CN109825469A - A kind of promotion abductive approach of the source of people umbilical cord mesenchymal stem cells to Chondrocyte Differentiation - Google Patents
A kind of promotion abductive approach of the source of people umbilical cord mesenchymal stem cells to Chondrocyte Differentiation Download PDFInfo
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Abstract
The present invention relates to a kind of promotion abductive approach of the source of people umbilical cord mesenchymal stem cells to Chondrocyte Differentiation, the induction step of the originally culture step of obtaining step, stem cell including umbilical cord tissue block, the subculture step of stem cell and stem cell to Chondrocyte Differentiation；The stem cell includes that P is made in the source of people umbilical cord mesenchymal stem cells resuspension of secondary culture to the 7th generation using serum free medium to the induction step of Chondrocyte Differentiation7For stem cell suspension, P7It is inoculated in for stem cell suspension in the serum free medium of the inducer containing cartilage differentiation and continues to cultivate, at an interval of three and half days the serum free medium of amount replacement inducer containing cartilage differentiation, arrive cartilage cell after induction 14 days.Source of people umbilical cord mesenchymal stem cells can be divided into cartilage cell by the present invention, be of great significance in terms of repairing human body cartilaginous tissue, treatment bone injury.
Description
Technical field
The present invention relates to biomedicine technical fields, more specifically, it to be related to a kind of promotion source of people umbilical cord mesenchyma dry
Abductive approach of the cell to Chondrocyte Differentiation.
Background technique
Knee articular cartilage defect is one of the Major research field of sports medical science clinical position, is the heat of Sports Scientific Research
Point and emphasis.The early stage of articular cartilage damage can cause the pain of injured joint and being limited for function of joint, and advanced joint may
There are retrogression pathological changes, non inflammatory osteoarthritic occurs, will seriously reduce patients ' life quality.After articular cartilage damage, cartilage
Cell supplies nutrition mainly by the matrix of knuckle synovia and cell peripheral, is energized with anerobic glycolysis, the repair ability of its own is very
It is limited, or even relying on itself is to repair again.
The appearance of mescenchymal stem cell (Mesenchymal stem cells, MSC) is the repairing and treating band of cartilage damage
To wish.Mescenchymal stem cell is the important member of stem cell line, derives from mesoderm growing early stage mesoderm and ectodermic one
Class multipotential stem cell has self-renewing, hyperproliferation and multi-lineage potential.In vivo and in vitro under specific inductive condition, MSC
The Various Tissues cell such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium, continuous passage can be divided into
Still there is multi-lineage potential after culture and freezen protective, can be used as injury repair of the ideal seed cell for histoorgan
With treatment, there is wide potential applicability in clinical practice.
MSCs initially has found in marrow, due to the MSCs cell collection palpus row bone marrow puncture to derived from bone marrow, source
It is restricted, it is very difficult to obtain a large amount of bone marrow cells.Furthermore the MSCs cell immunogenicity of derived from bone marrow is stronger, and with
The increase at donor age, cell quantity and differentiation potential occur being decreased obviously trend, and viral infection rate is higher, for a long time people
Be dedicated to finding a kind of mescenchymal stem cell for substituting derived from bone marrow always.
Umbilical cord mesenchymal stem cells refer to and are present in one of neonatal umbilical cord tissue versatile stem cell, main to divide
Cloth Tong Shi magnificent glue and umbilical cord perivascular.It is done carefully the study found that the MSCs in umbilical cord tissue source admirably maintains mesenchyma
The biological characteristics of born of the same parents and the ability of Multidirectional Differentiation.Furthermore compared with marrow and adipose-derived MSCs, umbilical cord tissue is being drawn materials
And very strong advantage is shown in immunogenicity, it specifically includes that the stem cell in a. umbilical cord is more original, there is stronger Proliferation, Differentiation
Ability；B. immunocyte is more inmature, and functional activity is low, will not trigger immune response and cause graft versus host disease(GVH disease)；C. it does
Cell is easily isolated, purity is high, negative for tumor cells pollution；D. the infection of latent virus and pathogenic microorganism and propagation probability ratio
It is lower；To puerpera and newborn without any harm and damage when e. acquiring；6. from a wealth of sources, acquisition is convenient, is easy to save and transport
Defeated, dispute of ethic is less.
