CN108753694A - A kind of people's gum stem cell subgroup and its dryness detection method by cell surface marker specific enrichment - Google Patents

A kind of people's gum stem cell subgroup and its dryness detection method by cell surface marker specific enrichment Download PDF

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CN108753694A
CN108753694A CN201810595972.8A CN201810595972A CN108753694A CN 108753694 A CN108753694 A CN 108753694A CN 201810595972 A CN201810595972 A CN 201810595972A CN 108753694 A CN108753694 A CN 108753694A
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徐随民
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Nanjing Tai Sheng Biotechnology Co Ltd
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Nanjing Tai Sheng Biotechnology Co Ltd
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Abstract

The present invention provides species specific people's gum stem cell subgroup and its enrichment methods.This method includes that the acquisition of dental tissue sample is transported, the separation of gum stem cell, and expresses the enrichment of CD18 stem cells.The present invention also provides a kind of detection methods of the gum stem cell subgroup biological characteristics of enrichment.Including being detected with the relevant Colony forming ability of dryness, proliferative capacity detection, external Osteoblast Differentiation ability detection, internal Osteoblast Differentiation ability detection.

Description

A kind of people's gum stem cell subgroup by cell surface marker specific enrichment and its Dryness detection method
Technical field
The present invention relates to one kind by cell surface marker, preferably people's gum stem cell subgroup of CD18 specific enrichments and Its dryness detection method.The present invention also relates to the purposes that the cell being enriched with through the invention is used for regenerative medicine.
Background technology
Gum is a kind of unique oral cavity tissue, is attached on the alveolar bone of alveolus, is oral cavity as a kind of mucosal barrier The important component part of mucous membrane.
The reparation of gum and oral mucosa lesion, with cicatrization common on skin on the contrary, to show as degree of inflammation bright Aobvious light, promoting epidermization is rapider, the Scarless wound healing of similar fetal skin.This may exist specifically with gum and oral mucosa The stem cell of biological activity is related.
Applicant is detached from gum by enzyme digestion and is obtained with Clone formation and high proliferation in previous studies The cell of ability.These cells have Multidirectional Differentiation ability in vitro, include to cell differentiations such as bone, fat, endothelium, nerves Ability.When by these cells and hydroxyapatite/tricalcium phosphate（HA/TCP）Particle mixes, and transplants when nude mice by subcutaneous, can shape At connective tissue spline structure.
But these cells are height heterogeneities, if there are stringent level, pedigree are current as stem cell Also it is not very clear.In addition, in default of special surface markers, it is unfavorable for in-vitro separation and the identification of cell, also influence pair The research of its clinical efficacy.
Therefore, specific cell subsets is extracted by special surface markers, not only to the life of these cells of Study on thinning Object characteristic, the overall picture for understanding gum stem cell are most important, and whether have different clinical effectiveness to different cell subsets Research be also it is very crucial, have huge basic research and clinical meaning.
The cell surface marker is selected from CD18, STRO-1, CD106/VCAM-1, CD146/MUC-18, HOP-26, CD49A/ integrin β_1s, one or more of SB-10/CD166.
CD18, also referred to as 2 subfamily of integrin beta are a member in cell adhesion molecule, mediated cell and iuntercellular, cell Interaction between matrix.Since CD18 is significantly expressed on the leukocytes, past many researchs all concentrate on CD18 white Effect in cell adherence, migration and immune response.
But in fact, CD18 is also expressed on bone marrow stroma stem cell, and key effect is played during ostosis.It lacks The mouse of weary CD18 shows certain features of osteoporosis, including bone density reduces, and trabecular bone number is reduced, and trabecular bone is thick Degree is reduced, and bone trabecula space increases etc..And by virus transfection, the formation of bone can be promoted by being overexpressed CD18.
Applicant recent studies have shown that, gum stem cell also expresses CD18, and CD18 can be used as it is one unique thin Born of the same parents' sorting indicia, for being enriched with a kind of people's gum stem cell subgroup.
