosure of Invention
In view of the above, the invention is expected to provide an evaluation method of the immunomodulatory correlation between self-protein and the mesenchymal stem cell Th1, which can further evaluate the immunomodulatory capacity correlation between self-protein and the mesenchymal stem cell Th1, and provide a basis for detecting the quality of the mesenchymal stem cell by using the self-protein expressed by the mesenchymal stem cell.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
an evaluation method for immune regulation correlation between self protein and mesenchymal stem cell Th1 comprises the following steps:
1) and (3) cell culture: culturing and amplifying eight MSCs (mesenchymal stem cells) from tissues, including gingival MSCs, fat MSCs, placental decidua MSCs, placental chorionic MSCs, placental amnion MSCs, dental pulp MSCs, bone marrow MSCs and umbilical cord MSCs, wherein each MSCs is cultured by three strains respectively and amplified to P5 generation;
2) gene sequencing: extracting transcriptome genes of each strain of MSCs P5 generation cell sample in the step 1) and sequencing;
3) screening for differential genes: screening a differential gene related to Th1 immune regulation capacity of the MSCs according to the sequencing result of the step 2);
4) the differential genes were assigned: appointing the differential gene screened out in the step 3), and constructing a siRNA vector of the appointed differential gene, wherein the expressed self protein of the differential gene is to be evaluated for Th1 immunoregulation correlation;
here, the subsequent steps will further evaluate the correlation between the self protein expressed by the designated differential gene and the immunological regulation capacity of the mesenchymal stem cell Th1, and provide a basis for detecting the cell quality by using the self protein.
5) Cell transfection: selecting any two of eight P5-generation MSCs, transfecting by using the siRNA vector of the differential gene constructed in the step 4), interfering the expression of the specified gene, setting as a transfection group, additionally setting as a transfection control group, and respectively culturing cells under the same condition;
6) detecting Th1 immune regulation ability: and (3) carrying out lymphocyte subset Th1 subset flow analysis on the interfered cells of the transfection group and the transfection control group, obtaining the proportion of Th1 cells of the two MSCs transfection groups and the transfection control group in the step 5) through software analysis, calculating to obtain the ratio of Th1 to inhibit proliferation, and comparing and determining the variation trend of Th1 immunosuppressive capacity of the transfection group and the transfection control group according to the ratio of Th1 to inhibit proliferation.
Here, the greater the proliferation inhibition ratio of Th1 of the step 6), the stronger the immunosuppressive ability of the mesenchymal stem cell Th 1.
Further, the step 4) designates the differential gene as TLR5, and the constructed vector is TLR 5-siRNA.
Here, the TLR5-siRNA vector was purchased from Equida Biometrics and has the following sequence:
F-CCUGCUGAGCUUCAACUAUTT
R-AUAGUUGAAGCUCAGCAGGAG
further, the two P5-generation MSCs selected in step 5) are dental pulp MSCs and umbilical cord MSCs, respectively.
Further, the cells collected after transfection in step 5) are subjected to qPCR or Western Blotting validation.
Further, the software of the step 6) is Flowjo.
Further, the proliferation inhibition ratio of Th1 in step 6) was calculated in the form of [ (proportion of Th1 cells in the transfection control group-proportion of Th1 cells in the transfection control group)/proportion of Th1 cells in the transfection control group ]%.
Further, the invention also comprises a step of evaluating the immune regulation and control ability of Th1, if the content of self protein expressed by the differential gene specified in the step 4) is higher, the step 6) transfection group has stronger immune suppression ability than the transfection control group Th 1; if the content of the self protein expressed by the differential gene specified in the step 4) is less, the Th1 immunosuppression capability of the transfection group of the step 6) is weaker than that of the transfection control group.
