CN107879921B - Double p-Coumaric acids, preparation method and the application in CIK cell culture of a kind of cyperaquinone and the like - Google Patents
Double p-Coumaric acids, preparation method and the application in CIK cell culture of a kind of cyperaquinone and the like Download PDFInfo
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Abstract
The invention discloses a kind of double p-Coumaric acids of cyperaquinone and the like, preparation method and the applications in CIK cell culture.Present invention discover that cyperaquinone and the like goes first cyperaquinone, hydroxyl cyperaquinone to have a variety of pharmacological activity; for example it can be used as the freezing protective agent of CIK cell and NK cell; it can promote CIK cell and NK cell proliferation in vitro, fat stem cell Osteoblast Differentiation can be promoted；The present invention also provides single p-Coumaric acid of cyperaquinone and the like, double p-Coumaric acids and preparation method thereof, single p-Coumaric acid, double p-Coumaric acids of cyperaquinone and the like can promote CIK cell in vitro to be proliferated.Through retrieving, the prior art is not disclosed cyperaquinone and the like and promotes CIK cell, NK cell Proliferation and the purposes for promoting stem cell Osteoblast Differentiation, without single furan nucleus of cyperaquinone and the like, the report of bifuran p-Coumaric acid yet.
Description
Technical field
The invention belongs to chemical field, it is related to the development and utilization of known natural products, and in particular to cyperaquinone and its similar
Object goes the application of first cyperaquinone, hydroxyl cyperaquinone in terms of promoting CIK cell, NK cell Proliferation and promotion stem cell differentiation, also
It is related to single p-Coumaric acid of cyperaquinone and the like, double p-Coumaric acids and preparation method thereof and in CIK cell culture
Application.
Background technique
It is isolated from the herb of not tally dichotomous fimbristylis herb that cyperaquinone and the like, which removes first cyperaquinone, hydroxyl cyperaquinone,
Three kinds of quinones, the information such as chemical structure are as follows.
Research about these three compounds is few, and related activity report is almost blank.Applicants have found that this three
Kind compound has the function of excellent promotion CIK cell, NK cell Proliferation and promotes stem cell differentiation.Moreover, for promoting
When the purposes being proliferated into CIK cell, activity is significantly further enhanced after the furan nucleus open loop on parent nucleus.
Through retrieving, the prior art is not disclosed cyperaquinone and the like and promotes CIK cell, NK cell Proliferation and promotion
The purposes of stem cell differentiation, also without single furan nucleus of cyperaquinone and the like, the report of bifuran p-Coumaric acid.
Summary of the invention
It is an object of the invention to fill up the deficiencies in the prior art, cyperaquinone and the like is provided and removes first cyperaquinone, hydroxyl
Application of the base cyperaquinone in terms of promoting CIK cell, NK cell Proliferation and promotion stem cell differentiation, also offer cyperaquinone and its class
Application like single p-Coumaric acid of object, double p-Coumaric acids and preparation method thereof and in CIK cell culture.
Above-mentioned purpose of the invention is achieved by following technical solution:
A kind of single p-Coumaric acid of cyperaquinone and the like, chemical structure are as follows:
The preparation method of above-mentioned list p-Coumaric acid, includes the following steps:
It is water-soluble to be added to the acidity for being adjusted to pH value 6.2-6.4 with volatile acid by step S1 for the raw material of following chemical structure
In liquid；
Above-mentioned solution is moved into reaction kettle, carries out hydro-thermal reaction at a temperature of 170-190 DEG C after closed, instead by step S2
It is 8-12h between seasonable, naturally rings to room temperature after reaction；
Step S3, heating concentration make volatile acid volatilize, and remaining aqueous solution is freeze-dried to obtain the final product.
Preferably, the preferred hydrochloric acid of volatile acid, nitric acid, formic acid.
Preferably, 1mL acidic aqueous solution adds 3-5mg raw material.
Application of the above-mentioned list p-Coumaric acid in terms of CIK cell in vitro culture.
