CN104693075B - P18 micromolecular inhibitor and the purposes in human hematopoietic stem cell amplification in vitro - Google Patents
P18 micromolecular inhibitor and the purposes in human hematopoietic stem cell amplification in vitro Download PDFInfo
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- 0 *C(c(cc1)ccc1S(NC1CCCCC1)(=O)=O)=O Chemical compound *C(c(cc1)ccc1S(NC1CCCCC1)(=O)=O)=O 0.000 description 1
- PAFZNILMFXTMIY-UHFFFAOYSA-N NC1CCCCC1 Chemical compound NC1CCCCC1 PAFZNILMFXTMIY-UHFFFAOYSA-N 0.000 description 1
- PTCSSXYPZOFISK-UHFFFAOYSA-N OC(c(cc1)ccc1S(Cl)(=O)=O)=O Chemical compound OC(c(cc1)ccc1S(Cl)(=O)=O)=O PTCSSXYPZOFISK-UHFFFAOYSA-N 0.000 description 1
- VFCZRGPHFBZNRC-UHFFFAOYSA-N OC(c(cc1)ccc1S=O)=O Chemical compound OC(c(cc1)ccc1S=O)=O VFCZRGPHFBZNRC-UHFFFAOYSA-N 0.000 description 1
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Abstract
The present invention relates to p18 micromolecular inhibitor and the purposes in human hematopoietic stem cell amplification in vitro, its structure is:The present invention also provides the Preparation method and use of this p18 micromolecular inhibitor, and p18 micromolecular inhibitor of the present invention has good facilitation for human hematopoietic stem cell self, can be clinical treatment in the future new method is provided.
Description
Technical field
The present invention relates to field of medicaments, particularly p18 micromolecular inhibitor and at human cord blood source hemopoietic stem cellPurposes in amplification in vitro.
Background technology
Stem cell treat clinically because its ability with self and Multidirectional Differentiation becomes various diseases effective way itOne, this has also represented the arrival of regenerative medicine New Times. Wherein candidate stem cell research start the earliest, research the most deeply andBe most widely used. The malignant hematologic disease history of existing more than 50 year such as application bone-marrow transplantation treatment leukaemia. But make in marrowHemocytoblast quantity is lower, is difficult in patient body, effectively carry out hematopoietic reconstitution after transplanting, and this also becomes stem cell clinical practiceA bottleneck.
Candidate stem cell has multiple choices in vivo, such as self, differentiation, migration (going back to the nest), tranquillization, apoptosis etc.,There is the different fate of a very complicated signal path network adjustment candidate stem cell in these different selections behind. ExampleAs the adjusting albumen of the related to developments such as Wnt, Notch, Shh/BMP, TGF-β, IGF, the chromatin correlation factors such as Bmi,The transcription factors such as HoxB, NF κ, the cyclins such as INK4, KIP, PTEN etc. have all participated in this Signal RegulationNetwork. For candidate stem cell, its of paramount importance characteristic is self. Have at present multinomial research and point out,The self of candidate stem cell regulates with the adjusting of cell cycle closely bound up. Entering the cell cycle is that candidate stem cell carries outThe final step of self, therefore cell cycle, the especially adjusting of G1 phase may be to the selfs of candidate stem cellProduce vital effect.
Cell cycle mainly regulates and controls by the activity change of continuous cell cycle protein dependent kinase (CDKs), and CDKActivity must depend on again the contents level of Cyclin. In vivo, except phosphorylation/dephosphorization acid of Cyclin and catalytic subunitThe regulation and control of changing, CDK is also subject to the regulation and control of CKIs (CDKIs) to a great extent. Known at presentTwo class low-molecular-weight family proteins in CKI, Cip/Kip and INK4 can react with CDK, and the G1 that passes through capable of inhibiting cellPhase. Wherein, Cip/Kip family, mainly comprises p21, and p27 and p57 can react with a lot of Cyclin-CDK compounds;INK family, mainly comprises p16, p15, and p18 and p19, can specific inhibition CDK4,6.
More and more research shows, for other CKI, p18 does the self regulation and control of candidate stem cellWith more powerful. For example, the disappearance of p18 can cause the remarkable rising of mouse hemopoietic implantation rate, and this kind of effect is by carryingThe ratio of high candidate stem cell symmetry self division is realized, compared to the increasing that improved merely candidate stem cell in the pastGrow efficiency, improve differentiation and exhaustion that its symmetry division can avoid causing candidate stem cell. Therefore, p18 is a special shadowRing unique INK albumen of candidate stem cell self.