Here, the proliferation inhibition ratio was calculated by [ (Th 1 cell ratio of transfection control group-Th 1 cell ratio of transfection control group)/Th 1 cell ratio of transfection control group ]%, and the larger the proliferation inhibition ratio of Th1, the stronger the immunosuppressive ability of mesenchymal stem cell Th 1. When the expression quantity of the self-protein is higher, namely the expression of the corresponding differential gene is higher, the smaller the proportion of the Th1 cells in the transfection group is, the larger the proliferation inhibition ratio is, the stronger the Th1 immune inhibition capability is, and the better the cell quality is, and at the moment, the self-protein and the mesenchymal stem cell Th1 immune regulation capability are in positive correlation. When the kit relates to immune regulation and control treatment of diseases, a standard is provided for indirectly evaluating the immune regulation capacity of the mesenchymal stem cells by detecting the expression quantity of self protein.
The invention has the following beneficial effects: 1) according to the method, the heterogeneity of MSCs in the aspect of Th1 immune regulation and inhibition is further explored to evaluate the correlation between the self-protein and the Th1 immune regulation capacity of the mesenchymal stem cells, and a basis is provided for detecting the cell quality by using the self-protein expressed by the mesenchymal stem cells; 2) the method provides a standard for evaluating the immunoregulation capability of the mesenchymal stem cells by detecting the expression quantity of self protein when the method relates to immunoregulation for treating diseases.
Drawings
FIG. 1 is a PCA diagram showing the sequencing results of eight MSCs in the example of the present invention;
FIG. 2 shows the results of screening out the differential genes from eight MSCs according to the present invention;
FIG. 3 is a KEGG enrichment analysis of dental pulp MSCs and umbilical cord MSCs according to an embodiment of the present invention;
FIG. 4 shows the result of Th1 immunomodulation following TLR5 interference between dental pulp MSCs and umbilical cord MSCs in accordance with the present invention.
Detailed Description
So that the manner in which the features and aspects of the present invention can be understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings.
An evaluation method of the immunomodulatory relevance of self protein and mesenchymal stem cell Th1 comprises the following steps:
1) cell culture: culturing and amplifying eight MSCs (mesenchymal stem cells) from tissues, including gingival MSCs, fat MSCs, placental decidua MSCs, placental chorionic MSCs, placental amnion MSCs, dental pulp MSCs, bone marrow MSCs and umbilical cord MSCs, wherein each MSCs is cultured by three strains respectively and amplified to P5 generation;
here, the number of MSCs derived from different tissues in this example was 8, and each MSCs had 3 different donor sources for 24 MSCs of different sex, different age, and different tissues.
Here, there is heterogeneity in Th1 immunoregulation between MSCs of different sources, and MSCs of different tissue sources were used for cell culture up to P5 generation, so that suitable MSCs were selected for different differential genes in the following steps.
2) Gene sequencing: extracting transcriptome genes of each strain of MSCs P5 generation cell sample in the step 1) and sequencing;
here, the transcriptome gene extraction and sequencing steps are as follows:
i leave a P5 cell sample for each cell line, the cell amount is 2X 106；
II, extracting total RNA of each cell sample, and digesting DNA by using DNase;
III enriching eukaryotic mRNA by magnetic beads with oligo (dT);
IV, adding an interrupting reagent to interrupt the mRNA into short segments of about 300bp, synthesizing the first-strand cDNA by using the interrupted mRNA as a template by using a reverse transcription kit, preparing a second-strand synthesis reaction system to synthesize second-strand cDNA, and purifying the double-strand cDNA; and (3) carrying out end repair on the purified double-stranded cDNA by adopting a library building kit, adding A tail, connecting a sequencing joint, then carrying out fragment size selection, and finally carrying out PCR amplification.
The interrupting agent is VAHTS 2 xFrag/Prime Buffer produced by Novozam, and the commodity number is N402-01;
the reverse transcription kit is produced by Invitrogen
III kit, with the product number of 18080-;
the library establishing kit is a Collibri ES DNAlibrary Prep Kits for Illumina Systems kit produced by Invitrogen, and the product number is A43605024.
The constructed library is qualified by the Agilent 2100Bioanalyzer, then is sent to Shanghai Europe and easy biomedical science and technology Limited, and is sequenced by using an Illumina HiSeq X Ten sequencer, wherein the sequencing program is PE150, and double-end data of 125bp or 150bp are generated.