Summary of the invention
It is dry thin in view of the deficiencies of the prior art, the present invention intends to provide a kind of promotion source of people umbilical cord mesenchyma
Source of people umbilical cord mesenchymal stem cells can be divided into cartilage cell to the abductive approach of Chondrocyte Differentiation by born of the same parents, repair people
It is of great significance in terms of body cartilaginous tissue, treatment bone injury.
Above-mentioned technical purpose of the invention has the technical scheme that
A kind of promotion abductive approach of the source of people umbilical cord mesenchymal stem cells to Chondrocyte Differentiation, the acquisition including umbilical cord tissue block
Step, the originally culture step of stem cell, the subculture step of stem cell and stem cell walk to the induction of Chondrocyte Differentiation
Suddenly；The stem cell includes that secondary culture is resuspended to the 7th generation using serum free medium to the induction step of Chondrocyte Differentiation
Source of people umbilical cord mesenchymal stem cells P is made7For stem cell suspension, P7Inducer containing cartilage differentiation is inoculated in for stem cell suspension
Serum free medium in continue to cultivate, at an interval of three and half days amount replacement the inducer containing cartilage differentiation serum free medium, induction 14
After it to get arrive cartilage cell.
Further, the serum free medium of the inducer containing cartilage differentiation includes that the sugared culture solution of DMEM high, serum replace
For object, penicillin, streptomysin, TGF-β 1, ferritin, ascorbic acid, dexamethasone and Sodium Pyruvate；DMEM high sugar culture solution and
The weight ratio of serum substitute is 95:5,100U/mL penicillin, 100 μ g/mL streptomysins, 1, the 5 μ g/mL of TGF-β of 10ng/mL
Transferrins, 50 μ g/mL ascorbic acid, 10-7Mol/L dexamethasone, 6.25 μ g/mL Sodium Pyruvates.
Further, the obtaining step of the umbilical cord tissue block include both ends ligation umbilical cord it is sterilized after, be placed in and include
In 1wt% dual anti-physiological saline；It removing umbilical cord two and ligatures end, umbilical cord interlude is cut into the umbilical cord tissue section of 0.8-1.2cm long,
Removal bloodstain is rinsed repeatedly to umbilical cord tissue section using the dual anti-physiological saline of 1wt% is included；Umbilical cord tissue section is splitted, blood is removed
Pipe and epidermal tissue obtain China's Tong Shi glue tissue, are floated repeatedly using the dual anti-physiological saline of 1wt% Tong Shi glue tissue to China is included
Wash, shred the mm × 1 of 1 mm × 1 mm umbilical cord tissue block.
Further, the originally culture step of the stem cell includes being seeded to umbilical cord tissue block is adherent in culture dish,
The CO that temperature is 37 DEG C, volume fraction is 5%21-2 h is stood in incubator, complete culture solution culture is added into culture dish, often
Complete culture solution is replaced every 3 days half amounts, after stationary culture 14 days, cell is cleaned using physiological saline, is added 0.5wt%'s
Tryple-Express digestive juice, temperature be 37 DEG C standings 2-3 minute, be fully retracted to pseudopodium, cell rounding, complete trains be added
Nutrient solution terminates digestion, and piping and druming obtains P1For stem cell suspension.
Further, the subculture step of the stem cell includes to PnWhen reaching 80-90% convergence degree for stem cell, abandon
Supernatant is removed, the Tryple-Express digestive juice of 0.5wt% is added, after digesting completely, complete culture solution termination is added and disappears
Change, liquid is centrifuged and discards supernatant, with physiological saline to PnIt is cleaned for stem cell, and P is resuspended to obtain in serum free mediumnDai Gan
Cell suspension, by PnIt is inoculated in Tissue Culture Flask for stem cell suspension, and Tissue Culture Flask is placed in temperature as 37 DEG C, volume point
The CO that number is 5%2It is cultivated in incubator, obtains Pn+1For stem cell.Wherein, n=1,2,3,4,5.