At present amplifying candidate stem cell in vitro mainly relies on to adding the cells such as serum and SCF, TPO, Flt3L in culture mediumThe factor, but expanding effect is still undesirable, and candidate stem cell just breaks up in vitro soon very much, exhaustion. In research before onceEmploying add cytokine profiles fluid nutrient medium, with stroma cell co-incubation or in bioreactor, cultivate etc. method withProliferation of hematopoietic stem/progenitor cell in vitro, carries out but said method still can not obtain the sufficient candidate stem cell with transfer abilityClinical treatment.
Micromolecular compound has shown the advantage that it is unique in cultivating in vitro. First, the action effect of compound can lead toStructure, the concentration of overregulating compound change, and its process is simply controlled; Secondly, micromolecular compound compared to cell because ofIts character such as son or transcription factor is more stable, and processing in early stage and the administration of compound are very convenient. Therefore, expand in traditionIncrease in the method for candidate stem cell and add micromolecular compound, adopt even completely micromolecular compound substitute existing serum,The materials such as cell factor, may play a significant role in vitro culture candidate stem cell.
The present invention is intended to research and makes a kind of new p18 micromolecular inhibitor, and by the external training of this p18 micromolecular inhibitorSupport human hematopoietic stem cell, to reach the object of amplification in vitro of candidate stem cell.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, and a kind of p18 micromolecular inhibitor and dry thin at artificial blood is providedPurposes in born of the same parents' amplification in vitro.
The technical solution used in the present invention is:
A kind of p18 micromolecular inhibitor, its structure is:
The present invention also provides the preparation method of this p18 micromolecular inhibitor, comprises the steps:
Under condition of ice bath, be dissolved in carrene by cyclohexylamine with to carboxyl benzene sulfonyl chloride, add triethylamine, stir 1Hour, rise to after room temperature, continue to stir, after TLC monitoring has been reacted, rotary evaporation is removed reaction solution, residue purified,Obtain intermediate;
Under condition of ice bath, gained intermediate is dissolved in water, add NaOH, rise to after room temperature, stir 30 minutes,Gained reactant liquor vacuum freeze-drying, obtains finished product.
The reaction equation relating to is:
The present invention also provides the purposes of this p18 micromolecular inhibitor in human hematopoietic stem cell amplification in vitro.
Particularly, in described purposes, the concentration of p18 micromolecular inhibitor in amplification culture medium is 10-50nM.
Particularly, the present invention also provides a kind of human hematopoietic stem cell amplification in vitro method, comprises the steps:
By the human cord blood CD 34 of fresh separated+Cell uses amplification culture medium (StemSpanSFEM culture medium+[100ng/mL]SCF/TPO/Flt3L/IL-6) resuspended to cell concentration be 5 × 104Cell/mL, is then laid in 96 orifice plates, and every hole is 190 μ LCell suspension, adds the p18 micromolecular inhibitor that 10 μ L use amplification culture medium of the same race to dilute subsequently wherein, and p18 is littleThe final concentration of molecule inhibitor is 20nM. At 37 DEG C, 5%CO2In constant incubator, cultivate 7 days.
The beneficial effect that the present invention has:
P18 micromolecular inhibitor of the present invention has good facilitation for human hematopoietic stem cell self, can be in the futureClinical treatment provides new method.
Brief description of the drawings
In Fig. 1: embodiment 3, after in vitro culture, cell phenotype ratio changes statistical analysis figure;
In Fig. 2: embodiment 3, after in vitro culture, cell phenotype absolute quantity changes statistical analysis figure;
Fig. 3: embodiment 4 cobble sample regions form cell Poisson statistics distribution map;
In Fig. 4: embodiment 5, flow cytometer showed detects and transplants people CD45 in rear marrow+Cell content scatter diagram;
In Fig. 5: embodiment 5, statistical analysis derives from the variation of people's cell content in each group mouse bone marrow cells after transplanting.
Detailed description of the invention
In order to understand the present invention, below in conjunction with specific embodiment, the invention will be further described, but do not limit guarantor of the present inventionProtect scope.