Invention content
We have creatively invented a kind of method by CD18 specific enrichment people's gum stem cell subgroups, carry significantly The high efficiency of external gum stem cell separation and identification.What is more important, the gum stem cell being enriched with by this method are sub- Group has stronger clonality, more preferably Osteoblast Differentiation ability, has superior scientific research and potential applicability in clinical practice.
Specifically, the present invention is achieved through the following technical solutions：
The present invention provides the enrichment methods of species specific people's gum stem cell subgroup.This method includes adopting for dental tissue sample The enrichment of collection transport, the separation of gum stem cell, and expression CD18 cells.
This method uses the collection liquid combined containing Multiple Classes of Antibiotics and collects gingiva tissue sample, including but not limited to green Mycin, streptomysin, amphomoronal etc..The collection liquid can also be used for the acquisition of other tissue samples, such as fat, umbilical cord, placenta Deng, can substantially reduce sample pollution possibility.
The use concentration of penicillin can be 50U/mL, 100U/mL, 150U/mL, 200U/mL, 250U/mL in this method, 300U/mL.The use concentration of streptomysin can be 50 μ g/mL, 100 μ g/mL, 150 μ g/mL, 200 μ g/mL, 250 μ g/mL, 300 μg/mL.The use concentration of amphomoronal can be 1 μ g/mL, 2 μ g/mL, 2.5 μ g/mL 3 μ g/mL, 4 μ g/mL, 5 μ g/mL.
The preservation transport temperature of sample can be 2 DEG C, 4 DEG C, 6 DEG C, 8 DEG C, 10 DEG C, 15 DEG C, 25 DEG C in this method.
The time interval that since sample extract collecting in this method can be 4 hours, and 8 hours, 12 hours, 24 is small When, 48 hours, 72 hours, 96 hours.
This method is used containing there are many combinations of digestive ferment to come chorista sample, acquisition individual cells suspension.Including But be not limited to clostridiopetidase A, dispase, hyaluronidase, pancreatin, EDTA etc., the combination of these digestive ferments is greatly improved obtaining for cell Rate.
The use concentration of clostridiopetidase A can be 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ in this method Ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml.The use concentration of dispase can be 1 mg/ml, 2 Mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml.
May be used also the present invention is not limited to use CD18 to be enriched with gum stem cell subgroup although this method is preferred CD18 So as to it is enriched with gum stem cell subgroup, including STRO-1, CD106/VCAM-1 to the combination of next or multiple markers, CD146/MUC-18, HOP-26, CD49A/ integrin β_1, SB-10/CD166.
CD18 can use fluorescent marker or marked by magnetic bead.Correspondingly, accurate cell separation technology includes fluidic cell Instrument sorts or immunological magnetic bead sorting.
The present invention also provides a kind of detection methods of the gum stem cell subgroup biological characteristics of enrichment.Including with dryness Relevant Colony forming ability detection, proliferative capacity detection, external Osteoblast Differentiation ability detection, the detection of differentiation in vivo ability.? In terms of confirming cell dryness and biological efficacy, the combination of this set detection method is abundant and suitable.
This method is by measuring CFU-F in enrichment of cell（Colony Forming Unit-Fibroblast）Number, inspection Survey the clonality of CD18 Positive Stem Cells.
This method detects the in-vitro multiplication ability of CD18 Positive Stem Cells by BrdU incorporation methods.
The external basal medium of CD18 Positive Stem Cells can be α-MEM, DMEM, D/F-12, RPMI- in this method 1640 etc..Complete medium is addition fetal calf serum, ascorbic acid 2- phosphates, glutamy on the basis of basal medium Amine, penicillin, streptomysin etc..
The concentration of fetal calf serum can be 5%, 10%, 15%, 20% in complete medium.The concentration of ascorbic acid 2- phosphates It can be 50 μM, 100 μM, 150 μM, 200 μM.Other additive concentration are added according to art-recognized prioritization scheme.
In this programme Osteogenic Induction Medium be on the basis of basal medium, add dexamethasone, vitamin C and Sodium β-glycerophosphate etc..
