CN112391444A - Application of mesenchymal stem cells with different functional characteristics in treating different diseases - Google Patents
Application of mesenchymal stem cells with different functional characteristics in treating different diseases Download PDFInfo
- Publication number
- CN112391444A CN112391444A CN202011312847.5A CN202011312847A CN112391444A CN 112391444 A CN112391444 A CN 112391444A CN 202011312847 A CN202011312847 A CN 202011312847A CN 112391444 A CN112391444 A CN 112391444A
- Authority
- CN
- China
- Prior art keywords
- single cell
- magnetic beads
- sequencing
- stem cells
- 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.)
- Pending
Links
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 27
- 201000010099 disease Diseases 0.000 title claims abstract description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 9
- 210000004027 cell Anatomy 0.000 claims abstract description 59
- 239000011324 bead Substances 0.000 claims abstract description 37
- 238000012163 sequencing technique Methods 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 30
- 238000006243 chemical reaction Methods 0.000 claims abstract description 11
- 238000012545 processing Methods 0.000 claims abstract description 9
- 239000002184 metal Substances 0.000 claims abstract description 6
- 238000012408 PCR amplification Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 31
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 28
- 239000000047 product Substances 0.000 claims description 28
- 239000006228 supernatant Substances 0.000 claims description 24
- 239000007788 liquid Substances 0.000 claims description 21
- 235000019441 ethanol Nutrition 0.000 claims description 14
- 239000011534 wash buffer Substances 0.000 claims description 13
- 230000003321 amplification Effects 0.000 claims description 11
- 239000006285 cell suspension Substances 0.000 claims description 11
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 6
- 239000011159 matrix material Substances 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 3
- 239000012139 lysis buffer Substances 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 abstract description 11
- 230000006870 function Effects 0.000 abstract description 10
- 238000005516 engineering process Methods 0.000 abstract description 9
- 238000003745 diagnosis Methods 0.000 abstract description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 10
- 230000008569 process Effects 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 7
- 239000000463 material Substances 0.000 description 5
- 230000029087 digestion Effects 0.000 description 4
- 210000000130 stem cell Anatomy 0.000 description 4
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 210000003954 umbilical cord Anatomy 0.000 description 4
- 238000007664 blowing Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000009168 stem cell therapy Methods 0.000 description 3
- 238000009580 stem-cell therapy Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000012089 stop solution Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000019771 cognition Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000010219 correlation analysis Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 108091008053 gene clusters Proteins 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 201000010065 polycystic ovary syndrome Diseases 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 208000037920 primary disease Diseases 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
Images
Classifications
-
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Cell Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of medical diagnosis, and particularly relates to application of mesenchymal stem cells with different functional characteristics in treating different diseases. The invention firstly discloses a preparation method of a single-cell transcriptome library, which comprises the following steps: processing by adopting an automatic single cell processing system, carrying out PCR amplification after reaction in a metal bath, adding purified magnetic beads, centrifuging, amplifying, and sequencing by utilizing an XNovagNS sequencing platform to obtain a single cell transcriptome library. The invention provides a method for diagnosing different diseases by using a single-cell transcriptome sequencing technology. The function of the mesenchymal stem cells from the same source is identified and the subgroup is subdivided by using a single-cell transcriptome sequencing technology, and a diagnosis and treatment system for performing accurate treatment by using the mesenchymal stem cells with different functions is initially established.
Description
Technical Field
The invention belongs to the technical field of medical diagnosis, and particularly relates to application of mesenchymal stem cells with different functional characteristics in treating different diseases.
Technical Field
Mesenchymal stem cells are pluripotent stem cells that have the ability to self-renew and differentiate in multiple ways, such as bone, cartilage, fat, bone marrow hematopoietic tissues, and the like. The mesenchymal stem cells can secrete a plurality of cytokines for inhibiting the occurrence of diseases, and have positive effects on inhibiting inflammatory reaction in a body and promoting cell regeneration. In addition, mesenchymal stem cells have low immunogenicity, have small differences among different individuals, have basically no adverse reaction when carrying out allograft, and have safety and reliability which are widely verified, so that the mesenchymal stem cells are widely used in cell therapy. The existing evidence also indicates that the mesenchymal stem cells have better diagnosis and treatment effects on various primary diseases, such as atherosclerosis, arthritis, polycystic ovary syndrome and the like. However, basic research related to stem cell therapy is weak, and deep mechanisms of stem cell therapy need to be further researched.