Embodiment 1
A kind of p18 micromolecular inhibitor, its structure is:
The preparation method of this p18 micromolecular inhibitor, comprises the steps:
Under condition of ice bath, by cyclohexylamine (495 milligrams, 5.0 mMs) and to carboxyl benzene sulfonyl chloride (1100 milligrams, 5MM) be dissolved in carrene (100 milliliters), adding triethylamine (707 milligrams, 7 mMs), stirring 1 is littleTime, rise to after room temperature, continue to stir, after TLC monitoring has been reacted, rotary evaporation is removed reaction solution, and residue is with quickPreparative column purifying, obtains 300 milligrams of finished products (intermediate), yield 21%.
1HNMR(400MHz,DMSO-d6):δ7.94(d,J＝8.4Hz,2H),7.68(d,J＝8.4Hz,2H),7.51(bs,1H),2.90-2.91(m,1H),1.42-1.55(m,5H),0.99-1.11(m,5H).LC-MS(ESI):m/z284.0(M+H)+.
Under condition of ice bath, above-mentioned intermediate (103 milligrams, 0.36 mM) is dissolved in 5 ml waters, add hydrogenSodium oxide molybdena (14.56 milligrams, 0.36 mM), rises to after room temperature, stirs 30 minutes, and gained reactant liquor vacuum freeze-drying,Obtain 100 milligrams of finished products, yield 90%.
Sodium4-(N-cyclohexylsulfamoyl)benzoate(005A).
1HNMR(400MHz,DMSO-d6):δ7.74(d,J＝8.4Hz,2H),7.41(d,J＝6.8Hz,2H),3.60-3.61(m,1H),1.46-1.58(m,5H),0.99-1.16(m,5H).
LC-MS(ESI):m/z283.1(M+H)+。
Embodiment 2
A kind of human hematopoietic stem cell amplification in vitro method, comprises the steps:
By the human cord blood CD 34 of fresh separated+Cell uses amplification culture medium (StemSpanSFEM culture medium+[100ng/mL]SCF/TPO/Flt3L/IL-6) resuspended to cell concentration be 5 × 104Cell/mL, is then laid in 96 orifice plates, and every hole is 190 μ LCell suspension, adds the p18 micromolecular inhibitor that 10 μ L use amplification culture medium of the same race to dilute subsequently wherein, and p18 is littleThe final concentration of molecule inhibitor is 20nM. At 37 DEG C, 5%CO2In constant incubator, cultivate 7 days.
Embodiment 3: cell phenotype analysis
Adopt flow cytometer detection CD34+，CD34+CD49f+The ratio of cell and absolute quantity are for p18 micromolecular inhibitor bodyThe effect of outer amplifying candidate stem cell verifies, result shows the external expansion of p18 micromolecular inhibitor for candidate stem cellIncreasing effect is obvious.
Flow cytometer showed detects the experimental technique of p18 micromolecular inhibitor to the effect of human hematopoietic stem cell amplification in vitro:
1. by HES for umbilical cord blood (5:1) sedimented red cell of collecting, after 60 minutes, collect supernatant liquid,With the ACK of 4-5 times of volume in 37 DEG C of water-bath splitting erythrocyte 15 minutes, centrifugal 1800rpm, 10 minutes;
2. abandon supernatant, observe erythrocyte splitting situation, by cell harvesting to a 1 50mL centrifuge tube, use 40mlACKAgain in 37 DEG C of water-bath cracking 15 minutes, centrifugal 1800rpm, 10 minutes;
3. by 20mlPE buffer solution washing one time for cell, filter centrifugal 1500rpm, 8 minutes. Cell is entered simultaneouslyRow counting, adds CD34 magnetic bead according to MNCs number, fully mixes rear 4 DEG C of lucifuges and hatches 30 minutes;
4. hatch after end, every effective 20mlPE buffer solution is washed one time, if there is precipitation, need to filter, and centrifugal 1500rpm,5 minutes. Abandon supernatant, resuspended with 1mlPE buffer solution;
5. LS post is installed, is used 1mlPE buffer solution profit post 3 times. Dropwise add cell suspension, in magnetic field, pass through;
6. wash post with 2mlPE buffer solution, 2 times;
7. pillar is shifted out to magnetic field, be placed on collecting pipe, add 3mlPE buffer solution, release fast cell with piston, get10ul counting.
