A kind of promotion abductive approach of the source of people umbilical cord mesenchymal stem cells to Chondrocyte DifferentiationTechnical 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 dryAbductive 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 ResearchPoint 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 mayThere are retrogression pathological changes, non inflammatory osteoarthritic occurs, will seriously reduce patients ' life quality.After articular cartilage damage, cartilageCell supplies nutrition mainly by the matrix of knuckle synovia and cell peripheral, is energized with anerobic glycolysis, the repair ability of its own is veryIt 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 damageTo wish.Mescenchymal stem cell is the important member of stem cell line, derives from mesoderm growing early stage mesoderm and ectodermic oneClass multipotential stem cell has self-renewing, hyperproliferation and multi-lineage potential.In vivo and in vitro under specific inductive condition, MSCThe Various Tissues cell such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium, continuous passage can be divided intoStill there is multi-lineage potential after culture and freezen protective, can be used as injury repair of the ideal seed cell for histoorganWith 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, sourceIt 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 withThe increase at donor age, cell quantity and differentiation potential occur being decreased obviously trend, and viral infection rate is higher, for a long time peopleBe 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 divideCloth 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 mesenchymaThe 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 materialsAnd 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, DifferentiationAbility；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 doesCell is easily isolated, purity is high, negative for tumor cells pollution；D. the infection of latent virus and pathogenic microorganism and propagation probability ratioIt 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 transportDefeated, 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 mesenchymaSource 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 peopleIt 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 blockStep, the originally culture step of stem cell, the subculture step of stem cell and stem cell walk to the induction of Chondrocyte DifferentiationSuddenly；The stem cell includes that secondary culture is resuspended to the 7th generation using serum free medium to the induction step of Chondrocyte DifferentiationSource of people umbilical cord mesenchymal stem cells P is made7For stem cell suspension, P7Inducer containing cartilage differentiation is inoculated in for stem cell suspensionSerum 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 14After 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 replaceFor object, penicillin, streptomysin, TGF-β 1, ferritin, ascorbic acid, dexamethasone and Sodium Pyruvate；DMEM high sugar culture solution andThe 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/mLTransferrins, 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 includeIn 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 removedPipe 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 includedWash, 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, oftenComplete culture solution is replaced every 3 days half amounts, after stationary culture 14 days, cell is cleaned using physiological saline, is added 0.5wt%'sTryple-Express digestive juice, temperature be 37 DEG C standings 2-3 minute, be fully retracted to pseudopodium, cell rounding, complete trains be addedNutrient 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, abandonSupernatant is removed, the Tryple-Express digestive juice of 0.5wt% is added, after digesting completely, complete culture solution termination is added and disappearsChange, 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 GanCell 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 pointThe 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 cellCryopreservation step includes to PmWhen reaching 80-90% convergence degree for stem cell, liquid is discarded supernatant, the Tryple- of 0.5wt% is addedExpress digestive juice is added complete culture solution and terminates digestion, be centrifuged and discard supernatant liquid, use physiological saline after digesting completelyTo 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 suspensionIt 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 refrigeratorIn freeze 12-24 hours, cryopreservation tube taken out and is placed in liquid nitrogen immediately later freeze；The recovery and cultivation of the stem cellStep 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 cellIt 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 cellLiquid, 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 fractionPart, 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, P7GenerationStem 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 cordThe proliferative capacity and differentiation capability of matter stem cell still have without influence, source of people umbilical cord mesenchymal stem cells to Chondrocyte DifferentiationAbility.
