CN108384752A - Application at Cartilage culture base and its in micro- splitting technique in knee cartilage differentiation - Google Patents

Application at Cartilage culture base and its in micro- splitting technique in knee cartilage differentiation Download PDF

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CN108384752A
CN108384752A CN201810153231.4A CN201810153231A CN108384752A CN 108384752 A CN108384752 A CN 108384752A CN 201810153231 A CN201810153231 A CN 201810153231A CN 108384752 A CN108384752 A CN 108384752A
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cartilage
knee
culture base
micro
differentiation
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邵擎东
严旭
王浩
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No455 Hospital Pla
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No455 Hospital Pla
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1384—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from adipose-derived stem cells [ADSC], from adipose stromal stem cells

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Abstract

The present invention provides a kind of application at Cartilage culture base and its in micro- splitting technique in knee cartilage differentiation, including fat stem cell into chondrocyte induction differentiation basal medium, dexamethasone, ascorbic acid, ITS, Sodium Pyruvate, proline, TGF β 3.It is handled by hypoxemia, and the injury of knee joint reparation relatively non-hypoxia group of situation of hypoxic preconditioning group is more preferable, implies that hypoxemia processing has more repair of cartilage ability to the cartilage cell that fat stem cell differentiates.

Description

Application at Cartilage culture base and its in micro- splitting technique in knee cartilage differentiation
Technical field
The present invention relates to biocytology fields, more particularly at Cartilage culture base and its knee joint in micro- splitting technique Application in cartilage differentiation.
Background technology
Knee articular cartilage defect is clinically very common, is often accompanied by pain, swelling, influences normal activity and lead to bone Arthritis.It is difficult regeneration after damage since cartilaginous tissue does not have blood supply, source of nutrition to be moistened in periarticular synovia, Therefore, final therapeutic purpose is to restore normal knee joint function by the hyaline cartilage of regenerated defect.In this purpose Under, people have started surgical intervention of the various methods for cartilage damage, including micro fractures operation (MFX), knee close under arthroscope Save the soft transplanting of recovery technique, autologous bone, inserted plasty and the soft transplanting of frozen homologous bone, by articular hyaline cartilage be transplanted to by Damage position.Wherein MFX because its minimal invasive and it is easy to operate due to become a kind of most popular treatment means.However, repair tissue The overwhelming majority is fibrocartilage, rather than hyaline cartilage, and the technology is to be based on pluripotent stem cell differentiation technology, is suffered to old age The effect is relatively poor for person.Have confirmed that early and middle portion treatment results are good in spite of research, but due to regenerated fiber cartilage Biomethanics defect and scar generation, can cause that sb.'s illness took a turn for the worse over time.Therefore find have excellent differentiation potential and The stem cell seed resource of safety is most important.Wherein adipose-derived mescenchymal stem cell cell (ADSCs) seemed at The ideal seed material of people's selection, the reason is as follows that：It is easy to get and detaches, and to small (liposuction separation) for bulk damage； Autotransplantation is without rejection；The environmental condition that in-vitro multiplication needs is relatively easy；Still people can maintain this its in succeeding generations The potential of cartilage differentiation.In recent years, it has increasingly been paid close attention to by brainstrust using ADSCs treatment knee articular cartilage defects, and Most of experts, which think to treat using ADSCs, can promote wound healing and Regeneration of Articular Cartilage.As shown in Figure 1, traditionally People are the external evoked chondroblast of autologous stem cells using separation in terms of the stem-cell therapy of knee articular cartilage defect Row fixed-point injection again afterwards, but influenced by vivo environment, therapeutic effect is often bad.
To find out its cause, in the environment of a hypoxic-ischemic, the cell survival of transplanting is affected.For increased cell It is resistant to the ability of this anaerobic environment.
Invention content
In order to overcome the above-mentioned deficiencies of the prior art, one aspect of the present invention provides a kind of at Cartilage culture base, and feature exists In, including following component, fat stem cell is at chondrocyte induction differentiation basal medium, dexamethasone, ascorbic acid, ITS, acetone Sour sodium, proline, TGF-β 3.
Second aspect of the present invention provides a kind of above-mentioned knee cartilage differentiation in micro- splitting technique at Cartilage culture base In application induced 21 days under normal operation using described at Cartilage culture base, change a not good liquor every three days, and every 12 small Hypoxia Stress induces Shi Jinhang twice, each stress-inducing half an hour.
Preferably, the hypoxemia ingredient is 1% O2,5% CO2,94% N2.
Compared with prior art, beneficial effects of the present invention are as follows：
Present invention discover that no matter hypoxemia processing or normally cultivate under, fat stem cell can be promoted at cartilage by 21 days Differentiation (Safranin O dyeing), implies that fat stem cell is broken up without influence in hypoxemia processing.After treatment, knee cartilage is taken Tissue carries out masson dyeing and finds, the injury of knee joint reparation relatively non-hypoxia group of situation of hypoxic preconditioning group is more preferable, implies Hypoxemia processing has more repair of cartilage ability to the cartilage cell that fat stem cell differentiates.
Description of the drawings：
Fig. 1 is micro- splitting technique fixed-point injection figure in the prior art.
Fig. 2 is fat stem cell separation and identification in the embodiment of the present invention.
Fig. 3 is in the embodiment of the present invention into cartilage differentiation qualification figure.
Fig. 4 is that masson dyeing detection knee cartilage is damaged recovery situation figure in the embodiment of the present invention.
Specific implementation mode
The present invention is further described with reference to specific embodiment, to more fully understand the present invention.1, fatty Stem cell detaches：