Further, further include stem cell cryopreservation step and stem cell recovery and cultivation step；The stem cell
Cryopreservation step includes to PmWhen reaching 80-90% convergence degree for stem cell, liquid is discarded supernatant, the Tryple- of 0.5wt% is added
Express digestive juice is added complete culture solution and terminates digestion, be centrifuged and discard supernatant liquid, use physiological saline after digesting completely
To PmIt cleans for stem cell, and is resuspended in serum-free mesenchymal stem cell cryopreserving liquid, by PmIt is sub-packed in and freezes for stem cell suspension
It in pipe and seals, cryopreservation tube is placed in after program temperature reduction box and is placed in immediately by program temperature reduction box immediately -80 DEG C of refrigerator
In freeze 12-24 hours, cryopreservation tube taken out and is placed in liquid nitrogen immediately later freeze；The recovery and cultivation of the stem cell
Step includes being removed from liquid nitrogen P to be recoveredmFor stem cell cryopreserving pipe, being put into temperature is in 37 DEG C of water-baths, to PmFor stem cell
It is transferred in physiological saline after thawing, is centrifuged and discards supernatant liquid, and P is resuspended to obtain in serum free mediummIt is outstanding for stem cell
Liquid, by PmBe inoculated in Tissue Culture Flask for stem cell suspension, and by Tissue Culture Flask be placed in temperature be 37 DEG C, volume fraction 5%
CO2P is cultivated to obtain in incubatorm+1For stem cell.
Further, the serum-free mesenchymal stem cell cryopreserving liquid includes the serum free medium 80 in terms of mass fraction
Part, 10 parts of dimethyl sulfoxide, 5 parts of L- menthyl pyranoside, 4 parts of glycerol, 1 part of N-Acetyl-D-glucosamine.
Further, the serum free medium is HY STEMCELL human mesenchymal stem cell serum free medium.
Further, the concentration of the stem cell suspension is 1 × 107A/mL.
In conclusion the invention has the following advantages:
The first, a kind of promotion abductive approach of the source of people umbilical cord mesenchymal stem cells to Chondrocyte Differentiation through the invention, P7Generation
Stem cell can be to Chondrocyte Differentiation.And it is of great significance in terms of repairing human body cartilaginous tissue, treatment bone injury.
The second, it after the recovery and cultivation step of the cryopreservation step of stem cell and stem cell, is filled between source of people umbilical cord
The proliferative capacity and differentiation capability of matter stem cell still have without influence, source of people umbilical cord mesenchymal stem cells to Chondrocyte Differentiation
Ability.
Third combines the present invention to provide the jelly of stem cell using serum-free mesenchymal stem cell cryopreserving liquid provided by the invention
Step is deposited, freezing to play and preferably freeze effect to source of people umbilical cord mesenchymal stem cells.Cell after recovery is not only being survived
It is significantly improved in rate, ability of cell proliferation is also superior to conventional cryopreservation methods, and source of people umbilical cord mesenchymal stem cells
Specific surface marker does not also significantly decrease, and has higher expression rate.
Detailed description of the invention
Fig. 1 is the obtained P of embodiment 16The growth curve of culture 7 days is carried out for stem cell and to 2 gained of embodiment
The P arrived67 days growth curves are cultivated after being recovered for stem cell.
Specific embodiment
Invention is further described in detail with reference to embodiments.
Embodiment 1
A kind of promotion abductive approach of the source of people umbilical cord mesenchymal stem cells to Chondrocyte Differentiation, sequentially including umbilical cord tissue block
Obtaining step, the originally culture step of stem cell, the subculture step of stem cell and stem cell luring to Chondrocyte Differentiation
Lead step.
The obtaining step of umbilical cord tissue block includes being placed in after the umbilical cord of both ends ligation is sterilized and including the dual anti-physiology of 1wt%
In salt water；It removes umbilical cord two and ligatures end, umbilical cord interlude is cut into the umbilical cord tissue section of 0.8-1.2cm long, bis- using 1wt% is included
Anti- physiological saline rinses removal bloodstain to umbilical cord tissue section repeatedly；Umbilical cord tissue section is splitted, blood vessel and epidermal tissue is removed, obtains
Get Hua Tongshi glue tissue is rinsed repeatedly using the dual anti-physiological saline of 1wt% Tong Shi glue tissue to China is included, shreds to obtain 1 mm × 1
The umbilical cord tissue block of the mm of mm × 1.