Single cell transcriptome sequencing technology, one of the emerging biotechnology technologies of this century, has been considered for a considerable period of time as an important research tool for exploring inter-cell heterogeneity. The rise of single cell sequencing technology also provides a new idea for omics research. The single cell transcriptome sequencing technology developed by Tang et al was published in 2009, and the number of genes which can be detected by the method far exceeds that of gene microarrays, so that the transcriptional characterization at the development stage can be more thoroughly detected. The methods greatly expand the cognition of human beings on life activities and accelerate the process of exploring life science of human beings. In subsequent studies, various improved and innovative approaches were developed in succession, including Smart-seq2, MARS-seq, CEL-seq2, and Drop-seq, among others. Based on different database construction methods, the methods can be roughly classified into two types, one is a full-length transcriptome sequencing method, the other is a sequencing method based on independent molecular marker (UMI), and the two types of methods have advantages and disadvantages. The main representatives of the full-length transcriptome sequencing are Smart-seq, Smart-seq2 and the like, and the advantages of the method are that the preference of the 3' end is lower and the sequencing depth is higher. However, this type of method is cumbersome and low in throughput because the banking process requires manual separation of cells into separate tubes. The sequencing method based on UMI is simpler than the former method in process flow, so that single cell library building can be carried out by a machine sorting mode. The UMI sequencing method represented by Drop-seq can sort tens of thousands of cells into single cell suspension in a short time, and wrap related reagents of reverse transcription into single cell droplets together, thereby realizing rapid and high-throughput sequencing.
Disclosure of Invention
Although existing studies have demonstrated that mesenchymal stem cells have some therapeutic effect in the treatment of various diseases, their specific mechanism is still unknown. In the process of stem cell therapy, the inventors also found that the curative effect of stem cells prepared under the same conditions is greatly different among different populations, and the reasons for the difference are still unknown. The inventor guesses that the functions of the mesenchymal stem cells from the same source are likely to be different, and even the mesenchymal stem cells from the same source are likely to have different secreted cytokines, proteins, transcripts and the like under different culture conditions. Therefore, the invention utilizes the single-cell transcriptome sequencing technology to identify the functions of the mesenchymal stem cells from the same source and subdivide the subgroups, and initially establishes a diagnosis and treatment system for carrying out accurate treatment by utilizing the mesenchymal stem cells with different functions.
The technical scheme is as follows:
1. performing transcriptome sequencing on the mesenchymal stem cells by using a single-cell transcriptome sequencing technology;
2. analyzing the single-cell transcription group data, and grouping the cells according to different gene functions;
3. and by excavating marker genes of cells of different subgroups, mesenchymal stem cells with specific functions are screened out, so that different groups of people are treated accurately.
Specifically, the technical scheme of the invention is as follows:
the first aspect of the invention discloses a preparation method of a single-cell transcriptome library, which comprises the following steps:
s1: preparing a single cell suspension, wherein the single cell suspension is mesenchymal stem cells;
s2: centrifuging the strip-shaped bead solution, and then resuspending the strip-shaped bead solution by using a buffer solution for later use;
s3: processing by adopting an automatic single cell processing system, sequentially adding a single cell suspension, a resuspended strip-shaped bead solution, a washing buffer solution 1, a lysis buffer solution and a PBS buffer solution, and starting the system to obtain a product;
s4: centrifuging the product obtained in S3, only leaving strip-shaped beads, adding RT mix, placing the product in a metal bath for reaction, washing the product with washing buffer solution 2, washing buffer solution 3 and nuclease-free water in sequence, adding PCR mix, and performing PCR amplification to obtain an amplification product;
s5: and collecting an amplification product, adding purified magnetic beads, centrifuging to obtain a supernatant, adding purified magnetic beads rinsed with ethanol into the supernatant, centrifuging again, constructing a PCR system, amplifying, and sequencing by using an XNovagNS sequencing platform to obtain a single-cell transcriptome library.
Preferably, before S3, the method comprises the following steps: injecting 100% absolute ethyl alcohol into a sample inlet of the chip, and removing redundant liquid at the sample outlet; repeating for 2-3 times until no air bubbles appear in the chip.