A kind of double p-Coumaric acids of cyperaquinone and the like, chemical structure are as follows:
The preparation method of above-mentioned double p-Coumaric acids, includes the following steps:
It is water-soluble to be added to the acidity for being adjusted to pH value 5.3-5.5 with volatile acid by step S1 for the raw material of following chemical structure
In liquid；
Above-mentioned solution is moved into reaction kettle, carries out hydro-thermal reaction at a temperature of 170-190 DEG C after closed, instead by step S2
It is 8-12h between seasonable, naturally rings to room temperature after reaction；
Step S3, heating concentration make volatile acid volatilize, and remaining aqueous solution is freeze-dried to obtain the final product.
Preferably, the preferred hydrochloric acid of volatile acid, nitric acid, formic acid.
Preferably, 1mL acidic aqueous solution adds 3-5mg raw material.
Application of the above-mentioned double p-Coumaric acids in terms of CIK cell in vitro culture.
Application of the cyperaquinone of following chemical structure and the like in terms of CIK cell cryopreservation resuscitation and in vitro culture:
Preferably, the application are as follows: cyperaquinone and the like is added in frozen stock solution and improves CIK cell recovery
Rate.
Preferably, the application are as follows: cyperaquinone and the like is added in culture solution and improves CIK cell proliferation
Rate.
Application of the cyperaquinone of following chemical structure and the like in terms of NK cryopreservation and in vitro culture:
Preferably, the application are as follows: cyperaquinone and the like is added to raising NK cell recovery rate in frozen stock solution.
Preferably, the application are as follows: cyperaquinone and the like is added to raising NK cell proliferation rate in culture solution.
Application of the cyperaquinone of following chemical structure and the like in terms of inducing stem cell Osteoblast Differentiation:
Preferably, the stem cell is fat stem cell.
Present invention discover that: cyperaquinone and the like goes first cyperaquinone, hydroxyl cyperaquinone to have a variety of pharmacological activity, such as
It can be used as the freezing protective agent of CIK cell and NK cell, CIK cell and NK cell proliferation in vitro can be promoted, can promote
Fat stem cell Osteoblast Differentiation；The present invention also provides single p-Coumaric acids of cyperaquinone and the like, double p-Coumaric acids
And preparation method thereof, single p-Coumaric acid, double p-Coumaric acids of cyperaquinone and the like can promote CIK cell in vitro to increase
It grows.
Detailed description of the invention
Fig. 1 is the influence that single p-Coumaric acid of cyperaquinone and the like is proliferated CIK cell in vitro；
Fig. 2 is the influence that double p-Coumaric acids of cyperaquinone and the like are proliferated CIK cell in vitro；
Fig. 3 is influence of the cyperaquinone and the like to CIK cell cryopreservation resuscitation (A) and in-vitro multiplication (B)；
Fig. 4 is influence of the cyperaquinone and the like to NK cryopreservation (A) and in-vitro multiplication (B)；
Fig. 5 is influence of the cyperaquinone and the like to fat stem cell Osteoblast Differentiation.
Specific embodiment
It is specific with reference to the accompanying drawings and examples to introduce essentiality content of the present invention, but guarantor of the invention is not limited with this
Protect range.
Embodiment 1: the preparation of single p-Coumaric acid of cyperaquinone and the like
Firstly, by cyperaquinone, first cyperaquinone, hydroxyl cyperaquinone is gone to be respectively added to acidic aqueous solution (deionized water salt
Acid for adjusting pH value is to 6.3, naturally it is also possible to other volatile acids such as nitric acid or formic acid substitution) in, the addition of 1mL acidic aqueous solution
4mg raw material.Then the solution is moved into reaction kettle, carries out hydro-thermal reaction at a temperature of 180 DEG C after closed, the reaction time is
10h naturally rings to room temperature after reaction.Finally, heating concentration, is freeze-dried up to cyperaquinone list when pH value of solution weakly acidic pH
P-Coumaric acid removes first cyperaquinone list p-Coumaric acid, hydroxyl cyperaquinone list p-Coumaric acid, and purity is 95% or more.Sha
