CN102793722A - Application of human mesenchymal stem cells in preparing medicament for treating M3 leukemia - Google Patents
Application of human mesenchymal stem cells in preparing medicament for treating M3 leukemia Download PDFInfo
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- CN102793722A CN102793722A CN2012103104107A CN201210310410A CN102793722A CN 102793722 A CN102793722 A CN 102793722A CN 2012103104107 A CN2012103104107 A CN 2012103104107A CN 201210310410 A CN201210310410 A CN 201210310410A CN 102793722 A CN102793722 A CN 102793722A
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Abstract
The invention discloses an application of human mesenchymal stem cells in preparing a medicament for treating M3 leukemia. The human mesenchymal stem cells capable of promoting differentiation of M3 leukemia cells into mature granulocytes can be used independently or jointly used with all-trans retinoic acid (ATRA). According to the application of human mesenchymal stem cells in preparing the medicament for treating M3 leukemia disclosed by the invention, a technical means with low cost, low adverse effect and better effect is provided for development of medicaments for treating the leukemia.
Description
Technical field
The present invention relates to the application of a kind of human mesenchymal stem cell in preparation treatment M3 type leukemia medicament.
Background technology
Acute leukemia is the different phase that ability that the leukocyte owing to pathology loses further differentiation and maturation is stuck in cubhood.Acute promyelocytic leukemia (acute promyelocytic leukemia, APL) blocked in the unusual promyelocyte stage, is present unique clinically leukemia of carrying out induction-differential therapy by the differentiation of the leukocyte of M3.Use all-trans-retinoic acid (ATRA) perhaps can obtain complete remission rate separately up to 90% ~ 95% with the chemicals therapeutic alliance; But have the toxicity such as the retinoic acid syndrome that have deadly risk, it possibly cause that high leukocytic appears in the patient, FUO; Respiratory distress; Interstitial pneumonia, thoracic cavity or pericardial effusion, intermittent hypotension and acute renal failure.And the ATRA single therapy M3 type leukemia of persistence will cause carrying out property acquired drug-resistance, and recurrence all appears in nearly all patient in 3～June usually.Repetitively administered causes the medicine removing to accelerate to make the PC of ATRA be lower than effective treatment concentration thereby a possible reason of acquired drug-resistance is.
Address the above problem, possible strategy is to seek other to have the medicine or the Pharmaceutical composition of inducing leukemia cell differentiation ability, single with or with the ATRA combined induction, on the basis that does not lessen the curative effect, weaken ATRA and use the problem of being brought separately.
There are many researchs to report that (mesenchymal stem cells MSC) can promote all that in vivo and in vitro the normal hematopoiesis stem cell is differentiation to medullary system with drenching to mescenchymal stem cell.Originally, have and discover that the mice mesenchymal cell is that MS-5 and S17 all can promote the hematopoietic stem/progenitor cells in people's umbilical blood source to break up to bone-marrow-derived lymphocyte and granulocyte.Subsequently, people's bone marrow MSC is proved and can promotes the hematopoietic stem/progenitor cells megalokaryocyte to form in vivo and in vitro.Research recently shows that people MSC can promote that the hematopoietic stem/progenitor cells in bone marrow and Cord blood source is a cell differentiation to medullary system and pouring.But Shang Weiyou research shows that MSC can promote that the leukaemia breaks up.
The MSC in non-human source is because the variety of problems that exists in biology and the ethics should not be used for the treatment of human diseases.The MSC of people's derived from bone marrow can separate with fairly large preparation and obtains, and what on industry, possess use maybe.Mescenchymal stem cell (the umbilical cord-derived mesenchymal stem cells in umbilical cord source; UC-MSC) be the side-product of normal pregnancy birth process; Multiple ripe means such as available enzyme digestion method are obtained from the neonatal umbilical cord tissue easily; The mescenchymal stem cell basically identical in biological characteristics and other sources; Because popularity, the non-invasive and simplification of drawing materials, the low incidence rate of infection and the good amplification in vitro ability in its source have broad application prospects on industry.
Summary of the invention
Induce differentiation medicament high, defectives such as toxicity is serious, drug combination cost height of existing relapse rate in treatment M3 type leukemia for solving existing in prior technology; The present invention can promote acute promyelocytic leukemia cell to be tied to form ripe differentiation to grain through research proof MSC; Its short differentiation has the associating enhancing when using simultaneously with ATRA, and a kind of technical scheme that human mesenchymal stem cell is applied to prepare the leukemic medicine of treatment M3 type is provided on this basis.