The use concentration of dexamethasone can be 5nM, 10nM, 15nM, 20nM, 25nM, 30nM in Osteogenic Induction Medium. The use concentration of vitamin C can be 50 μ g/mL, 100 μ g/mL, 150 μ g/mL, 200 μ g/mL, 250 μ g/mL, 300 μ g/ mL.The use concentration of sodium β-glycerophosphate can be 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM。
This method is by by enrichment of cell and hydroxyapatite/tricalcium phosphate（HA/TCP）Carrier mixes, and transplanting is in immune Deficient animals are subcutaneous, and preferred immunodeficient animals are nude mices, and the differentiation in vivo ability of cell is detected by histotomy.This One internal implant system, and goldstandard art-recognized at present.
The CD18 Positive Stem Cells that the present invention is enriched with can be directly used for regenerative medicine, preferably tooth and relevant disease Regenerative medicine.
In addition, in view of the key effect that CD18 is played during ostosis, the present invention also relates to pass through various transgenosis Technology, it is preferable that by way of retrovirus-mediated method, CD18 genes are transferred to various stroma stem cells, including but not It is limited to the tissue-derived stroma stem cell such as tooth, fat, marrow, umbilical cord, placenta, bleeding of the umbilicus, is used for bone and relevant disease again Raw medicine and organizational project.
Description of the drawings
The phenotypic analysis of Fig. 1 CD18 Positive Stem Cells subgroups
The phenotypic analysis of Fig. 2 CD18 feminine gender stem cells compares
The Colony forming capability analysis of Fig. 3 CD18 Positive Stem Cells
The Colony forming capability analysis of Fig. 4 CD18 feminine gender stem cells compares
The proliferative capacity of Fig. 5 CD18 Positive Stem Cells is analyzed
The external Osteoblast Differentiation of Fig. 6 CD18 Positive Stem Cells
The differentiation in vivo of Fig. 7 CD18 Positive Stem Cells.
Specific implementation mode
The enrichment of 1 CD18 positive gum stem cells of embodiment
Materials and methods
Remaining or be dropped healthy gingiva tissue 1. after collecting conventional dental operation, be immediately placed on 4 DEG C of precoolings contains 50- In the α-MEM culture mediums of 300U/mL penicillin, 50-300 μ g/mL streptomysins and 1-5 μ g/mL amphomoronals, 2 DEG C of -8 DEG C of items It is transported to Clean Operating Lab under part.Since the time interval that gum stem cell is extracted being collected into be no more than 72 hours；
2. being transferred to Biohazard Safety Equipment, gingiva tissue is taken out to culture dish, with the sterile phosphate buffer of 4 DEG C of precoolings （Phosphate Buffer Saline, PBS）Cleaning；
3. gingiva tissue is placed in the PBS of the dispases of mg/ml containing 1-10,4 DEG C overnight；
4. taking out gingiva tissue, is shredded, be placed in the PBS of the IV Collagenase Types of mg/ml containing 1-10,37 DEG C with aseptic operation Digestion 2 hours；
5. digestive juice is filtered by 70 microns of cell sieve, single cell suspension is obtained；
6. 900rpm centrifuges 5min, it is primary to wash cell with PBS；
7. cell is resuspended with appropriate PBS, CD18 monoclonal antibodies, mixing is added, room temperature is protected from light incubation 30 minutes；
8. after washing, 900rpm centrifuges 5min, cell is resuspended with PBS；
9. being sorted by fluidic cell, the CD18 positives and negative cells are obtained respectively.
Analysis result shows that the Positive Stem Cells CD18 of enrichment expresses strong positive（Fig. 1）, negative stem cell CD18 expression the moon Property（Fig. 2）, which is significantly effective.
The Colony forming ability of 2 CD18 positive gum stem cells of embodiment
Materials and methods
1. taking the CD18 Positive Stem Cells subgroups of sorting, count；
2. 900rpm centrifuges 5min；
3. with fetal calf serum containing 10%-20%, 50 μM -200 μM of ascorbic acid 2- phosphates, 2mM glutamine, 100U/mL α-MEM culture mediums resuspension the cell of penicillin, 100 μ g/mL streptomysins；
4. by 104A CD18 Positive Stem Cells are inoculated in T25 Tissue Culture Flasks, are placed in 37 DEG C, 5% CO2Incubator in train It supports；
5. per liquid is changed within 3-4 days；
6. after 14-16 days, outwelling culture solution, PBS cleans attached cell, then uses and contains 0.1% toluidine blue and 2% poly first The PBS solution of aldehyde is fixed cell, dyes.Cell mass more than 50 cells is designated as a colony（Colony Forming Unit-Fibroblast, CFU-F）.