Careless quinone list p-Coumaric acid goes first cyperaquinone list p-Coumaric acid, hydroxyl cyperaquinone list p-Coumaric acid nuclear magnetic data to be shown in Table 1-
3。
Embodiment 2: the preparation of double p-Coumaric acids of cyperaquinone and the like
Firstly, by cyperaquinone, first cyperaquinone, hydroxyl cyperaquinone is gone to be respectively added to acidic aqueous solution (deionized water salt
Acid for adjusting pH value is to 5.4, naturally it is also possible to other volatile acids such as nitric acid or formic acid substitution) in, the addition of 1mL acidic aqueous solution
4mg raw material.Then the solution is moved into reaction kettle, carries out hydro-thermal reaction at a temperature of 180 DEG C after closed, the reaction time is
10h naturally rings to room temperature after reaction.Finally, heating concentration, is freeze-dried double up to cyperaquinone when pH value of solution weakly acidic pH
P-Coumaric acid removes the double p-Coumaric acids of first cyperaquinone, the double p-Coumaric acids of hydroxyl cyperaquinone, and purity is 95% or more.Sha
The double p-Coumaric acids of careless quinone go the double p-Coumaric acids of first cyperaquinone, the double p-Coumaric acid nuclear magnetic datas of hydroxyl cyperaquinone to be shown in Table 1-
3。
The nuclear magnetic data of the double p-Coumaric acids of 1 cyperaquinone of table, cyperaquinone list p-Coumaric acid, cyperaquinone compares
The nuclear magnetic data that table 2 removes first cyperaquinone, removes first cyperaquinone list p-Coumaric acid, removes the double p-Coumaric acids of first cyperaquinone
Compare
The nuclear magnetic data of the double p-Coumaric acids of 3 hydroxyl cyperaquinone of table, hydroxyl cyperaquinone list p-Coumaric acid, hydroxyl cyperaquinone
Compare
Embodiment 3: cyperaquinone and the like improves CIK cell cryopreservation resuscitation rate
1, experimental material
AIM-VCTSTMCulture medium is purchased from Gibco company, and recombinated interleukin-2 (rh IL-2) is purchased from double aigret medicine companies；
Lymphocyte separation medium is purchased from Nycomed Pharma company (Nycomed PharmaAS, Oslo, Norway), APC label
CD3 (CD3-PerCP), the APC-H7 label for CD4 (CD4-PE), the PerCP label that anti-CD56 (CD56-APC), PE are marked
CD8 (CD8-APC-H7) monoclonal antibody is purchased from BD company.
2, the separation, culture amplification, flow cytometer detection of CIK cell
It extracts in peripheral blood to the centrifuge tube containing heparin sodium of healthy volunteer, centrifugation obtains serum and whole blood cells,
Whole blood cells are obtained into mononuclearcell (PBMCs) through density gradient centrifugation.The PBMCs of collection is resuspended in oneself containing 10% inactivation
The AIM-VCTS of body blood plasmaTMCulture medium adjusts cell density 1.5 × 106/ ml or so, and IFN-γ (1000U/ml) is added, carefully
Born of the same parents set 37 DEG C, 5%CO2Cultivated in incubator, for 24 hours afterwards be added AntiCD3 McAb McAb (50ng/ml), rhIL-2 (500U/ml) and
rhIL-1α(300U/ml).Later every 2 days AIM-VCTSs of the addition containing 5% autologous plasma, 1000U/mlrhIL-2TMCulture
Base, controlling cell density after each fluid infusion is 1.5 × 106/ml。
Flow cytometer detection: taking the CIK cell of culture 14 days, and 1000r/min is centrifuged after 5min and washes 1 time with 1 × PBS, 1500r/
Min is centrifuged 5min, and adjustment cell number is about 7.5 × 105/ pipe/100 μ l, add CD4-PE, CD3-PerCP, CD8-APC-H7,
Each 2 μ l of CD56-APC, blows even, is protected from light and is incubated for 20min, PBS washes 1 time, finally with PBS400 μ l resuspension, upper machine testing.Detection training
The ratio of CIK cell, flow cytomery result are analyzed using BDCellQuest software after supporting 14 days.
Streaming the results show that culture 14 days after CIK cell height express, content 58.5%.
3, the freezing, recover of CIK cell, Cell viability measurement and flow cytometer detection
The CIK cell of amplification cultivation to the 14th day is taken, 1000r/min is centrifuged 10min, supernatant is abandoned, according to following grouping
The frozen stock solution of different compositions is added, adjusts separately cell concentration to 2 × 107/ ml places 1h in 4 DEG C of refrigerators first, then-
2h is placed in 20 DEG C of refrigerators, -80 DEG C of low temperature refrigerators is gone to and places 2 months.