The application of human mesenchymal stem cell of the present invention in preparation treatment M3 type leukemia medicament, wherein human mesenchymal stem cell can be used separately, also can with all-trans-retinoic acid (ATRA) Combined application.
The application of human mesenchymal stem cell of the present invention in preparation treatment M3 type leukemia medicament, wherein human mesenchymal stem cell can use the piece of tissue enzyme digestion in the middle of the neonatal umbilical cord Placenta Hominis, to obtain.
The application of human mesenchymal stem cell of the present invention in preparation treatment M3 type leukemia medicament; Solved independent use all-trans-retinoic acid (ATRA) possibly cause the acquired drug-resistance problem, improved the efficient that the M3 leukemia is induced differentiation, reduced the untoward reaction that possibly exist.
The application of human mesenchymal stem cell of the present invention in preparation treatment M3 type leukemia medicament; The source of wherein said human mesenchymal stem cell is not limited only to depleted neonatal umbilical cord placenta tissue; At present along with the technological development of correlative technology field; All can obtain competent human mesenchymal stem cell in the tissues such as adult bone marrow, fat, endometrium, and the existing mescenchymal stem cell in this type of source and the instance that storage is used to set up the adult mesenchyma stem cell of obtaining in a large number.Based on this, the human mesenchymal stem cell that can know above-mentioned source all can be applicable in the technical scheme of the present invention.
The application of human mesenchymal stem cell of the present invention in preparation treatment M3 type leukemia medicament, wherein the practical application mode of human mesenchymal stem cell should be with intravenous means, separately or with other short differentiation medicament Combined application.Though the embodiment among the present invention does not comprise the actual amount that M3 leukaemic end user mescenchymal stem cell is treated; But instance through disclosed a large amount of mescenchymal stem cell clinical treatment damages of prior art and immune disease; Employed mescenchymal stem cell consumption can be (0.5 ~ 5) * 10 with reference to the consumption in the disclosed other diseases treatment among the present invention
6/ kg.
The invention has the beneficial effects as follows the application of the disclosed human mesenchymal stem cell of the present invention in preparation treatment M3 type leukemia medicament, for this leukemoid medicine exploitation provides low-cost, low untoward reaction and the better technological means of effect.
Description of drawings
Fig. 1 be morphology and NBT reduction test show UC-MSC induce the NB4 cell to the differentiation of grain system and and ATRA used inducing action to unite enhancing simultaneously.
Fig. 2 is that the regulation and control of cell surface differentiation antigen show that UC-MSC induces the NB4 cell to the differentiation of grain system but not to the mononuclear cell differentiation, and has used inducing action to unite enhancing simultaneously with low dose of ATRA.
Fig. 3 is the simultaneous cell G0/G1 phase Cycle Arrest that UC-MSC promotes the NB4 cell differentiation.
Fig. 4 is that UC-MSC induces the leukaemia in APL patient source to break up to grain system.