Analysis result shows and CD18 feminine gender stem cells（Fig. 4）Than CD18 Positive Stem Cells（Fig. 3）There is stronger clone Forming ability.
The in-vitro multiplication ability of 3 CD18 positive gum stem cells of embodiment
Materials and methods
1. taking the CD18 Positive Stem Cells subgroups of sorting, count；
2. 900rpm centrifuges 5min；
3. with fetal calf serum containing 10%-20%, 50 μM -200 μM of ascorbic acid 2- phosphates, 2mM glutamine, 100U/mL α-MEM culture mediums resuspension the cell of penicillin, 100 μ g/mL streptomysins；
4. the cell that step 3 is obtained is with 104It is inoculated in 12 orifice plates per hole, is placed in 37 DEG C, 5% CO2Incubator in cultivate；
5. after culture 1-2 days, BrdU reagents are added（1:100）, it is incubated for 24 hours；
6. BrdU staining kits dye cell；
7. haematoxylin is redyed；
8. counting Positive Stem Cells number under mirror, the proliferation rate of cell is measured.
BrdU can be incorporated into the newly synthesized DNA of cell as a kind of analog of thymidine, reflect cell Proliferative capacity.After CD18 Positive Stem Cells culture 1-2 days, BrdU is added, is dyed through BrdU, it is as a result visible（Fig. 5）, 80% or more Cell is positive, and shows that the CD18 Positive Stem Cells of enrichment have stronger proliferative capacity.
The external Osteoblast Differentiation ability of 4 CD18 positive gum stem cells of embodiment
Materials and methods
1. taking the CD18 Positive Stem Cells subgroups of sorting, count；
2. 900rpm centrifuges 5min；
3. with fetal calf serum containing 10%-20%, 50 μM -200 μM of ascorbic acid 2- phosphates, 2mM glutamine, 100U/mL α-MEM culture mediums resuspension the cell of penicillin, 100 μ g/mL streptomysins；
4. inoculation 4 × 105A CD18 Positive Stem Cells are placed in 37 DEG C, 5% CO in 6 orifice plates2Incubator in cultivate；
5. when cell is expanded to 80% degrees of fusion, it is replaced with Osteogenic Induction Medium（Containing 5-30nM dexamethasone, α-MEM the culture mediums of 50-300 μ g/mL vitamin Cs and 1-10 mM sodium β-glycerophosphates）；
6. per liquid is changed within 3-4 days；
7. after induction in 3-4 weeks, microscopic observation cell is shown in that dark yellow calcium tubercle is formed；
8. discarding culture solution, after PBS is cleaned 2 times, PBS is discarded；
9. changing 60% isopropanol into be fixed, after room temperature 1min, isopropanol is discarded, is cleaned 2 times using distilled water, With 1% Alizarin red staining, room temperature 3min.After PBS is cleaned 2 times, take pictures after exhausting liquid.
In vitro after osteogenic induction culture in 3-4 weeks, pass through Alizarin red staining（Fig. 6）, it is seen that iuntercellular has mineral deposition, Show that CD18 Positive Stem Cells can be to osteoblast differentiation.
The differentiation in vivo ability of 5 CD18 positive gum stem cells of embodiment
Materials and methods
1. by 2 × 106The hydroxyapatite/tricalcium phosphate of a CD18 Positive Stem Cells and 15mg（HA/TCP）Particle is mixed It closes, and is transplanted to the nude mice dorsal sc of 6-8 week old；
2. post-transplantation is drawn materials for 8 weeks；
After 3. euthanasia puts to death mouse, careful separation subcutaneous cell agglomerate and skin are fixed with 4% paraformaldehyde, 4 DEG C of mistakes Night；
4. discarding fixer, after PBS is cleaned up, the EDTA for changing 10% into carries out decalcification, changes a decalcifying Fluid within every 3 days, After decalcification 21 days, dehydration embedding；
5. after slice, carrying out haematoxylin/Yihong（H&E）Dyeing and Masson ' s trichrome stains；
6. microscopy.