Routine control group: AIM-VCTSTMCulture medium, autologous plasma and dimethyl sulfoxide volume ratio 5:4:1；
Cyperaquinone group: the cyperaquinone of 5 μ g/mL is added on the basis of Routine control group；
Go first cyperaquinone group: that 5 μ g/mL are added on the basis of Routine control group removes first cyperaquinone；
Hydroxyl cyperaquinone group: the hydroxyl cyperaquinone of 5 μ g/mL is added on the basis of Routine control group；
Recovery: taking out the CIK cell frozen from -80 DEG C of refrigerators, is put into rapidly in 37 DEG C of water-baths and melts, uses AIM-
VCTSTMCulture medium washing, is resuspended in AIM-VCTSTMIn culture medium, autologous plasma culture 4h is added, measures motility rate.
Cell viability measurement: 2g/L trypan blue dye liquor respectively with freeze before and recovery after CIK cell suspension 1:1 mixing,
After dyeing 2min, Cell viability measurement is carried out using full-automatic cell calculating instrument, each sample counting 3 times is averaged.
Flow cytomery is carried out to the cell for cultivating 4h after recovery using above-mentioned stream measuring method.
As a result as shown in the A in Fig. 3, Cell viability is (97.5 ± 2.1) % before freezing；It is conventional right compared with before freeze
Increase according to cell fragment after group recovery, Cell viability is substantially reduced, and is (70.4 ± 2.3) %, statistically significant (the P < of difference
0.05)；Compared with Routine control group, cyperaquinone group goes cell fragment after first cyperaquinone group and the recovery of hydroxyl cyperaquinone group obvious
Less, Cell viability is higher, respectively (93.5 ± 2.2) %, (94.4 ± 1.9) %, (93.9 ± 2.3) %.Flow cytometer
Testing result shows cyperaquinone group, go first cyperaquinone group and hydroxyl cyperaquinone group to recover after CIK cell content still 50% with
On.
Therefore, by the visible cyperaquinone of above-mentioned experiment, go first cyperaquinone and hydroxyl cyperaquinone to CIK cell freeze have protect
Shield effect, anabiosis rate is high, and does not influence CIK cell phenotype, may be used as CIK cell freezing protective agent.
Embodiment 4: cyperaquinone and the like improves NK cryopreservation rate
1, experimental material
NK cell culture medium is purchased from Germany CellGroSCGM, and recombinated interleukin-2 (rhIL-2) is purchased from double aigret medicines
Industry；Lymphocyte separation medium is purchased from NycomedPharma company (NycomedPharmaAS, Oslo, Norway), PerCP-
The CD56 monoclonal antibody of CD3, FITC label of Cy5.5 label is purchased from BD company.
2, the separation, culture amplification, flow cytometer detection of NK cell
It extracts in peripheral blood to the centrifuge tube containing heparin sodium of healthy volunteer, centrifugation obtains serum and whole blood cells,
Whole blood cells are obtained into mononuclearcell (PBMCs) through density gradient centrifugation.The PBMCs of collection is resuspended in containing 500U/mlrhIL-
2 NK cell culture medium, adjustment cell density are 3.5 × 105/ ml is inoculated in 6 well culture plates, every hole 5ml, in 37 DEG C,
5%CO2Incubator culture, every 2 days half amounts are changed liquid 1 time, and adjusting cell number is 5 × 104/ ml collects the NK cell of culture 10d,
The CD56 that CD3, FITC label of PerCP-Cy5.5 label is added carries out cell surface marker detection.
NK cell phenotype is CD3-CD56+；It collects the cell through rhIL-2 Fiber differentiation 10d and carries out flow cytometer detection, PBMC
NK cell (CD3-CD56+) ratio is 6.5% before not cultivating, and NK cell proportion increases to 78.5% after cultivating 10d.
3, the freezing, recover of NK cell, Cell viability measurement and flow cytometer detection
The NK cell of amplification cultivation to the 10th day is taken, 1000r/min is centrifuged 10min, abandons supernatant, adds according to following grouping
The frozen stock solution for entering different compositions adjusts separately cell concentration to 2 × 107/ ml places 1h in 4 DEG C of refrigerators first, and then -20
2h is placed in DEG C refrigerator, -80 DEG C of low temperature refrigerators is gone to and places 2 months.