The originally culture step of stem cell includes being seeded to umbilical cord tissue block is adherent in culture dish, and temperature is 37 DEG C, body
The CO that fraction is 5%21-2 h is stood in incubator, complete culture solution culture is added into culture dish, at an interval of three and half days amount replacement
Complete culture solution after stationary culture 14 days, discards culture solution and umbilical cord tissue block, cleans cell using physiological saline, is added
The Tryple-Express digestive juice of 0.5wt%, temperature be 37 DEG C standing 2-3 minutes, be fully retracted to pseudopodium, cell rounding adds
Enter complete culture solution and terminate digestion, piping and druming obtains P1For stem cell suspension, P is adjusted1It is 1 × 10 for stem cell suspension concentration7A/
mL。
The subculture step of stem cell includes to PnWhen reaching 80-90% convergence degree for stem cell, liquid is discarded supernatant, is added
The Tryple-Express digestive juice of 0.5wt% is added complete culture solution and terminates digestion, be centrifuged and discard after digesting completely
Clear liquid, with physiological saline to PnIt is cleaned for stem cell, and the weight in HY STEMCELL human mesenchymal stem cell serum free medium
Hang to obtain PnFor stem cell suspension, P is adjustednIt is 1 × 10 for stem cell suspension concentration7A/mL, by PnIt is inoculated in for stem cell suspension
T-175 Tissue Culture Flask, and T-175 Tissue Culture Flask is placed in the CO that temperature is 37 DEG C, volume fraction is 5%2It is trained in incubator
It supports, obtains Pn+1For stem cell, wherein n=1,2,3,4,5,6.
Stem cell includes using HY STEMCELL human mesenchymal stem cell serum-free to the induction step of Chondrocyte Differentiation
P is made in the source of people umbilical cord mesenchymal stem cells that secondary culture to the 7th generation is resuspended in culture medium7For stem cell suspension, P is adjusted7Dai Gan
Concentration of cell suspension is 1 × 107A/mL, P7It is inoculated in the serum free medium of the inducer containing cartilage differentiation for stem cell suspension
Continue to cultivate, at an interval of three and half days the serum free medium of amount replacement inducer containing cartilage differentiation, arrives cartilage after induction 14 days
Cell.The serum free medium of the inducer containing cartilage differentiation includes DMEM high sugared culture solution, serum substitute, penicillin, strepto-
Element, TGF-β 1, ferritin, ascorbic acid, dexamethasone and Sodium Pyruvate；The weight of DMEM high sugar culture solution and serum substitute
Than for 95:5,100U/mL penicillin, 100 μ g/mL streptomysins, 1, the 5 μ g/mL transferrins of TGF-β of 10ng/mL, 50 μ g/mL
Ascorbic acid, 10-7Mol/L dexamethasone, 6.25 μ g/mL Sodium Pyruvates.
Embodiment 2
A kind of promotion abductive approach of the source of people umbilical cord mesenchymal stem cells to Chondrocyte Differentiation, sequentially including umbilical cord tissue block
Obtaining step, the originally culture step of stem cell, the subculture step of stem cell, the cryopreservation step of stem cell, stem cell
The induction step of recovery and cultivation step and stem cell to Chondrocyte Differentiation.
The obtaining step of umbilical cord tissue block includes being placed in after the umbilical cord of both ends ligation is sterilized and including the dual anti-physiology of 1wt%
In salt water；It removes umbilical cord two and ligatures end, umbilical cord interlude is cut into the umbilical cord tissue section of 0.8-1.2cm long, bis- using 1wt% is included
Anti- physiological saline rinses removal bloodstain to umbilical cord tissue section repeatedly；Umbilical cord tissue section is splitted, blood vessel and epidermal tissue is removed, obtains
Get Hua Tongshi glue tissue is rinsed repeatedly using the dual anti-physiological saline of 1wt% Tong Shi glue tissue to China is included, shreds to obtain 1 mm × 1
The umbilical cord tissue block of the mm of mm × 1.