By CD18 Positive Stem Cells and hydroxyapatite/tricalcium phosphate（HA/TCP）Particle mixes, and is transplanted to 6-8 weeks The nude mice dorsal sc in age.Histotomy is shown after 8 weeks（Fig. 7）, CD18 Positive Stem Cells formed connective tissue spline structure, have The histologic characteristics of early stage connective tissue phenotype, there are collagenous fibres.

Claims (12)

1. a kind of people's gum stem cell subgroup by cell surface marker specific enrichment, which is characterized in that the stem cell For people gum stem cell, the cell surface marker is selected from CD18, STRO-1, CD106/VCAM-1, CD146/MUC-18, HOP- 26, CD49A/ integrin β_1s, one or more of SB-10/CD166.
2. people's gum stem cell subgroup described in claim 1, which is characterized in that described using CD18 as cell surface marker CD18 uses fluorescent marker or marked by magnetic bead.
3. the enrichment method of people's gum stem cell subgroup described in a kind of claims 1 or 2, which is characterized in that the tool of the method Steps are as follows for body：
Step 1, after collecting conventional dental operation, the remaining or healthy gingiva tissue that is dropped is immediately placed on 4 DEG C of precoolings In the α-MEM culture mediums of penicillin containing 50-300U/mL, 50-300 μ g/mL streptomysins and 1-5 μ g/mL amphomoronals, 2 DEG C -8 Clean Operating Lab is transported under the conditions of DEG C, since the time interval that gum stem cell is extracted being collected into be no more than 72 hours；
Step 2, it is transferred to Biohazard Safety Equipment, takes out wisdom tooth to culture dish, the sterile phosphate buffer being pre-chilled with 4 DEG C cleans, That is PBS cleans its outer surface；
Step 3, gingiva tissue is placed in the PBS of the dispases of mg/ml containing 1-10,4 DEG C overnight；
Step 4, gingiva tissue is taken out, is shredded, is placed in the PBS of the IV Collagenase Types of mg/ml containing 1-10,37 with aseptic operation DEG C digestion 2 hours；
Step 5, digestive juice is filtered by 70 microns of cell sieve, obtains the single cell suspension of the stem cell of gum containing someone, 900rpm centrifuges 5min, and it is primary to wash cell with PBS；
Step 6, cell is resuspended with PBS, CD18 monoclonal antibodies, mixing is added, room temperature is protected from light incubation 30 minutes；
Step 7, after washing, 900rpm centrifuges 5min, and cell is resuspended with PBS；
Step 8, by selected by flow cytometry apoptosis or immunological magnetic bead sorting, CD18 Positive Stem Cells subgroups are obtained.
4. the dryness detection method of people's gum stem cell subgroup described in a kind of claims 1 or 2, which is characterized in that the method It is as follows：
Step 1, the CD18 Positive Stem Cells subgroups of enrichment are taken, are counted；
Step 2,900rpm centrifuges 5min；
Step 3, with fetal calf serum containing 10%-20%, 50 μM -200 μM of ascorbic acid 2- phosphates, 2mM glutamine, Cell is resuspended in the α-MEM culture mediums of 100U/mL penicillin and 100 μ g/mL streptomysins；
Step 4, by 104A CD18 Positive Stem Cells are inoculated in T25 Tissue Culture Flasks, are placed in 37 DEG C, 5% CO2Incubator in Culture；
Step 5, per liquid is changed within 3-4 days, after 14-16 days, culture solution is outwelled, PBS cleans attached cell, then uses and contains 0.1% toluene Amine indigo plant and the PBS solution of 2% paraformaldehyde are fixed, dye.