Routine control group: NK cell culture medium and dimethyl sulfoxide volume ratio 9:1；
Cyperaquinone group: the cyperaquinone of 5 μ g/mL is added on the basis of Routine control group；
Go first cyperaquinone group: that 5 μ g/mL are added on the basis of Routine control group removes first cyperaquinone；
Hydroxyl cyperaquinone group: the hydroxyl cyperaquinone of 5 μ g/mL is added on the basis of Routine control group；
Recovery: taking out the NK cell frozen from -80 DEG C of refrigerators, is put into rapidly in 37 DEG C of water-baths and melts, with NK cell
Culture medium washing, is resuspended in NK cell culture medium and cultivates 4h, measures motility rate.
Cell viability measurement: 2g/L trypan blue dye liquor respectively with freeze before and recovery after NK cell suspension 1:1 mix, contaminate
After color 2min, Cell viability measurement is carried out using full-automatic cell calculating instrument, each sample counting 3 times is averaged.
Flow cytomery is carried out to the cell for cultivating 4h after recovery using above-mentioned stream measuring method.
As a result as shown in the A in Fig. 4, Cell viability is (96.7 ± 2.5) % before freezing；It is conventional right compared with before freeze
Increase according to cell fragment after group recovery, Cell viability is substantially reduced, and is (73.5 ± 2.4) %, statistically significant (the P < of difference
0.05)；Compared with Routine control group, cyperaquinone group goes cell fragment after first cyperaquinone group and the recovery of hydroxyl cyperaquinone group obvious
Less, Cell viability is higher, respectively (92.3 ± 2.5) %, (94.6 ± 2.4) %, (92.8 ± 2.5) %.Flow cytometer
NK cell content is still 75% or more after testing result shows cyperaquinone group, first cyperaquinone group and hydroxyl cyperaquinone group is gone to recover.
Therefore, by the visible cyperaquinone of above-mentioned experiment, go first cyperaquinone and hydroxyl cyperaquinone to NK cell cryopreservation have protect
Shield effect, anabiosis rate is high, and does not influence NK cell phenotype, may be used as NK cell cryopreservation protective agent.
Embodiment 5: cyperaquinone and the like, single p-Coumaric acid, double p-Coumaric acids promote CIK cell in vitro proliferation
People's CIK cell is grown with CCK8 method detection cyperaquinone and the like, single p-Coumaric acid, double p-Coumaric acids
Influence.Method particularly includes: amplification cultivation uses AIM-VCTS to the 14th day CIK cell in Example 3TMCulture medium is resuspended
At 5 × 104The cell suspension of/ml；96 orifice plates are inoculated in, the cyperaquinone containing 2.0 μM is separately added into, removes first cyperaquinone, hydroxyl Sha
Careless quinone and its single p-Coumaric acid, double p-Coumaric acids, control group do not add cyperaquinone and the like, single p-Coumaric acid,
Double p-Coumaric acids, every group sets 3 multiple holes, in 37 DEG C, 5%CO2Continue after cultivating 48h in incubator, CCK820 μ is added in every hole
L continues to discard supernatant after cultivating 4h, and each hole light absorption value (OD) of detection records as a result, be averaged in microplate reader 450nm at,
Finally each administration group CIK cell growing multiplication rate (%) is calculated according to following formula.
CIK cell proliferation rate (%)=[(administration group OD value)-(control group OD value)]/(control group OD value) × 100%.
The facilitation that cyperaquinone and the like grows people's CIK cell B institute such as in table 4-6 and Fig. 1, Fig. 2 and Fig. 3
Show.
The CIK cell proliferation rate (%) of 4 cyperaquinone of table and the like administration group
The CIK cell proliferation rate (%) of single p-Coumaric acid administration group of 5 cyperaquinone of table and the like
The CIK cell proliferation rate (%) of double p-Coumaric acid administration groups of 6 cyperaquinone of table and the like
Therefore, by the visible cyperaquinone of above-mentioned experiment, remove first cyperaquinone, hydroxyl cyperaquinone and its single p-Coumaric acid, double
P-Coumaric acid can promote the proliferation of CIK cell, and cyperaquinone, go drug effect after first cyperaquinone, hydroxyl cyperaquinone open loop stronger.