The originally culture step of stem cell includes being seeded to umbilical cord tissue block is adherent in culture dish, and temperature is 37 DEG C, body
The CO that fraction is 5%21-2 h is stood in incubator, complete culture solution culture is added into culture dish, at an interval of three and half days amount replacement
Complete culture solution after stationary culture 14 days, discards culture solution and umbilical cord tissue block, cleans cell using physiological saline, is added
The Tryple-Express digestive juice of 0.5wt%, temperature be 37 DEG C standing 2-3 minutes, be fully retracted to pseudopodium, cell rounding adds
Enter complete culture solution and terminate digestion, piping and druming obtains P1For stem cell suspension, P is adjusted1It is 1 × 10 for stem cell suspension concentration7A/
mL。
The subculture step of stem cell includes to PnWhen reaching 80-90% convergence degree for stem cell, liquid is discarded supernatant, is added
The Tryple-Express digestive juice of 0.5wt% is added complete culture solution and terminates digestion, be centrifuged and discard after digesting completely
Clear liquid, with physiological saline to PnIt is cleaned for stem cell, and the weight in HY STEMCELL human mesenchymal stem cell serum free medium
Hang to obtain PnFor stem cell suspension, P is adjustednIt is 1 × 10 for stem cell suspension concentration7A/mL, by PnIt is inoculated in for stem cell suspension
T-175 Tissue Culture Flask, and T-175 Tissue Culture Flask is placed in the CO that temperature is 37 DEG C, volume fraction is 5%2It is trained in incubator
It supports, obtains Pn+1For stem cell；Wherein, n=1,2,3,4,5.
The cryopreservation step of stem cell includes to P6When reaching 80-90% convergence degree for stem cell, liquid is discarded supernatant, is added
The Tryple-Express digestive juice of 0.5wt% is added complete culture solution and terminates digestion, be centrifuged and discard after digesting completely
Clear liquid, with physiological saline to P6It cleans for stem cell, and is resuspended in serum-free mesenchymal stem cell cryopreserving liquid, adjust P6Dai Gan
Cell concentration is 1 × 107/mL, by P6It is sub-packed in cryopreservation tube and seals for stem cell suspension, be immediately placed in cryopreservation tube
After program temperature reduction box and program temperature reduction box is placed in -80 DEG C of refrigerator immediately and is frozen 12-24 hours, later takes cryopreservation tube
It out and is placed in liquid nitrogen and freezes immediately.Serum-free mesenchymal stem cell cryopreserving liquid includes the HY in terms of mass fraction
It is 80 parts of STEMCELL human mesenchymal stem cell serum free medium, 10 parts of dimethyl sulfoxide, 5 parts of L- menthyl pyranoside, sweet
4 parts, 1 part of N-Acetyl-D-glucosamine of oil.
The recovery and cultivation step of stem cell includes being removed from liquid nitrogen P to be recovered6For stem cell cryopreserving pipe, it is put into temperature
Degree is in 37 DEG C of water-baths, to P6It is transferred in physiological saline after melting for stem cell, is centrifuged and discards supernatant liquid, and in HY
P is resuspended to obtain in STEMCELL human mesenchymal stem cell serum free medium6For stem cell suspension, by P6It is inoculated with for stem cell suspension
The CO that temperature is 37 DEG C, volume fraction is 5% is placed in T-175 Tissue Culture Flask, and by T-175 Tissue Culture Flask2In incubator
Cultivate to obtain P7For stem cell.