Preferably, in S4, the reaction conditions of the metal bath are: the reaction temperature is 42 ℃, the rotation speed is 1000rpm, and the reaction time is 90 min.
Preferably, in S5, the purified magnetic beads are Ampure XP purified magnetic beads; more preferably, the Ampure XP purified magnetic beads are removed from 4 ℃ in advance for 30min, and allowed to return to room temperature.
Preferably, in S5, purified magnetic beads are added to the amplification products, and the volume of the purified magnetic beads and the amplification products is 0.6: 1, uniformly mixing the two, incubating at room temperature for 4-6min, centrifuging, standing for 4-6min until the liquid is transparent and clear, and removing the supernatant to a new container to obtain the supernatant.
Preferably, in S5, 80% ethanol is added to the supernatant to rinse the purified magnetic beads, the mixture is incubated at room temperature for 30S, and the step is repeated once after the supernatant is removed.
Preferably, the automated single cell processing system is a Singleron Matrix instrument system.
In a second aspect, the invention discloses a single cell transcriptome library obtained according to the above method.
The third aspect of the invention discloses a single cell transcriptome sequencing method, which comprises the following steps: sequencing was performed using the single cell transcriptome library described above.
The fourth aspect of the invention discloses the following applications:
(a) the above single cell transcriptome library is used to analyze cell subsets and identify cell types;
(b) the single cell transcriptome sequencing method described above is used to detect different diseases.
Compared with the prior art, the invention has at least the following distinguishing technical characteristics:
according to the invention, the function of the mesenchymal stem cells from the same source is identified and the subgroups are subdivided by using a single-cell transcriptome sequencing technology, and a diagnosis and treatment system for performing accurate treatment by using the mesenchymal stem cells with different functions is initially established.
Drawings
FIG. 1 is a graph of the distribution of single cell clusters (left) and different generations of single cells in different clusters (right);
FIG. 2 is a schematic diagram showing the correlation analysis between gene clusters formed by differential genes between respective clusters.
Detailed Description
The present application is further illustrated by the following detailed examples, which should be construed to be merely illustrative and not limitative of the remainder of the disclosure.
The instruments, equipment, reagents used in the examples are available from various sources, for example, purchased, or may be prepared.
Example 1
The embodiment discloses a method for analyzing the function of mesenchymal stem cells by using a single-cell transcriptome sequencing method, which specifically comprises the following steps:
1. stem cell preparation
1.1 Collection treatment
1) Before operation, the experimental environment is checked, equipment and reagent information is confirmed, and required consumables and instruments are transferred into a sterile area of a cell preparation workshop according to regulations.
2) The superclean bench is opened and ventilated, waits for the pilot lamp to turn green to evenly clean whole superclean bench with aseptic gauze that contains 75% alcohol, required utensil, consumptive material etc. need can put into superclean bench behind 75% alcohol disinfection.
3) The petri dish, forceps and scissors were prepared.
4) The umbilical cord was removed with forceps and placed in a petri dish, and the umbilical cord tissue was washed with physiological saline.
5) The washed harvest was cut open, the vessels in the umbilical cord were peeled off, the umbilical cord tissue was cut into small tissue pieces and planted in a petri dish.
6) The prepared culture solution No. 1 is added into the culture dish.
7) The dish is marked with the number of the collection/cell, the date of inoculation, and the cell type, and then placed in an incubator.
1.2 Stem cell culture Medium
1) Before operation, the experimental environment is checked, equipment and reagent information is confirmed, and required consumables and instruments are transferred into a sterile area of a cell preparation workshop according to regulations.
2) The superclean bench is opened and ventilated, waits for the pilot lamp to turn green to evenly clean whole superclean bench with aseptic gauze that contains 75% alcohol, required utensil, consumptive material etc. need can put into superclean bench behind 75% alcohol disinfection.
3) And taking out primary cells needing to be changed in liquid from the incubator, and putting the primary cells into a super clean bench.
4) And sucking the culture solution in the culture dish by using a liquid transfer device and abandoning the culture solution in a waste liquid cylinder.
5) The prepared culture solution was added to each dish.
6) After all the culture dishes are filled with liquid, the date of liquid change is marked on the culture dishes, and the culture dishes are placed in an incubator.
7) And (6) clearing the field.
1.3 Stem cell subculture
1) Before operation, the experimental environment is checked, equipment and reagent information is confirmed, and required consumables and instruments are transferred into a sterile area of a cell preparation workshop according to regulations.