Embodiment 6: cyperaquinone and the like promotes NK cell proliferation in vitro
Influence with CCK8 method detection cyperaquinone and the like to NK cells of human beings growth.Method particularly includes:
The NK cell of amplification cultivation to the 10th day in Example 4 is resuspended with NK cell culture medium at 5 × 104/ ml's is thin
Born of the same parents' suspension；96 orifice plates are inoculated in, the cyperaquinone containing 2.0 μM is separately added into, removes first cyperaquinone and hydroxyl cyperaquinone, control group does not add
Add cyperaquinone and the like, every group sets 3 multiple holes, in 37 DEG C, 5%CO2Continue after cultivating 48h in incubator, every hole is added
CCK820 μ l, continues to discard supernatant after cultivating 4h, and each hole light absorption value (OD) record is detected at microplate reader 450nm as a result, asking it
Average value finally calculates each administration group NK growth and proliferation of cell rate (%) according to following formula.
NK cell proliferation rate (%)=[(administration group OD value)-(control group OD value)]/(control group OD value) × 100%.
The facilitation that cyperaquinone and the like grows NK cells of human beings is as shown in B in table 7 and Fig. 4.
The NK cell proliferation rate (%) of 7 cyperaquinone of table and the like administration group
Therefore, by the visible cyperaquinone of above-mentioned experiment, go first cyperaquinone and hydroxyl cyperaquinone that can promote the increasing of NK cell
It grows.
Embodiment 7: cyperaquinone and the like promotes fat stem cell Osteoblast Differentiation
1, experimental material
Cleaning grade health new zealand white rabbit, 3 monthly ages, weight 2-3kg；H-DMEM (HyClone company, the U.S.)；Tire ox
Serum FBS (Hangzhou Chinese holly)；Dexamethasone, ascorbic acid, sodium β-glycerophosphate (Sigma company).
2, fat stem cell (ADSCs) is separately cultured
Fat stem cell (various concentration induced by dexamethasone fat stem cell Osteoblast Differentiation is separately cultured using literature method
Experimental study, Chinese Reconstructive surgery magazine the 12nd phase of volume 25 in December, 2011), concrete operations are as follows: New Zealand great Bai
Rabbit takes dorsal position with 3% yellow Jackets (30mg/kg) auricular vein injecting anesthetic, makees groin middle part longitudinal incision about
3cm cuts 4~6mL of subcutaneous fat, and rejecting blood vessel, fascia, PBS (pH7.4) are rinsed 5 times as far as possible, removes red blood cell, sufficiently cuts
It is broken to 1mm × 1mm × 1mm size tissue block, is placed in 50mL conical flask, be added 0.1% Type I collagen enzyme of 3~5 times of volumes, 37
DEG C shaking table concussion digestion 60min, is added isometric H-DMEM (containing 10%FBS and 100U/mL penicillin, 100 μ g/mL streptomysins)
Culture solution terminates digestion.To be centrifuged radius 20cm, 1200r/min centrifugation 10min under room temperature, supernatant and upper-layer fat are abandoned.5mLH-
DMEM (containing 10%FBS and dual anti-) culture solution suspends precipitating, the filtering of 200 mesh nylon leaching nets, and filtrate is same as above method centrifugation, abandons supernatant,
3mLH-DMEM (containing 10%FBS and dual anti-) culture solution suspends precipitating, is inoculated in 50mL Tissue Culture Flask, 37 DEG C, 5%CO2And it is full
With humidified incubator culture.48h changes liquid for the first time, removes non-attached cell, and 2~3d is changed liquid 1 time later, to cell up to 70%~
It is passed on when 80% fusion.
3, ADSCs Osteoblast Differentiation Fiber differentiation and Alizarin red staining
H-DMEM osteogenic induction basic culture solution: 10%FBS, 100U/mL penicillin, 100 μ are added in H-DMEM culture solution
G/mL streptomysin, 10mmol/L β-phosphoglycerol sodium, 50 μm of ol/L ascorbic acid.
It is resuspended with above-mentioned H-DMEM osteogenic induction basic culture solution at 1 × 10 after taking the 3rd generation ADSCs cell to be centrifuged5A/
ML, administration group is separately added into the cyperaquinone containing 5.0 μM, removes first cyperaquinone and hydroxyl cyperaquinone, control group do not add cyperaquinone and
Its analog, 37 DEG C, 5%CO2And saturated humidity incubator culture 7d.