5. the dryness detection method of people's gum stem cell subgroup described in a kind of claims 1 or 2, which is characterized in that the method It is as follows：
Step 1, the CD18 Positive Stem Cells subgroups of enrichment are taken, are counted；
Step 2,900rpm centrifuges 5min；
Step 3, with fetal calf serum containing 10%-20%, 50 μM -200 μM of ascorbic acid 2- phosphates, 2mM glutamine, α-MEM culture mediums resuspension the cell of 100U/mL penicillin, 100 μ g/mL streptomysins；
Step 4, cell step 3 obtained is with 104It is inoculated in 12 orifice plates per hole, is placed in 37 DEG C, 5% CO2Incubator in train It supports；
Step 5, after cultivating 1-2 days, Brdu reagents are added, the Brdu reagents are incubated for 24 hours；
Step 6, Brdu staining kits dye cell；Haematoxylin is redyed；Positive Stem Cells number is counted under mirror, measures institute State the proliferation rate of stem cell.
6. the dryness detection method of people's gum stem cell subgroup described in a kind of claims 1 or 2, which is characterized in that the method It is as follows：
Step 1, the CD18 Positive Stem Cells subgroups of enrichment are taken, are counted；
Step 2,900rpm centrifuges 5min；
Step 3, with fetal calf serum containing 10%-20%, 50 μM -200 μM of ascorbic acid 2- phosphates, 2mM glutamine, α-MEM culture mediums resuspension the cell of 100U/mL penicillin, 100 μ g/mL streptomysins；
Step 4,4 × 10 are inoculated with5A CD18 Positive Stem Cells are placed in 37 DEG C, 5% CO in 6 orifice plates2Incubator in cultivate；
Step 5, when the expansion of stem cells is to 80% degrees of fusion, be replaced with Osteogenic Induction Medium, the culture medium be containing There are 5-30nM dexamethasone, the α-MEM cultures of 50-300 μ g/mL vitamin Cs and 1-10 mM sodium β-glycerophosphates Base；
Step 6, every to change liquid within 3-4 days；After induction in 3-4 weeks, the stem cell, sees that dark yellow calcium tubercle is formed under the microscope；
Step 7, it discards culture solution and discards PBS after PBS is cleaned 2 times；
Step 8, it changes 60% isopropanol into be fixed, after room temperature 1min, discards isopropanol, use distilled water cleaning 2 Time, with 1% Alizarin red staining, room temperature 3min；After PBS is cleaned 2 times, take pictures after exhausting liquid.
7. a kind of dryness detection method of people's gum stem cell subgroup as claimed in claim 1 or 2, which is characterized in that the side Method is as follows：
Step 1, by 2 × 106The hydroxyapatite/tricalcium phosphate of a CD18 Positive Stem Cells and 15mg, i.e. HA/TCP particles Mixing, and it is transplanted to the immunodeficient animals dorsal sc of 6-8 week old；
Step 2, post-transplantation is drawn materials for 8 weeks；
Step 3, after euthanasia puts to death the immunodeficient animals, careful separation subcutaneous cell agglomerate and skin, with 4% poly Formaldehyde is fixed, and 4 DEG C overnight；
Step 4, fixer is discarded, after PBS is cleaned up, the EDTA for changing 10% into carries out decalcification, changes a decalcification within every 3 days Liquid, after decalcification 21 days, dehydration embedding；
Step 5, after slice, haematoxylin/eosin stains and Masson ' s trichrome stains are carried out；
Step 6, microscopy.
8. the dryness detection method of people's gum stem cell subgroup described in claim 7, the immunodeficient animals are nude mice.
9. the purposes of people's gum stem cell subgroup described in a kind of claims 1 or 2, which is characterized in that be used for the regeneration of bone disease The preparation of medicine and tissue engineering material.
10. the purposes of people's gum stem cell subgroup described in a kind of claims 1 or 2, which is characterized in that will by transgenic technology CD18 genes are transferred to stroma stem cell, the preparation of regenerative medicine and tissue engineering material for bone disease.
11. the purposes of people's gum stem cell subgroup described in claim 10, which is characterized in that the stromal stem cell source choosing From tooth, fat, marrow, umbilical cord, placenta or bleeding of the umbilicus.
12. the purposes of people's gum stem cell subgroup described in claim 10, which is characterized in that the transgenic technology is by inverse The mode that Retroviral mediates.
CN201810595972.8A 2018-06-11 2018-06-11 A kind of people's gum stem cell subgroup and its dryness detection method by cell surface marker specific enrichment Pending CN108753694A (en)

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