Stem cell includes using HY STEMCELL human mesenchymal stem cell serum-free to the induction step of Chondrocyte Differentiation
P is made in the source of people umbilical cord mesenchymal stem cells that secondary culture to the 7th generation is resuspended in culture medium7For stem cell suspension, P is adjusted7Dai Gan
Concentration of cell suspension is 1 × 107A/mL, P7It is inoculated in the serum free medium of the inducer containing cartilage differentiation for stem cell suspension
Continue to cultivate, at an interval of three and half days the serum free medium of amount replacement inducer containing cartilage differentiation, arrives cartilage after induction 14 days
Cell.The serum free medium of the inducer containing cartilage differentiation includes DMEM high sugared culture solution, serum substitute, penicillin, strepto-
Element, TGF-β 1, ferritin, ascorbic acid, dexamethasone and Sodium Pyruvate；The weight of DMEM high sugar culture solution and serum substitute
Than for 95:5,100U/mL penicillin, 100 μ g/mL streptomysins, 1, the 5 μ g/mL transferrins of TGF-β of 10ng/mL, 50 μ g/mL
Ascorbic acid, 10-7Mol/L dexamethasone, 6.25 μ g/mL Sodium Pyruvates.
To cartilage cell obtained by embodiment 1 and embodiment 2, fixed after 15 minutes using 4% paraformaldehyde room temperature execute Ah
Sharp Xinlan's dyeing identification, whether the cell detected is cartilage cell.
The dyeing identification of A Li Xinlan is to suck paraformaldehyde, and after being rinsed 2 times with PBS, the A Li Xinlan dye of pH 2.5 is added
Liquid dyes 30 minutes, distilled water flushing 2 minutes, staining conditions is observed under inverted phase contrast microscope, if visible under microscope
There is cell pellet generation, and is blue by A Li Xinlan dye liquor dye, the expression of sulphation glycosaminoglycan is positive as in cell cytosol,
Show the biological characteristics of cartilage cell.
The result shows that the either obtained cartilage cell of the obtained cartilage cell of embodiment 1 or embodiment 2, aobvious
It is visible under micro mirror to have cell pellet generation, and the expression of sulphation glycosaminoglycan is positive (by A Li Xinlan dye liquor dye in cell cytosol
For blue), show the biological characteristics of cartilage cell.
To the obtained P of embodiment 16The P obtained after recovering for stem cell and to embodiment 26For the increasing of stem cell
Situation is grown to compare.Draw the obtained P of embodiment 16The growth curve chart of culture 7 days is carried out for stem cell and to implementation
The obtained P of example 267 days growth curve charts are cultivated after being recovered for stem cell, as a result as shown in Figure 1.It is provided by the invention
Serum-free mesenchymal stem cell cryopreserving liquid combines the present invention to provide the cryopreservation step of stem cell, to source of people umbilical cord mesenchymal stem cells
Freeze to play and preferably freeze effect, the proliferative capacity of the stem cell after recovery is slightly worse than without the proliferation of the stem cell frozen
Ability.It follows that compared to the prior art, the stem cell after recovery is not only significantly improved in survival rate, cell increases
Ability is grown also superior to conventional cryopreservation methods.
To the obtained P of embodiment 16The P obtained after recovering for stem cell and to embodiment 26For the table of stem cell
The Comparative result of face label expression rate.The specific surface marker of source of people umbilical cord mesenchymal stem cells have CD34-, CD44+,
CD45-,CD90+.Respectively to the obtained P of embodiment 16The P obtained after recovering for stem cell and to embodiment 26Dai Gan
Cell, stem cell suspension is made after collecting in digestion after culture 3 days, and the antibody with immunofluorescence is added and is incubated for, carries out
The flow cytometer detection of source of people umbilical cord mesenchymal stem cells surface marker, the specific antibody of addition are respectively that (be negative CD34 table
Up to), CD45 (be negative expression), CD44 (positive expression), CD90 (positive expression).The obtained P of embodiment 16Dai Gan
Cell is after culture 3 days, and CD34-CD44+ expression rate is 99.9%, CD45-CD90+ expression rate to streaming as the result is shown is 99.9%；
The P that embodiment 2 obtains after being recovered6For stem cell after culture 3 days, CD34-CD44+ expression rate is streaming as the result is shown
99.5%, CD45-CD90+ expression rate are 98.7%.It follows that by using serum-free mescenchymal stem cell provided by the invention
After the cryopreservation step that frozen stock solution combines the present invention to provide stem cell, the specific surface marker of source of people umbilical cord mesenchymal stem cells
It does not significantly decrease, there is higher expression rate.