2) The superclean bench is opened and ventilated, waits for the pilot lamp to turn green to evenly clean whole superclean bench with aseptic gauze that contains 75% alcohol, required utensil, consumptive material etc. need can put into superclean bench behind 75% alcohol disinfection.
3) And taking out the cells to be passaged from the incubator, putting the cells into a super clean bench, sucking the culture solution in the culture dish by using a liquid transfer machine, and abandoning the culture solution in a waste liquid tank.
4) Each dish was washed with 0.9% physiological saline.
5) Pancreatin was added to each dish and digested at 37 ℃.
6) After digestion of all cells in the dish was complete, stop solution was added to stop digestion.
7) The cell suspension was transferred to a 15mL centrifuge tube and centrifuged to discard the supernatant.
8) The cells were resuspended in the appropriate medium and counted.
9) The cell suspension was seeded into a new dish according to the counting results.
10) The serial number of the collected material/cell, the passage date and the cell generation number are marked on the culture dish, and then the culture dish is put into an incubator.
2. Single cell transcriptome library preparation and sequencing
2.1 Single cell suspension preparation
1) Before operation, the experimental environment is checked, equipment and reagent information is confirmed, and required consumables and instruments are transferred into a sterile area of a cell preparation workshop according to regulations.
2) The superclean bench is opened and ventilated, waits for the pilot lamp to turn green to evenly clean whole superclean bench with aseptic gauze that contains 75% alcohol, required utensil, consumptive material etc. need can put into superclean bench behind 75% alcohol disinfection.
3) And taking out the mesenchymal stem cells to be passaged from the incubator, putting the mesenchymal stem cells into a super clean bench, sucking the culture solution in the culture dish by using a liquid transfer machine, and abandoning the culture solution in a waste liquid tank.
4) Each dish was washed with 0.9% physiological saline.
5) Pancreatin was added to each dish and digested at 37 ℃.
6) After digestion of all cells in the dish was complete, stop solution was added to stop digestion.
This example uses
Single cell transcriptome library kit Singleron Matrix.
2.2 Single cell transcriptome library preparation and sequencing
1) Before the experiment, the bench top was wiped with RNase away. And in the experimental process, the experiment table top is wiped (for a plurality of times) by using RNaseaway. The consumables used in this experiment all need to be sterile and free of nuclease.
2) Pipette 900 μ L of Barcode Beads into a 1.5mL centrifuge tube, place on a magnetic rack, and after the solution is clarified, carefully remove the supernatant.
3) The tube was removed, 1mL PBS was added, mixed and centrifuged instantly, placed on a magnetic stand, and after the solution was clear, the supernatant was carefully removed and repeated twice.
4) The centrifuge tube was removed, 40. mu.L PBS was added, resuspended and placed in a 4 ℃ freezer for use.
5) The separated cells were resuspended in precooled PBS (cell concentration 1X 10)5One/ml),
6) 200 μ L of 100% absolute ethanol was slowly injected into the sample inlet of the chip (200 μ L over 10 seconds) and the excess liquid from the sample outlet was removed (repeat step wash process 2-3 times until no more bubbles appeared in the chip).
7) 200 μ L of 0.02% PBST (0.02% Tween-20 in PBS, as-prepared) was injected into the chip inlet (10 seconds) (the washing process was repeated 2 times), and the excess liquid was removed from the outlet and allowed to stand at room temperature for 30 min.
8) And (3) turning on a Singleron Matrix instrument power supply, confirming that a green indicator light of the power supply is normally turned on, and displaying interface contents normally on a display screen. Slightly pushing the upper cover buckle of the instrument, opening the upper cover of the instrument, using a pipette, adding 500 μ L of Wash Buffer 1 (New cell (Nanjing) Biotechnology Limited, Cat #1100011) into the connecting module slot-1 according to the label, using the pipette, adding 100 μ L of processed cell suspension into the connecting module slot-2 according to the label, using the pipette, adding 40 μ L of Barcode Beads into the connecting module slot-3 according to the label, using the pipette, adding 100 μ L of lysine Buffer (New cell (Nanjing) Biotechnology Limited, Cat #1100011) into the connecting module slot-4 according to the label, using the pipette, and adding 1400 μ L of PBS Buffer into the connecting module slot-5 according to the label.