The adipose tissue at new zealand white rabbit groin is taken first, is put into the Du Shi phosphate-buffereds containing mycillin Blood stains are cleaned in liquid (DPBS).It is transferred in a new culture dish, fully shreds, addition digestive juice (10% fetal calf serum, 0.09% clostridiopetidase A I, remaining is DMEM/F12), pipettor is fully beaten, until tissue can be unobstructed discrepancy suction nozzle, by fat Fat tissue is transferred in centrifuge tube, and 37 DEG C of digestion 1h periodically rock, tissue and digestive juice is made to mix well.After having digested, blow repeatedly Digestive juice is beaten, cell suspension is made in dispersion tissue's block.1200rpm centrifuges 5min, the adipose tissue on upper layer is blown and beaten repeatedly minimum 1min is centrifuged again.The digestive juice of the adipose tissue and middle level of upper layer maturation is discarded, then the thin of bottom is resuspended with basal medium Born of the same parents.First by 250 μM of nylon leaching net filtration cell suspension of cell suspension, then be 80 μM by gained filtrate aperture nylon mistake Filter removes most mature fat cell, collects filtrate, pays attention to washing filter with suitable basal medium again after filtering every time Net reduces the cell residue on strainer.The filtrate finally obtained is centrifuged, the adipocyte for abandoning floating is inhaled, uses basal medium It washs 3 times repeatedly.Cell culture is resuspended with complete medium for the last time, and inoculation is placed in routine culture in incubator.For the first time Liquid is changed, culture medium is discarded, PBS is washed 5-6 times repeatedly, and fresh complete medium is added and continues to cultivate.Be repeated 3 times change liquid until Cell face is substantially clean.It digests and expands culture identification.
Relatively special molecule CD29, the CD44 of ADSCs is detected using routine immunization fluorescent method and low cytometric analysis, CD90, CD105 etc..Low cytometric analysis detects the cell cycle, calculates the cell proportion in the G0/G1 phases.It is lured by difference Drain carries out induction differentiation to the ADSCs of in vitro culture, makes ADSCs to cartilage cell, adipocyte and endothelial cell direction point Change, using colouring methods such as alizarin Suo Hong, ALP, oil red, sarranine-O- dyeing and CD31.
2, at cartilage differentiation：Using cartilage differentiation inducing culture (formula table 1), (5%CO under normal condition2, 37 DEG C) Induction 21 days, changes a not good liquor in every 3 days.
Table 1 is at Cartilage culture based formulas：
3, hypoxic preconditioning：Hypoxemia (1%O twice is carried out per 12h2, 5%CO2, 94%N2) stress-inducing 30min, directly Until injecting the previous day.
4, sarranine-O- dyes (Safranin O)
For sample after the fixation of 4% paraformaldehyde solution, 20% sucrose is dehydrated 4-6h, and 30% sucrose solution dehydrated overnight enters O.T.C embedding mediums impregnate 2h, and -20 DEG C are overnight, enter -20 DEG C after room temperature rewarming 2h again overnight, 8 μm of freezing microtome slice, and 37 It DEG C is dried overnight.0.02% fast green dyeing liquors dye 4min, tap water rinse, and 1% acetic acid color separation several seconds was added 0.1% sarranine-O- dyeing liquors dye 3min, tap water rinse, and neutral gum mounting after drying moves to microscopically observation
5, masson is dyed：Rabbit is killed after 4 weeks, knee cartilage tissue is taken to carry out a group change detection.
Step：Paraffin embedding after dehydration, every serial section 6 are opened, 4 μm of tissue thickness, in slice 70 DEG C of insulating boxs of merging 15min, dewaxing are toasted, PBS is rinsed 3 times, every all over 2min.Sample is put in antigen repairing box, is dipped in 0.01mol citron acid solutions In, it is put into micro-wave oven and heats 15min, be cooled to room temperature.PBS is rinsed 3 times, every all over 2min.Remove PBS liquid, slice plus goat Serum is closed, and room temperature, which is protected from light, is incubated 20min.Unnecessary serum is removed, is not washed, 50 μ l primary antibodies are added dropwise in slice.Remove PBS liquid, slice 50 μ l secondary antibodies are added dropwise, are placed in 40min in 37 DEG C of insulating boxs, PBS concussions washing 4 times is every to dye 30min all over 5min, masson3, Negative staining, hematoxylin pathological staining liquid dye 40min, are dehydrated transparent processing, neutral gum mounting, and room temperature is dried, micro- sem observation Film making.
6, rabbit knee cartilage damage model and the treatment of micro- splitting technique：Take new zealand white rabbit 6,6g/L amobarbitals Ear edge intravenous is anaesthetized, and by retreating the stock facies artieularis malleolaris position electric bone drill in joint in experimental rabbit, is controlling its diameter 4.5mm, depth 3m cause its defects of knee, and differential is carried out after 2 weeks and splits technology, as shown in Fig. 2, injection proper splitting Cartilage cell and cartilage cell through hypoxic preconditioning kill rabbit after 1 × 106,4 weeks, take knee cartilage tissue to carry out Situation is repaired in massoon dyeing identifications.
As a result
1, the separation and identification of fat stem cell：Separation and qualification result are carried out to ADSCs as shown in Fig. 2, being marked to surface Note object carries out Immunofluorescence test and finds that ADSCs specific marker proteins CD29, CD44, CD90 and CD105 have expression, and interior Endothelial cell surface marker vWF and CD34 illustrates that fat stem cell detaches successfully without expression.
2, as shown in figure 3, the study found that research find no matter hypoxemia processing or normally cultivate under, can by 21 days Promote fat stem cell at cartilage differentiation (Safranin O dyeing), implies that fat stem cell is broken up without influence in hypoxemia processing.
3, it as shown in figure 4, after treatment in 4 weeks, takes knee cartilage tissue to carry out masson dyeing and finds, hypoxic preconditioning The cartilage that the relatively non-hypoxia group of situation is more preferable, and hint hypoxemia processing differentiates fat stem cell is repaired in the injury of knee joint of group Cell has more repair of cartilage ability.
Specific embodiments of the present invention are described in detail above, but it is intended only as example, the present invention is simultaneously unlimited It is formed on particular embodiments described above.To those skilled in the art, it is any to the equivalent modifications that carry out of the present invention and It substitutes also all among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by impartial conversion and Modification, all should be contained within the scope of the invention.