The group of cells creep plate of osteogenic induction culture 7d is taken, PBS is rinsed 3 times, each 5min；95% 4 DEG C of ethyl alcohol fixation
30min；Tri-distilled water rinses 3 times, each 5min；0.1% alizarin red dye liquor, 37 DEG C of effect 30min are added；Tri-distilled water rinses 3 times,
Each 5min；It spontaneously dries, neutral gum mounting, extracellular Ca2+ mineralization situation is observed under inverted phase contrast microscope.
Alizarin red staining result is as shown in Figure 5.Osteoblast will appear calcification phenomenon, can dye red by alizarin red agent, not
The cell of calcification will not be colored.Without obvious calcification, there is different degrees of calcification and dyes control group in each administration group, cyperaquinone,
Go first cyperaquinone group calcium scoring point less, fragmentary to be distributed, hydroxyl cyperaquinone group large area calcium scoring then occurs, illustrates nutgrass flatsedge
Quinone, go first cyperaquinone and hydroxyl cyperaquinone can effective induced lipolysis stem cell Osteoblast Differentiation, and hydroxyl cyperaquinone drug effect is most strong.
Bone defect caused by wound or tumour is the common disease of the departments such as orthopaedics, Plastic renovation section, Oral and Maxillofacial Surgery,
Seriously affect the shape and function of patient.Bone cement or other artificial materials can promote the healing of slight bone defect, biggish bone
Defect then needs the bone collection reparation of vascularization.Although common ilium or fibular flap transplanting can repair most of bone defect,
But donor site damage is larger.Organizational project and regenerative medicine are planted careful to repair or replacement damaged tissues provide new thinking
Born of the same parents are keys therein.The source of fat stem cell is sufficient, it is high to obtain simplicity, yield, proliferation is fast, has multi-lineage potential,
Can the various cell factors of paracrine, promote vascularization, play immunoregulation effect, and can recruit other cells participate in rebuild,
Therefore the always choosing of the hot topic of seed cell.Cyperaquinone goes first cyperaquinone and hydroxyl cyperaquinone that can effectively do induced lipolysis
Cell Osteoblast Differentiation, makes full use of the resources advantage of fat stem cell, provides osteoblast for bone collection.
The effect of above-described embodiment is specifically to introduce essentiality content of the invention, but those skilled in the art should know
Protection scope of the present invention should not be confined to the specific embodiment by road.
Claims (5)
1. a kind of double p-Coumaric acids of cyperaquinone and the like, chemical structure are as follows:
R1=CH3R2=CH3, which is the double p-Coumaric acids of cyperaquinone；
Or R1=CH3R2=H, double p-Coumaric acids are to remove the double p-Coumaric acids of first cyperaquinone；
Or R1=CH2OHR2=CH3, which is the double p-Coumaric acids of hydroxyl cyperaquinone.
2. the preparation method of double p-Coumaric acids described in claim 1, which comprises the steps of:
The raw material of following chemical structure is added in the acidic aqueous solution for being adjusted to pH value 5.3-5.5 with volatile acid by step S1；
R1=CH3R2=CH3, which is cyperaquinone, is used to prepare the double p-Coumaric acids of cyperaquinone；
Or R1=CH3R2=H, the raw material are to remove first cyperaquinone, are used to prepare the double p-Coumaric acids of first cyperaquinone；
Or R1=CH2OHR2=CH3, which is hydroxyl cyperaquinone, is used to prepare the double p-Coumaric acids of hydroxyl cyperaquinone；
Above-mentioned solution is moved into reaction kettle, carries out hydro-thermal reaction after closed at a temperature of 170-190 DEG C by step S2, when reaction
Between be 8-12h, naturally ring to room temperature after reaction；
Step S3, heating concentration make volatile acid volatilize, and remaining aqueous solution is freeze-dried to obtain the final product.
3. preparation method according to claim 2, it is characterised in that: volatile acid is hydrochloric acid, nitric acid, formic acid.
4. preparation method according to claim 2, it is characterised in that: 1mL acidic aqueous solution adds 3-5mg raw material.
5. application of the double p-Coumaric acids described in claim 1 in terms of CIK cell in vitro culture.
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