9) After confirming that the sample and the reagent are placed in the connection module, clicking an initialization button on the screen of the instrument, wherein the instrument enters an initialization process, the bottom display lamp displays 'initialization', and the display lamp icon is converted from 'initialization' to 'running' after the initialization is completed (about 10 seconds). After initialization is completed, the system is automatically switched to an operation page, at the moment, the instrument upper cover is lightly covered, and the cover closing completion is confirmed after the sound of the instrument upper cover buckle is heard. And clicking a screen to prepare a sample, starting the operation of the instrument, and performing sample preparation, wherein the operation time is about 34 min.
10) After running for about 34min, the screen displays "complete" indicating that mRNA capture has been completed.
11) The product is recovered into a precooled 1.5mL centrifuge tube, and 200 μ L wash buffer 1 is sucked by a 200 μ L pipette to repeatedly wash the product tank for 2-3 times until the magnetic beads in the product tank are completely taken out, and the product tank is placed on ice to wait for subsequent reaction.
12) RT Mix was formulated on ice as in table 1 below, vortexed and briefly centrifuged, and the following reagents were purchased under the serial No. #1100011 from new gyby (nanjing) biotechnology limited.
TABLE 1
13) The centrifuge tube for the product collected in step 10 was placed on a magnetic rack and after the solution cleared, the supernatant was carefully removed. And (3) taking the centrifuge tube off the magnetic frame, adding 1mL of Wash Buffer 1, uniformly mixing, centrifuging for a short time, placing on the magnetic frame, and carefully removing the supernatant after the solution is clarified.
14) The tube was removed from the magnetic stand, 500. mu.L of 1 × Wash Buffer (10 × Wash Buffer to 1 ×, New cell (Nanjing) Biotechnology Ltd., Cat #1100011 diluted with Nuclear-free Water in advance) was added, mixed well, centrifuged briefly, placed on the magnetic stand, and the supernatant carefully removed after the solution was clarified.
15) The tube was then briefly centrifuged to remove the liquid as completely as possible, leaving only the Barcode Beads at the bottom of the tube. 200 μ L of prepared RT Mix was added into a centrifuge tube containing Barcode Beads, and placed in a metal bath set in advance, at 42 ℃ and 1000rpm, for reaction for 90 min.
16) After the reaction is finished, the centrifuge tube is placed on a magnetic frame immediately, and after the solution is clarified, the supernatant is carefully removed.
17) The centrifuge tube was removed from the magnetic stand, 1mL of Wash Buffer 2 (new glan bioscience ltd., cat #1100011) was added, mixed well, centrifuged briefly, placed on the magnetic stand, and the supernatant was carefully removed after the solution was clarified.
18) The centrifuge tube was removed from the magnetic stand, 1mL of Wash Buffer 3 (new glan bioscience ltd., cat #1100011) was added, mixed well, centrifuged briefly, placed on the magnetic stand, and the supernatant was carefully removed after the solution was clarified.
19) The centrifuge tube was removed from the magnetic stand, 1mL of Nuclear-free Water was added, the mixture was inverted and mixed, centrifuged briefly, placed on the magnetic stand, and the supernatant carefully removed after the solution was clarified. Repeat 1 time, centrifuge tube instantaneous separation, as far as possible to suck the liquid, only leave the centrifuge tube bottom Barcode Beads.
20) Preparing PCR Mix on ice according to the following table 2, mixing evenly by vortex and centrifuging briefly, adding the PCR Mix into a centrifuge tube with Barcode Beads left in the previous step, mixing evenly by blowing, adding into an 8-joint calandria simultaneously, dividing each sample into 8 tubes for amplification, wherein the liquid volume of each tube is 50 mu L, and the following reagent products are purchased from New Zeng Yuan (Nanjing) Biotech limited company with the product number of # 1100011.
TABLE 2
21) The tube caps were capped and placed in a PCR instrument for amplification, the PCR procedure is shown in table 3 below. After the PCR process is finished, the product can be stored at 4 ℃ for 48h, or directly subjected to cDNA amplification and purification.
TABLE 3
22) Ampure XP purified magnetic beads were removed from 4 ℃ 30min in advance and returned to room temperature.
Note: the beads are mixed well before use.