It follows that a kind of promotion source of people umbilical cord mesenchymal stem cells are to the induction side of Chondrocyte Differentiation through the invention
Method, P7It can be to Chondrocyte Differentiation for stem cell.Moreover, by the cryopreservation step of stem cell and the recovery of stem cell and training
After supporting step, on the proliferative capacity of source of people umbilical cord mesenchymal stem cells and differentiation capability without influence, source of people umbilical cord mesenchyma is dry thin
Born of the same parents still have the ability to Chondrocyte Differentiation.This is because using serum-free mesenchymal stem cell cryopreserving provided by the invention
Liquid combines the present invention to provide the cryopreservation step of stem cell, freezing to play and preferably freeze effect to source of people umbilical cord mesenchymal stem cells
Fruit.Cell after recovery is not only significantly improved in survival rate, ability of cell proliferation also superior to conventional cryopreservation methods, and
And the specific surface marker of source of people umbilical cord mesenchymal stem cells does not also significantly decrease, and has higher expression rate.
It should be understood that preparation method described in the embodiment of the present invention is only used for illustrating the present invention, rather than to this
The limitation of invention belongs to the simple modifications of preparation method of the present invention under concept thereof of the invention claimed
Range.
Claims (9)
1. a kind of promotion abductive approach of the source of people umbilical cord mesenchymal stem cells to Chondrocyte Differentiation, which is characterized in that including navel
The originally culture step of obtaining step, stem cell with tissue block, the subculture step of stem cell and stem cell are thin to cartilage
The induction step of born of the same parents' differentiation；The stem cell includes being resuspended to pass using serum free medium to the induction step of Chondrocyte Differentiation
It is commissioned to train to support to the source of people umbilical cord mesenchymal stem cells in the 7th generation and P is made7For stem cell suspension, P7It is inoculated in and contains for stem cell suspension
Continue to cultivate in the serum free medium of cartilage differentiation inducer, at an interval of three and half days amount replacement the inducer containing cartilage differentiation without blood
Clear culture medium arrives cartilage cell after induction 14 days.
2. a kind of promotion source of people umbilical cord mesenchymal stem cells according to claim 1 are to the induction side of Chondrocyte Differentiation
Method, which is characterized in that the serum free medium of the inducer containing cartilage differentiation includes DMEM high sugared culture solution, blood serum substituting
Object, penicillin, streptomysin, TGF-β 1, ferritin, ascorbic acid, dexamethasone and Sodium Pyruvate；DMEM high sugar culture solution and blood
The weight ratio of clear substitute is 95:5, and 1, the 5 μ g/mL of TGF-β of 100U/mL penicillin, 100 μ g/mL streptomysins, 10ng/mL turns
Ferritin, 50 μ g/mL ascorbic acid, 10-7Mmol/mL dexamethasone, 6.25 μ g/mL Sodium Pyruvates.
3. a kind of promotion source of people umbilical cord mesenchymal stem cells according to claim 1 are to the induction side of Chondrocyte Differentiation
Method, which is characterized in that the obtaining step of the umbilical cord tissue block includes being placed in after the umbilical cord of both ends ligation is sterilized and including 1wt%
In dual anti-physiological saline；It removes umbilical cord two and ligatures end, umbilical cord interlude is cut into the umbilical cord tissue section of 0.8-1.2cm long, uses
It includes the dual anti-physiological saline of 1wt% and rinses removal bloodstain repeatedly to umbilical cord tissue section；Split umbilical cord tissue section, remove blood vessel and
Epidermal tissue obtains China's Tong Shi glue tissue, is rinsed, cut repeatedly using the dual anti-physiological saline of 1wt% Tong Shi glue tissue to China is included
The broken umbilical cord tissue block for obtaining the mm × 1 of 1 mm × 1 mm.