And (3) carrying out PCA analysis on the sequencing result, investigating the distribution condition of the samples, and exploring the relation among the samples or verifying the experimental design.
FIG. 1 is a PCA diagram showing the sequencing results of eight MSCs, and as shown in FIG. 1, the closer the sample clustering distance or PCA distance is, the more similar the samples are. The samples of each group are distributed in different areas of two-dimensional or three-dimensional space, and the samples of the same group are more concentrated in spatial distribution.
3) Screening for differential genes: screening a differential gene related to Th1 immune regulation capacity of the MSCs according to the sequencing result of the step 2), wherein the process is as follows:
according to the sequencing results of the MSCs from 8 tissue sources, the gene sequencing results of different cells are compared in pairs to generate 28 groups of data, FIG. 2 shows the differential gene results screened by the eight MSCs, as shown in FIG. 2, the differential expression gene statistical histogram is shown, and the horizontal axis shows 28 comparison groups; the vertical axis is the number of differential genes in the comparison group.
The 28 sets of data are searched for the sequencing results of enrichment-KEGG, as shown in fig. 3, which is the results of KEGG enrichment analysis of dental pulp MSCs and umbilical cord MSCs, and the graph is screened for pathway entries corresponding to differential genes with a number greater than 2, and sorted from large to small according to-log 10Pvalue corresponding to each entry.
Searching genes related to Th1 and Th2 cell differentiation in a TERM list, listing all 28 groups of pairwise comparison genes, then searching the searched genes in a differential _ expressed _ gene folder of a sequencing result, screening the differential genes of padj <0.05 and fold Change >2.0 of each differential gene in two cells, sequencing the screened differential genes according to padj and fold Change values, and determining 9 genes related to Th1 and having obvious difference multiples, wherein the genes are respectively as follows: HLX, IL6, IL17B, IL1 α, IL1 β, IL27R α, TGF β R3, TLR3, TLR 5.
4) Specifying the differential genes: in the embodiment, the screened TLR5 protein expressed by the TLR5 gene is subjected to Th1 immune regulation correlation evaluation, and a basis is provided for detecting the quality of mesenchymal stem cells by using the self TLR5 protein;
construction of TLR5-siRNA for subsequent transfection of MSCs to interfere with TLR5 gene expression.
Here, the TLR5-siRNA vector was purchased from Equida Biometrics and has the following sequence:
F-CCUGCUGAGCUUCAACUAUTT
R-AUAGUUGAAGCUCAGCAGGAG
5) cell transfection: in this embodiment, two kinds of MSCs, namely dental pulp MSCs and umbilical cord MSCs, are selected, transfected with the TLR5-siRNA vector of the differential gene constructed in step 4), which interferes with TLR5 gene expression, and set as a transfection group, and a transfection control group is additionally set, and cell culture is performed under the same condition;
the specific transfection steps are as follows:
i, constructing a pair of TLR5-siRNA interference sequences, targeting and combining with TLR5 specific mRNA, inhibiting the expression of genes of the TLR5-siRNA interference sequences or silencing the genes, and blocking the expression of proteins.
In this example, the TLR5-siRNA vector was synthesized by the river mare bio corporation, with the following specific sequence:
F-CCUGCUGAGCUUCAACUAUTT
R-AUAGUUGAAGCUCAGCAGGAG
II, plating MSCs, and adding 800 μ L of complete culture medium (Lg-DMEM basal medium + 10% FBS + 1% streptomycin) during culture;
III12-16h later, the cell density is 60-70%, and transfection is carried out. siRNA: the volume ratio of RNAiMAX is 1: 3. Taking a 12-well plate as an example, the siRNA concentration is 50 nM;
IV, liquid preparation: SiRNA working solution 1: mixing 97.5 μ L basal medium +2.5 μ L siRNA, and standing at room temperature for 5 min; RNAiMAX working liquid 1: mixing 92.5 μ L of basal medium +7.5 μ L of RNAiMAX, and standing at room temperature for 5 min; transfection working solution 2: adding the RNAiMAX working solution 1 into the siRNA working solution 1, lightly mixing uniformly, and standing at room temperature for 20 min;
v, gently dripping 200 mu L of the transfection working solution 2 mixture into the transfected cells, and quickly and uniformly mixing;
VI, after transfection for 6 hours, the culture medium can be replaced by a normal complete culture medium for continuous culture or not; 24h after transfection, subsequent experiments can be carried out; cells can also be collected for qPCR or Western Blotting validation 48-72h after transfection.