8. by (StemSpanSFEM culture medium+[100ng/mL] of amplification culture medium for cell obtainingSCF/TPO/Flt3L/IL-6) re-suspended cell to concentration is 5.0 × 104Cells/mL, is laid in 96 orifice plates, immediately adds suitableWhen the testing compound (p18 micromolecular inhibitor) of concentration, control group does not add p18 micromolecular inhibitor;
9. cell is in 5%CO2, in 37 DEG C of cell culture incubators, cultivate 7 days;
10. after 7 days, flow cytometer showed detects the ratio of CD34 and CD49f;
According to the numerical value of the each group of cell quantities that draw and ratio, adopt Prism5 software to carry out statistical analysis.
Interpretation of result:
From Fig. 1,2, during cultivating in vitro, p18 micromolecular inhibitor 005A can significantly improve CD34+CD49f+CellRatio (005A group (20nM concentration) is 30.83%, and control group is 19.87%), 20nM concentration in 005A group after processingTime, its approximately 1.5 times of being control group, and there is significant difference (p < 0.05). Meanwhile, under this concentration, the little molecule of p18After inhibitor 005A processes, CD34+CD49f+The absolute quantity of cell has also improved approximately 1.4 times, and (005A group is5509/104Cells, control group is 3831/104Cells, p < 0.05). From above result, the processing of p18 micromolecular inhibitorAfter really can significantly improve the level of candidate stem cell.
Embodiment 4: cells in vitro functional analysis
Adopt cobblestone sample region to form cell analysis (Cobblestoneareaformingcellsanalysis) to little point of process p18The human hematopoietic stem cell of sub-inhibitor processing carries out functional analysis, and result shows that p18 micromolecular inhibitor extracorporeal treatment can be remarkableImprove the level of candidate stem cell.
1. recovery M2-10B4 cell, with the culture medium that goes down to posterity (high sugared 1640+10%FBS) re-suspended cell, and carries out cellCounting;
2. use collagen to be coated with 96 orifice plates. Every hole approximately adds 50 μ L collagen solutions, and jog orifice plate is laid in itAt the bottom of plate. Partly uncap and be placed in super-clean bench, room temperature is dried at least 1h;
3. irradiate stroma cell; Micro-Microscopic observation stroma cell is in good condition and reach required cell number time digestion is collected thinBorn of the same parents; The use culture medium that goes down to posterity is washed cell twice, and carries out cell count; Use long period culture medium (H5100+10-6MHC)Re-suspended cell, makes cell concentration be about 106-108/ mL, exposure dose is 8000cGy (X-ray);
4. use in PBS buffer solution or long period culture medium and the floating acid on collagen surface;
5. irradiate rear centrifugal collecting cell, and carried out cell count. Use long period culture medium re-suspended cell, make it denseDegree is 1.25 × 104/mL；
6. cell is inoculated in 96 coated orifice plates of collagen, every hole adds 100 μ LM2-10B4 cell suspensions,In incubator (37 DEG C, 5%CO2) middle cultivation, after at least spending the night, just can spread confluent monolayer cells (being cell to be measured).
7. collect cell to be measured, herein the detailed preparation method of cell to be measured with reference to step in embodiment 3 1.-9.. Carry out cellCounting, then uses long period culture medium re-suspended cell; Remove carefully the culture medium of orifice plate interior 90%, note not blottingOr stirring stroma cell layer;
8. every hole adds upper strata cell to be measured by calculating concentration, 100 μ L/ holes;
9. half amount is changed liquid (not containing p18 micromolecular inhibitor in fresh culture) weekly, does not carefully stir base while changing liquidCell plastid layer.
10. after 5 weeks, count the positive hole count that contains cobblestone sample region, and utilize Poisson distribution to add up, adopt Prism5Software is drawn.
Interpretation of result:
As shown in Figure 3, after p18 micromolecular inhibitor 005A (20nM) processes, in cell primitive hematopoietic cell ratio obviously onRising, is 1/397 (1/313～1/503), and control group is 1/1031 (1/745～1/1425), p=0.0174. Therefore can think p18After micromolecular inhibitor 005A processes, the cell proportion that can form original hematopoiesis region is increased significantly, and therefore thinks 005AAmplifying candidate stem cell in vitro.