Third combines the present invention to provide the jelly of stem cell using serum-free mesenchymal stem cell cryopreserving liquid provided by the inventionStep 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 survivedIt is significantly improved in rate, ability of cell proliferation is also superior to conventional cryopreservation methods, and source of people umbilical cord mesenchymal stem cellsSpecific 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 embodimentThe 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 blockObtaining step, the originally culture step of stem cell, the subculture step of stem cell and stem cell luring to Chondrocyte DifferentiationLead 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 includedAnti- physiological saline rinses removal bloodstain to umbilical cord tissue section repeatedly；Umbilical cord tissue section is splitted, blood vessel and epidermal tissue is removed, obtainsGet 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 × 1The 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, bodyThe 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 replacementComplete culture solution after stationary culture 14 days, discards culture solution and umbilical cord tissue block, cleans cell using physiological saline, is addedThe Tryple-Express digestive juice of 0.5wt%, temperature be 37 DEG C standing 2-3 minutes, be fully retracted to pseudopodium, cell rounding addsEnter 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 addedThe Tryple-Express digestive juice of 0.5wt% is added complete culture solution and terminates digestion, be centrifuged and discard after digesting completelyClear liquid, with physiological saline to PnIt is cleaned for stem cell, and the weight in HY STEMCELL human mesenchymal stem cell serum free mediumHang 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 suspensionT-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 incubatorIt 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 DifferentiationP 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 GanConcentration of cell suspension is 1 × 107A/mL, P7It is inoculated in the serum free medium of the inducer containing cartilage differentiation for stem cell suspensionContinue 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 daysCell.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 substituteThan 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/mLAscorbic 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 blockObtaining step, the originally culture step of stem cell, the subculture step of stem cell, the cryopreservation step of stem cell, stem cellThe 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 includedAnti- physiological saline rinses removal bloodstain to umbilical cord tissue section repeatedly；Umbilical cord tissue section is splitted, blood vessel and epidermal tissue is removed, obtainsGet 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 × 1The 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, bodyThe 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 replacementComplete culture solution after stationary culture 14 days, discards culture solution and umbilical cord tissue block, cleans cell using physiological saline, is addedThe Tryple-Express digestive juice of 0.5wt%, temperature be 37 DEG C standing 2-3 minutes, be fully retracted to pseudopodium, cell rounding addsEnter 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 addedThe Tryple-Express digestive juice of 0.5wt% is added complete culture solution and terminates digestion, be centrifuged and discard after digesting completelyClear liquid, with physiological saline to PnIt is cleaned for stem cell, and the weight in HY STEMCELL human mesenchymal stem cell serum free mediumHang 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 suspensionT-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 incubatorIt 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 addedThe Tryple-Express digestive juice of 0.5wt% is added complete culture solution and terminates digestion, be centrifuged and discard after digesting completelyClear liquid, with physiological saline to P6It cleans for stem cell, and is resuspended in serum-free mesenchymal stem cell cryopreserving liquid, adjust P6Dai GanCell concentration is 1 × 107/mL, by P6It is sub-packed in cryopreservation tube and seals for stem cell suspension, be immediately placed in cryopreservation tubeAfter 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 tubeIt out and is placed in liquid nitrogen and freezes immediately.Serum-free mesenchymal stem cell cryopreserving liquid includes the HY in terms of mass fractionIt is 80 parts of STEMCELL human mesenchymal stem cell serum free medium, 10 parts of dimethyl sulfoxide, 5 parts of L- menthyl pyranoside, sweet4 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 temperatureDegree 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 HYP is resuspended to obtain in STEMCELL human mesenchymal stem cell serum free medium6For stem cell suspension, by P6It is inoculated with for stem cell suspensionThe 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 incubatorCultivate to obtain P7For stem cell.
Stem cell includes using HY STEMCELL human mesenchymal stem cell serum-free to the induction step of Chondrocyte DifferentiationP 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 GanConcentration of cell suspension is 1 × 107A/mL, P7It is inoculated in the serum free medium of the inducer containing cartilage differentiation for stem cell suspensionContinue 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 daysCell.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 substituteThan 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/mLAscorbic 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 AhSharp 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 addedLiquid dyes 30 minutes, distilled water flushing 2 minutes, staining conditions is observed under inverted phase contrast microscope, if visible under microscopeThere 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, aobviousIt 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 cytosolFor 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 cellSituation 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 implementationThe 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 inventionSerum-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 cellsFreeze 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 frozenAbility.It follows that compared to the prior art, the stem cell after recovery is not only significantly improved in survival rate, cell increasesAbility 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 cellThe 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 GanCell, 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 outThe 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 tableUp to), CD45 (be negative expression), CD44 (positive expression), CD90 (positive expression).The obtained P of embodiment 16Dai GanCell 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 shown99.5%, CD45-CD90+ expression rate are 98.7%.It follows that by using serum-free mescenchymal stem cell provided by the inventionAfter 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 cellsIt 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 inventionMethod, P7It can be to Chondrocyte Differentiation for stem cell.Moreover, by the cryopreservation step of stem cell and the recovery of stem cell and trainingAfter 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 thinBorn of the same parents still have the ability to Chondrocyte Differentiation.This is because using serum-free mesenchymal stem cell cryopreserving provided by the inventionLiquid 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 cellsFruit.Cell after recovery is not only significantly improved in survival rate, ability of cell proliferation also superior to conventional cryopreservation methods, andAnd 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 thisThe limitation of invention belongs to the simple modifications of preparation method of the present invention under concept thereof of the invention claimedRange.