6) Detecting Th1 immune regulation ability: and (3) carrying out lymphocyte subset Th1 subset flow analysis on the interfered cells of the transfection group and the transfection control group, and analyzing by Flowjo software to obtain the proportion of Th1 cells of the transfection group and the transfection control group of the two MSCs (dental pulp MSCs and umbilical cord MSCs) in the step 5), and calculating to obtain the proliferation inhibition ratio of Th1, wherein the proliferation inhibition ratio of Th1 is calculated in the mode of [ (the proportion of Th1 cells of the transfection control group-the proportion of Th1 cells of the transfection group)/the proportion of Th1 cells of the transfection control group ]%.
Here, the flow antibody marker used in this example for testing the Th1 immunoregulatory ability was CD3+CD8-IFN-γ+Purchased from BD corporation; the Th1 subgroup flow analysis adopts high-end flow cytometry sorter, model BD FACSAria II.
According to the proliferation inhibition rate of Th1, the trend of Th1 immunosuppression ability of the transfection group and the transfection control group is comparatively determined. The larger the proliferation inhibition ratio of Th1, the stronger the immunosuppressive ability of the mesenchymal stem cell Th 1.
FIG. 4 shows the immunoregulation results of Th1 after the interference of dental pulp MSCs and umbilical cord MSCs, as shown in FIG. 4, the proliferation inhibition ratio of blank control group Th1 was 31.8%, the proliferation inhibition ratio of umbilical cord MSCs transfected control group was 20.4%, the proliferation inhibition ratio of umbilical cord MSCs transfected group was 24.3%, the proliferation inhibition ratio of dental pulp MSCs transfected control group was 16.2%, and the proliferation inhibition ratio of dental pulp MSCs transfected group was 23.0%. FIG. 4 shows that the proliferation inhibition rate of Th1 in the transfected group of dental pulp MSCs and umbilical cord MSCs is higher than that in the corresponding transfected control group, the proportion of Th1 cells in the transfected group is relatively small, and the expression capacity of TLR5 is stronger, i.e. the immunosuppressive capacity of Th1 in the transfected group of dental pulp MSCs and umbilical cord MSCs is stronger and the cell quality is better than that in the transfected control group. Therefore, the higher the content of the TLR5 protein, the stronger the Th1 immune suppression capability of the MSCs is, and the positive correlation exists between the two.
Therefore, when the expression amount of the self-protein is higher, namely the expression of the corresponding differential gene is higher, the smaller the proportion of the Th1 cells in the transfection group is, the larger the proliferation inhibition ratio is, the stronger the Th1 immune inhibition capability is, and the better the cell quality is, at this time, the self-protein is in positive correlation with the mesenchymal stem cell Th1 immune regulation capability. When the kit relates to immune regulation and treatment of diseases, the evaluation of the Th1 immune regulation correlation between self protein and mesenchymal stem cells can provide a standard for indirectly evaluating the immune regulation capability of the mesenchymal stem cells by detecting the expression quantity of the self protein.
The specific steps of the method of the invention are not limited and detailed, and the specific components of the reagents are not detailed, as would be understood by one of ordinary skill in the art as a matter of common general knowledge.
The above description is only exemplary of the present invention and should not be taken as limiting the scope of the present invention, and any modifications, equivalents, improvements, etc. that are within the spirit and principle of the present invention should be included in the present invention.