23) The liquid in the 8 rows of tubes was collected in a 1.5mL centrifuge tube, flash separated and the volume calculated. Adding 240 μ L Ampure XP purified magnetic beads (0.6 × product volume), blowing, mixing, incubating at room temperature for 5min, centrifuging for a short time, and standing on a magnetic frame for 5 min; until the liquid was clear, the supernatant was carefully removed to a new 1.5mL centrifuge tube and left for storage.
24) Keeping the PCR tube on the magnetic frame all the time, adding 800. mu.L of newly prepared 80% ethanol to rinse the Ampure XP purified magnetic beads. Incubate at room temperature for 30s, carefully remove the supernatant, repeat this step 1 time.
25) Taking off the centrifuge tube, centrifuging for a short time, placing on the magnetic frame again, removing excessive alcohol, and air drying.
26) Taking down the centrifuge tube, adding 20 mu L of Buffer EB, blowing and beating Ampure XP purified magnetic beads uniformly, incubating for 5min at room temperature, centrifuging for a short time, standing on a magnetic frame until the liquid is transparent and clear. The supernatant was aspirated and transferred to a new EP tube, which was the purified product.
27) Taking 1 uL sample to carry out the Qubit concentration detection, taking 2 uL sample to carry out the fragment size detection.
28) FR Buffer is unfrozen at room temperature and is uniformly mixed for later use, a reaction system shown in the following table 4 is configured in a sterilized PCR tube, and the following reagents are purchased from New Zengyuan (Nanjing) Biotechnology Co., Ltd., a product number of # 1100011.
TABLE 4
29) The mixture was gently pipetted and mixed and incubated at 55 ℃ for 10 minutes.
30) The fragmented product is purified, steps 22-26.
31) And amplifying and purifying the fragmentation product, and sequencing the transcriptome by using an XNovagNS sequencing platform. 3. Mesenchymal stem cell functional analysis and subgroup identification
1) Rstudio (R version 4.0.2) was turned on and the seruat single cell analysis package and phase dependent package were installed.
2) And reading the gene expression matrix obtained quantitatively after sequencing into an R environment.
3) Differences between mesenchymal stem cells were calculated according to codes provided by the authorities, and marker genes of different cell clusters were obtained, and experimental results are shown in fig. 1 and 2.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method of preparing a single cell transcriptome library, comprising:
s1: preparing a single cell suspension, wherein the single cell suspension is mesenchymal stem cells;
s2: centrifuging the strip-shaped bead solution, and then resuspending the strip-shaped bead solution by using a buffer solution for later use;
s3: processing by adopting an automatic single cell processing system, sequentially adding a single cell suspension, a resuspended strip-shaped bead solution, a washing buffer solution 1, a lysis buffer solution and a PBS buffer solution, and starting the system to obtain a product;
s4: centrifuging the product obtained in S3, only leaving strip-shaped beads, adding RT mix, placing the product in a metal bath for reaction, washing the product with washing buffer solution 2, washing buffer solution 3 and nuclease-free water in sequence, adding PCR mix, and performing PCR amplification to obtain an amplification product;
s5: and collecting an amplification product, adding purified magnetic beads, centrifuging to obtain a supernatant, adding purified magnetic beads rinsed with ethanol into the supernatant, centrifuging again, constructing a PCR system, amplifying, and sequencing by using an XNovagNS sequencing platform to obtain a single-cell transcriptome library.
2. The method of claim 1, wherein before S3, the automated single cell processing system is pretreated with absolute ethanol, and the method comprises the following steps: injecting 100% absolute ethyl alcohol into a sample inlet of the chip, and removing redundant liquid at the sample outlet; repeating for 2-3 times until no air bubbles appear in the chip.
3. The method according to claim 1, wherein in S4, the reaction conditions of the metal bath are: the reaction temperature is 42 ℃, the rotation speed is 1000rpm, and the reaction time is 90 min.
4. The method of claim 1, wherein in S5, the purified magnetic beads are Ampure XP purified magnetic beads; preferably, the Ampure XP purified magnetic beads are removed from 4 ℃ 30min in advance, and allowed to return to room temperature.
5. The method of claim 1, wherein in S5, purified magnetic beads are added to the amplification products, and the volume of the purified magnetic beads and the amplification products is 0.6: 1, uniformly mixing the two, incubating at room temperature for 4-6min, centrifuging, standing for 4-6min until the liquid is transparent and clear, and removing the supernatant to a new container to obtain the supernatant.