Embodiment 5: animal model test
Adopting immune deficiency mouse model is the goldstandard that detects the hematopoietic reconstitution ability of candidate stem cell, can examine by transplantation experimentsSurvey the long-term hematopoietic potential of transplanted cells, find by this experiment, p18 micromolecular inhibitor 005A is not damage after processingThe hematopoietic reconstitution ability of cell, and can effectively improve the ratio of hematopoietic reconstitution.
1. by the fresh human cord blood CD 34 of getting+Cell goes out CD34 through airflow classification+38-45RA-90+Cell;
2. get the CD34 of some+38-45RA-90+Cell is transplanted in Day0, contrasts as not cultivation group;
3. by the CD34 obtaining+38-45RA-90+(StemSpanSFEM culture medium+[100ng/mL] of amplification culture medium for cellSCF/TPO/Flt3L/IL-6) re-suspended cell to concentration is 5.0 × 104Cells/mL, is laid in 96 orifice plates, immediately adds suitableWhen the testing compound (p18 micromolecular inhibitor) of concentration, control group does not add p18 micromolecular inhibitor; Subsequently in5％CO2, in 37 DEG C of cell culture incubators, cultivate 7 days; Then transplant;
4. the mouse that adopts is 4-5 NOD-SCID mouse in age in week, and before transplanting, 8-10h carries out half lethal dose (220cGy) photographPenetrate;
5. transplant according to cultivating a front 500 cells/mouse, injection system adopts one-sided shin bone pulp cavity to transplant;
6. after 12 weeks, put to death mouse, get bilateral marrow and carry out respectively flow cytometer showed detection people CD45 cell proportion.
Interpretation of result:
As shown in Figure 4, utilize above experimental technique, after transplanting, in mouse, can successfully occur deriving from people's cell mass. AndAnd as shown in Figure 5, after p18 micromolecular inhibitor 005A processes, the hematopoietic reconstitution ability of cell is significantly higher than control group,In mouse bone marrow cells, derive from people's cell proportion apparently higher than control group (p < 0.05), the reconstruction water of candidate stem cell when not cultivatingFlat quite, and can be by migrating in injection side marrow (IF) not in injection side marrow (BM), and in control group mice, do not inject side seamIn marrow, substantially have no the cell (p<0.05) that derives from people. Meanwhile, after 005A processes, hematopoietic reconstitution level higher (>10%)Mouse number of elements will higher than not cultivation group (005A group is 6/9, and not cultivation group is 2/8,005A group be about not cultivation group 2.67Doubly). Therefore can think, after in vitro culture, cell is not lost the ability of its hematopoietic reconstitution, and has carried out effective oneselfUpgrade.
Claims (5)
1. a p18 micromolecular inhibitor, is characterized in that: its structure is:
2. the preparation method of p18 micromolecular inhibitor described in claim 1, is characterized in that: comprise the steps:
Under condition of ice bath, be dissolved in carrene by cyclohexylamine with to carboxyl benzene sulfonyl chloride, add triethylamine, stir 1 hour, rise to after room temperature, continue to stir, after TLC monitoring has been reacted, rotary evaporation is removed reaction solution, and residue purified obtains intermediate;
Under condition of ice bath, gained intermediate is dissolved in water, add NaOH, rise to after room temperature, stir 30 minutes, gained reactant liquor vacuum freeze-drying, obtains finished product.
3. the purposes of p18 micromolecular inhibitor in human hematopoietic stem cell amplification in vitro described in claim 1.
4. purposes according to claim 3, is characterized in that: in described purposes, the concentration of p18 micromolecular inhibitor in amplification culture medium is 10-50nM.
5. right to use requires a human hematopoietic stem cell amplification in vitro method for the p18 micromolecular inhibitor described in 1, it is characterized in that: comprise the steps: the human cord blood CD 34 of fresh separated+Cell use amplification culture medium, i.e. StemSpanSFEM culture medium+[100ng/mL] SCF/TPO/Flt3L/IL-6 resuspended to cell concentration be 5 × 104Cell/mL, is then laid in 96 orifice plates, and every hole is 190 μ L cell suspensions, adds wherein subsequently the p18 micromolecular inhibitor that 10 μ L use amplification culture medium of the same race to dilute, and the final concentration of p18 micromolecular inhibitor is 20nM, at 37 DEG C, and 5%CO2In constant incubator, cultivate 7 days.
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