4. a kind of promotion source of people umbilical cord mesenchymal stem cells according to claim 1 are to the induction side of Chondrocyte Differentiation
Method, which is characterized in that the originally culture step of the stem cell includes being seeded to umbilical cord tissue block is adherent in culture dish, temperature
The CO for being 5% for 37 DEG C, volume fraction21-2 h is stood in incubator, complete culture solution culture is added into culture dish, every 3
Its half amount replacement complete culture solution, after stationary culture 14 days, is discarded culture solution and umbilical cord tissue block, is cleaned using physiological saline thin
Born of the same parents, are added the Tryple-Express digestive juice of 0.5wt%, temperature be 37 DEG C standing 2-3 minutes, be fully retracted to pseudopodium, cell
It is rounded, complete culture solution is added and terminates digestion, piping and druming obtains P1For stem cell suspension.
5. a kind of promotion source of people umbilical cord mesenchymal stem cells according to claim 1 are to the induction side of Chondrocyte Differentiation
Method, which is characterized in that the subculture step of the stem cell includes to PnWhen reaching 80-90% convergence degree for stem cell, discard
The Tryple-Express digestive juice of 0.5wt% is added in clear liquid, after digesting completely, complete culture solution is added and terminates digestion, from
The heart simultaneously discards supernatant liquid, with physiological saline to PnIt is cleaned for stem cell, and P is resuspended to obtain in serum free mediumnIt is outstanding for stem cell
Liquid, by PnBe inoculated in Tissue Culture Flask for stem cell suspension, and by Tissue Culture Flask be placed in temperature be 37 DEG C, volume fraction 5%
CO2It is cultivated in incubator, obtains Pn+1For stem cell.
6. a kind of promotion source of people umbilical cord mesenchymal stem cells according to claim 1 are to the induction side of Chondrocyte Differentiation
Method, which is characterized in that further include the cryopreservation step of stem cell and the recovery and cultivation step of stem cell；The stem cell freezes
Step includes to PmWhen reaching 80-90% convergence degree for stem cell, liquid is discarded supernatant, the Tryple-Express that 0.5wt% is added disappears
Change liquid, after digesting completely, complete culture solution is added and terminates digestion, liquid is centrifuged and discards supernatant, with physiological saline to PmDai Gan
Cell cleaning, and be resuspended in serum-free mesenchymal stem cell cryopreserving liquid, by PmIt is sub-packed in cryopreservation tube simultaneously for stem cell suspension
Cryopreservation tube, is placed in after program temperature reduction box immediately and is placed in -80 DEG C of refrigerator immediately by program temperature reduction box and freeze by sealing
12-24 hours, cryopreservation tube is taken out later and is placed in liquid nitrogen freezes immediately；The recovery and cultivation step packet of the stem cell
It includes and is removed from liquid nitrogen P to be recoveredmFor stem cell cryopreserving pipe, being put into temperature is in 37 DEG C of water-baths, to PmAfter melting for stem cell
It is transferred in physiological saline, is centrifuged and discards supernatant liquid, and P is resuspended to obtain in serum free mediummFor stem cell suspension, by Pm
It is inoculated in Tissue Culture Flask for stem cell suspension, and Tissue Culture Flask is placed in the CO that temperature is 37 DEG C, volume fraction is 5%2Training
It supports in case and cultivates to obtain Pm+1For stem cell.
7. a kind of promotion source of people umbilical cord mesenchymal stem cells according to claim 6 are to the induction side of Chondrocyte Differentiation
Method, which is characterized in that the serum-free mesenchymal stem cell cryopreserving liquid include 80 parts of serum free medium in terms of mass fraction,
10 parts of dimethyl sulfoxide, 5 parts of L- menthyl pyranoside, 4 parts of glycerol, 1 part of N-Acetyl-D-glucosamine.
8. a kind of promotion source of people umbilical cord mesenchymal stem cells described according to claim 1 or 5 or 6 or 7 are to Chondrocyte Differentiation
Abductive approach, which is characterized in that the serum free medium be HY STEMCELL human mesenchymal stem cell free serum culture
Base.
9. a kind of promotion source of people umbilical cord mesenchymal stem cells described according to claim 1 or 4 or 5 or 6 are to Chondrocyte Differentiation
Abductive approach, which is characterized in that the concentration of the stem cell suspension be 1 × 107A/mL.
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