6. The method of claim 1, wherein in S5, the purified magnetic beads are rinsed with 80% ethanol and incubated at room temperature for 30S, and the step is repeated once after removing the supernatant.
7. The method of claim 1, wherein the automated single cell processing system is a Singleron Matrix instrument system.
8. A single cell transcriptome library obtained according to the method of claims 1-7.
9. A method for single cell transcriptome sequencing, comprising: sequencing using the single cell transcriptome library of claim 8.
10. The following applies:
(a) the single cell transcriptome library of claim 8 for analyzing cell subpopulations and identifying cell types;
(b) the single cell transcriptome sequencing method of claim 9 for detecting different diseases.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011312847.5A CN112391444A (en) | 2020-11-20 | 2020-11-20 | Application of mesenchymal stem cells with different functional characteristics in treating different diseases |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011312847.5A CN112391444A (en) | 2020-11-20 | 2020-11-20 | Application of mesenchymal stem cells with different functional characteristics in treating different diseases |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112391444A true CN112391444A (en) | 2021-02-23 |
Family
ID=74607107
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011312847.5A Pending CN112391444A (en) | 2020-11-20 | 2020-11-20 | Application of mesenchymal stem cells with different functional characteristics in treating different diseases |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112391444A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113373523A (en) * | 2021-08-02 | 2021-09-10 | 中南大学湘雅医院 | Myasthenia gravis peripheral blood single cell transcriptome library, preparation method and application |
-
2020
- 2020-11-20 CN CN202011312847.5A patent/CN112391444A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113373523A (en) * | 2021-08-02 | 2021-09-10 | 中南大学湘雅医院 | Myasthenia gravis peripheral blood single cell transcriptome library, preparation method and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105154314B (en) | A kind of gene assaying device for collecting extraction, amplification and detection integration | |
| CN111621466B (en) | Preparation method and application of pulmonary artery tissue single cell suspension | |
| CN106754313A (en) | Nucleic acid sequencing apparatus and system | |
| WO2018123220A1 (en) | Method of obtaining fetal cell nucleic acids | |
| CN105525033A (en) | Method and device for detecting microorganisms in blood | |
| CN108130323A (en) | A kind of banking process suitable for the analysis of plant transposase accessibility chromatin | |
| US20240043919A1 (en) | Method for traceable medium-throughput single-cell copy number sequencing | |
| CN113293196A (en) | Single cell nucleus extraction method suitable for frozen tissue | |
| CN115927565A (en) | Establishment and application of method for detecting pathogenic microorganisms in CAR-T cell product based on mNGS | |
| CN108220453A (en) | A kind of noninvasive antenatal method for paternity test and its kit | |
| CN112391444A (en) | Application of mesenchymal stem cells with different functional characteristics in treating different diseases | |
| CN105039569B (en) | A kind of breakpoint analysis method of mutual translocation chromosome | |
| CN110512011A (en) | A kind of intestinal flora determination method, device, system and storage medium | |
| CN113637668A (en) | Kit for simultaneously extracting pathogenic bacteria DNA of blood plasma and blood cells and application thereof | |
| CN116926218B (en) | Probe combination, gene chip, kit and method for detecting ascophyllum sp | |
| CN103103182A (en) | Haliotis discus hannai microsatellite molecular marker and preparation method thereof | |
| CN112376115A (en) | Method for detecting different diseases by using single cell transcriptome sequencing technology | |
| CN107988345A (en) | The method of HLA-A*02 subtype typings | |
| CN106191032B (en) | The Disease-causing gene model and its construction method of dysnoesia disease and application | |
| CN107058564A (en) | Analysis method for diversity of intestinal flora of autism of children and specific primer pair | |
| CN113322314B (en) | Novel tissue unicell space transcriptome technology | |
| CN115678852A (en) | Method for high-purity enrichment of single tumor cells in tumor tissue | |
| CN111484976A (en) | Lung cancer circulating tumor cell detection kit and detection system | |
| Mukae et al. | Development of RNA/DNA automated extraction and purification device for infectious disease diagnosis | |
| CN113025688A (en) | Method for detecting full-length non-poly (A) transcript based on customized nanopore direct RNA sequencing reverse transcription linker |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20210223 |