Claims (3)

1. a kind of at Cartilage culture base, which is characterized in that including following component, fat stem cell breaks up basis training at chondrocyte induction Support base, dexamethasone, ascorbic acid, ITS, Sodium Pyruvate, proline, TGF-β 3.
2. a kind of application described in claim 1 at Cartilage culture base in micro- splitting technique in knee cartilage differentiation, It is characterized in that, using described at Cartilage culture base, induces 21 days under normal operation, change a not good liquor every three days, and is every 12 small Hypoxia Stress induces Shi Jinhang twice, each stress-inducing half an hour.
3. the application according to claim 2 at Cartilage culture base in micro- splitting technique in knee cartilage differentiation, It is characterized in that, the hypoxemia ingredient is 1%O2, 5%CO2, 94%N2。
CN201810153231.4A 2018-02-13 2018-02-13 Application at Cartilage culture base and its in micro- splitting technique in knee cartilage differentiation Pending CN108384752A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111282018A (en) * 2020-05-13 2020-06-16 优赛生命科技有限公司 Adipose-derived stem cell composition for wound repair
WO2023245813A1 (en) * 2022-06-22 2023-12-28 广东省科学院生物与医学工程研究所 Composition and use thereof in preparation of cell membrane

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005001076A2 (en) * 2003-06-27 2005-01-06 Ethicon, Incorporated Postpartum cells derived from placental tissue, and methods of making and using the same
CN104546915A (en) * 2015-02-16 2015-04-29 黑龙江天晴干细胞股份有限公司 Composition for treating osteoarthritis as well as preparation method and application of composition
CN105695402A (en) * 2016-04-14 2016-06-22 安沂华 Composition and method for inducing mesenchymal stem cells to be differentiated to cartilage cells
CN106350483A (en) * 2016-10-14 2017-01-25 中卫华医（北京）生物科技有限公司 Culture method for inducing adipose tissue-derived stromal cells to differentiate to chondrocyte

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005001076A2 (en) * 2003-06-27 2005-01-06 Ethicon, Incorporated Postpartum cells derived from placental tissue, and methods of making and using the same
CN104546915A (en) * 2015-02-16 2015-04-29 黑龙江天晴干细胞股份有限公司 Composition for treating osteoarthritis as well as preparation method and application of composition
CN105695402A (en) * 2016-04-14 2016-06-22 安沂华 Composition and method for inducing mesenchymal stem cells to be differentiated to cartilage cells
CN106350483A (en) * 2016-10-14 2017-01-25 中卫华医（北京）生物科技有限公司 Culture method for inducing adipose tissue-derived stromal cells to differentiate to chondrocyte

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111282018A (en) * 2020-05-13 2020-06-16 优赛生命科技有限公司 Adipose-derived stem cell composition for wound repair
WO2023245813A1 (en) * 2022-06-22 2023-12-28 广东省科学院生物与医学工程研究所 Composition and use thereof in preparation of cell membrane

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