Sequence listing
<110> Nanjing drum building hospital
<120> evaluation method for immune regulation correlation between self-protein and mesenchymal stem cell Th1
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA/RNA
<213> TLR5-siRNA vector F (Artificial sequence)
<400> 1
ccugcugagc uucaacuaut t 21
<210> 2
<211> 21
<212> DNA/RNA
<213> TLR5-siRNA vector R (Artificial sequence)
<400> 2
auaguugaag cucagcagga g 21
Claims (7)
1. A method for evaluating the immunomodulatory relevance of self protein and mesenchymal stem cell Th1 is characterized by comprising the following steps:
1) cell culture: culturing and amplifying eight MSCs (mesenchymal stem cells) from tissues, including gingival MSCs, fat MSCs, placental decidua MSCs, placental chorionic MSCs, placental amnion MSCs, dental pulp MSCs, bone marrow MSCs and umbilical cord MSCs, wherein each MSCs is cultured by three strains respectively and amplified to P5 generation;
2) gene sequencing: extracting transcriptome genes of each strain of MSCs P5 generation cell sample in the step 1) and sequencing;
3) screening for differential genes: screening a differential gene related to Th1 immune regulation capacity of the MSCs according to the sequencing result of the step 2);
4) specifying the differential genes: appointing the differential gene screened out in the step 3), and constructing a siRNA vector of the appointed differential gene, wherein the expressed self protein of the differential gene is to be evaluated for Th1 immunoregulation correlation;
5) cell transfection: selecting any two of eight P5-generation MSCs, transfecting by using the siRNA vector of the differential gene constructed in the step 4), interfering the expression of the specified gene, setting as a transfection group, additionally setting as a transfection control group, and respectively culturing cells under the same condition;
6) detecting Th1 immune regulation ability: and (3) carrying out lymphocyte subset Th1 subset flow analysis on the interfered cells of the transfection group and the transfection control group, obtaining the proportion of Th1 cells of the two MSCs transfection groups and the transfection control group in the step 5) through software analysis, calculating to obtain the ratio of Th1 to inhibit proliferation, and comparing and determining the variation trend of Th1 immunosuppressive capacity of the transfection group and the transfection control group according to the ratio of Th1 to inhibit proliferation.
2. The method for evaluating the Th1 immunoregulation correlation between self-protein and mesenchymal stem cell according to claim 1, wherein the differential gene designated in step 4) is TLR5, and the construction vector is TLR 5-siRNA.
3. The method for evaluating the immunomodulatory relevance of self-protein and Th1 of claim 1, wherein the two P5 generation MSCs selected in step 5) are dental pulp MSCs and umbilical cord MSCs.
4. The method for evaluating the immunological regulation correlation between the self-protein and the Th1 mesenchymal stem cell of claim 1, wherein the software of step 6) is Flowjo.
5. The method for evaluating the immunomodulatory correlation between self-protein and the Th1 of claim 1, wherein the proliferation-suppressing ratio of Th1 in step 6) is calculated by [ (ratio of Th1 cells in transfection control group-ratio of Th1 cells in transfection control group)/ratio of Th1 cells in transfection control group ]%.
6. The method for evaluating the immunological regulation correlation between self-protein and the Th1 of claim 1, further comprising a step of evaluating the immunological regulation ability of Th1, wherein if the content of self-protein expressed by the differential gene specified in the step 4) is higher, the Th1 of the transfection group of the step 6) has stronger immunosuppression ability than that of the transfection control group; if the content of the self protein expressed by the differential gene specified in the step 4) is less, the Th1 immunosuppression capacity of the transfection group of the step 6) is weaker than that of the transfection control group; the self protein is positively correlated with the Th1 immunoregulation of the mesenchymal stem cells.
7. The method for evaluating the Th1 immunoregulatory correlation between self-protein and mesenchymal stem cell according to claim 1, wherein the cells collected after transfection in step 5) are subjected to qPCR or Western Blotting validation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111483872.4A CN114563330A (en) | 2021-12-07 | 2021-12-07 | Evaluation method for immune regulation correlation between self protein and mesenchymal stem cell Th1 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111483872.4A CN114563330A (en) | 2021-12-07 | 2021-12-07 | Evaluation method for immune regulation correlation between self protein and mesenchymal stem cell Th1 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114563330A true CN114563330A (en) | 2022-05-31 |
Family
ID=81711520
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111483872.4A Withdrawn CN114563330A (en) | 2021-12-07 | 2021-12-07 | Evaluation method for immune regulation correlation between self protein and mesenchymal stem cell Th1 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114563330A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113980896A (en) * | 2021-10-27 | 2022-01-28 | 中国人民解放军军事科学院军事医学研究院 | Application of IRF1 in regulating and controlling immune regulation effect of mesenchymal stem cells and product |
-
2021
- 2021-12-07 CN CN202111483872.4A patent/CN114563330A/en not_active Withdrawn
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113980896A (en) * | 2021-10-27 | 2022-01-28 | 中国人民解放军军事科学院军事医学研究院 | Application of IRF1 in regulating and controlling immune regulation effect of mesenchymal stem cells and product |
| CN113980896B (en) * | 2021-10-27 | 2023-10-20 | 中国人民解放军军事科学院军事医学研究院 | Application of IRF1 in regulation and control of mesenchymal stem cell immunoregulation and product |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chan et al. | Identification of the human skeletal stem cell | |
| Roson-Burgo et al. | Insights into the human mesenchymal stromal/stem cell identity through integrative transcriptomic profiling | |
| Seshi et al. | Human bone marrow stromal cell: coexpression of markers specific for multiple mesenchymal cell lineages | |
| Jansen et al. | Functional differences between mesenchymal stem cell populations are reflected by their transcriptome | |
| Wojakowski et al. | Cardiomyocyte differentiation of bone marrow-derived Oct-4+ CXCR4+ SSEA-1+ very small embryonic-like stem cells | |
| JP2001103963A (en) | Various mesodermal line differentiation potency regarding interstitial cells derived from adipose tissue and use of the same differentiation potency | |
| CN104046593A (en) | Human cell with low immunogenicity and preparation method thereof | |
| Nakayama et al. | The development of induced pluripotent stem cell-derived mesenchymal stem/stromal cells from normal human and RDEB epidermal keratinocytes | |
| Benayahu et al. | Differentiation of bone marrow stroma-derived mesenchymal cells | |
| Tubsuwan et al. | Parallel assessment of globin lentiviral transfer in induced pluripotent stem cells and adult hematopoietic stem cells derived from the same transplanted β-thalassemia patient | |
| Srinivasan et al. | Strategies to enhance immunomodulatory properties and reduce heterogeneity in mesenchymal stromal cells during ex vivo expansion | |
| CN109735541B (en) | ACADSB gene knockout dairy cow mammary gland epithelial cell line and construction method thereof | |
| CN114563330A (en) | Evaluation method for immune regulation correlation between self protein and mesenchymal stem cell Th1 | |
| CN111690686B (en) | Application of miRNA high expression in promoting in-vitro proliferation and osteogenic differentiation of umbilical cord mesenchymal stem cells | |
| CN113789333B (en) | Application of Chi3l1 in regulating and controlling hUC-MSCs to inhibit Th17 differentiation-mediated immunoregulation | |
| CN106244679A (en) | MiR 100 inhibitor purposes in reducing cancer metastasis | |
| CA3239019A1 (en) | Human inducibility controllable t-cell and method for preparing same | |
| Kanazawa et al. | Hematopoietic–mesenchymal signals regulate the properties of mesenchymal stem cells | |
| CN111893091B (en) | Method for inducing rat very small embryonic stem cells to differentiate into myocardial cells | |
| CN113801843B (en) | Method for enhancing human urine-derived stem cell stem property | |
| CN114214285A (en) | Application of lncRNA in inhibiting aging of umbilical cord mesenchymal stem cells through high expression | |
| CN104120106B (en) | Utilize pig to dedifferente adipose cell and induce the method being differentiated to form Skeletal Muscle Cell | |
| Mao et al. | RNA‐seq and ATAC‐seq analyses of multilineage differentiating stress enduring cells: Comparison with dermal fibroblasts | |
| CN113789296A (en) | Method for efficiently inducing human umbilical cord mesenchymal stem cells to differentiate into liver cells and application | |
| Wang et al. | Human lymphoid-neutrophil/monocyte restriction co-ordinately activates increased proliferation despite parallel heterogeneity in transcriptional changes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20220531 |