The specific embodiment
Below in conjunction with the accompanying drawing and the specific embodiment the present invention is done further explain:
1. utilize enzyme digestion digestion full-term pregnancy to cut open the umbilical cord that healthy newborn is produced in the palace, obtain the former foster umbilical cord mesenchymal stem cells of being commissioned to train, be incubated at and contain 10% hyclone, 10 through adherent method
-8The M dexamethasone, 10ng/ml EGF (peprotech company, the U.S.), 2ng/ml bFGF (peprotech company, the U.S.), the DMEM/F12 culture medium of 100 μ g/ml streptomycins and 100U/ml penicillin (Gibco company, the U.S.) is put 37 ℃, 5%CO
2In the saturated humidity incubator, cell grows to 80-90% and merges the back had digestive transfer culture, gets the cell experiment in P4 ~ P6 generation and uses.People APL cell line NB4 cell is frozen by this laboratory liquid nitrogen, is incubated at RPMI 1640 culture medium (Gibco company, the U.S.) that contain 10% hyclone, 100 μ g/ml streptomycins and 100U/ml penicillin, puts 37 ℃, 5%CO
2Suspension culture in the saturated humidity incubator went down to posterity 1 time in per 2 ~ 3 days.In 6 orifice plates, set up NB4 cell and UC-MSC co-culture system, set up 4 experimental grouies, be i.e. NB4 matched group, UC-MSC processed group, ATRA processed group and UC-MSC/ATRA Combined Treatment group.UC-MSC is inoculated in 6 orifice plates in advance, and 5 * 10
5/ hole places 37 ℃, 5%CO
2Cultivate in the saturated humidity incubator, reach 80～90% merge after, 30Gy irradiation in caesium source stops cell proliferation.NB4 cell initial concentration 10
5/ ml inoculation, the 4ml/ hole.After cultivating certain hour, blow and beat repeatedly gently when collecting the leukaemia, avoid destroying the UC-MSC cellular layer of lower floor.Through cellular morphology, nitro nitroblue tetrazolium reduction test (nitroblue tetrozolium reduction test, NBT test), leukaemia's differentiation state is estimated in the detection of cell surface medullary system differentiation sign CD11b and CD14.We find that UC-MSC can promote the NB4 cell can cause the NB4 cell cycle G0/G1 phase block simultaneously to the eventually last differentiation and maturation of grain system, and when uniting use with low dose of ATRA (10 nM), short differentiation has additive effect.Be specially:
(1) the NB4 cell, gets rid of cell to microscope slide through getting rid of sheet machine (Cytospin4, Shandong) or/and 10 nM ATRA handle to cultivate collecting cell after 72 hours at UC-MSC, the Rui Shi Giemsa staining after 30 minutes microscopically observe and respectively organize the NB4 cellular morphology.Shown in the A among Fig. 1, the NB4 cellular control unit is typical germinal cell form, and cell space is bigger, and endochylema is dark blue to be dyed, the big and circle of cell nucleus, and karyoplasmic ratio is bigger, and chromatin is loose, visible bigger kernel; UC-MSC cultivates the sign that differentiation is arranged on the later cellular morphology altogether, and cell volume before diminishes, and nucleus diminishes, and it is big that karyoplasmic ratio becomes, nuclear chromatin before dense gathering, karyon begins depression to occur, the cell of children's grain and children's grain in evening in occurring being similar on the form; ATRA processed group cell is than occurring young grain in more evening on the UC-MSC processed group form; More obvious on the UC-MSC/ATRA Combined Treatment group cellular morphology than the differentiation of ATRA processed group, more sophisticated further band form nucleus appear, the visible segmented granulocyte of idol.
(2) (reactive oxygen species, ROS) generation increases to follow intracellular active oxygen in the neutrophilic granulocyte maturation process.The hydrogen of in the ROS forming process, taking off can and make it be reduced to the bluish violet species precipitate in endochylema by the acceptance of nitro NBT.In this experiment, cultivating altogether 24 hours, after 48 hours and 72 hours; Collect respectively and respectively organize the NB4 cell, carry out the NBT test and weigh intracellular reactive oxygen species generation formation, promptly cell is resuspended in 0.1% NBT (Sigma company after phosphate buffer (PBS) washing; The U.S.); Add 400ng/ml Buddhist ripple ester (Sigma company, the U.S.), 37 ℃ of water-baths 1 hour.Through getting rid of the sheet machine cell is got rid of to microscope slide, safranin O (Sigma company, the U.S.) dyeing back microscopically observation of cell form is also counted the ratio that 400 cells obtain NBT reacting positive cell; Thereby judge granulocytic differentiation state; Shown in the B among Fig. 1, the NB4 cell NBT test positive cells ratio after UC-MSC and ATRA act on respectively obviously raises, and is time dependence; And further raise after the UC-MSC/ATRA synergy, effect has additive effect.
(3) detection of expressing about cell surface differentiation antigen; CD11b is a medullary system differentiation sign; When cell to granulocyte differentiation with to the mononuclear cell differentiation phase, positive rate all occurs and strengthen, express hardly at myeloblast and promyelocyte surface; Begin from myelocyte, positive rate strengthens gradually; CD14 is a mononuclear cell differentiation sign.Previously have research to show, different inducing under the differentiation agent effect, the NB4 cell can be that both direction breaks up with monokaryon to grain system, so the direction of cell differentiation is judged in our expression that detects CD11b and CD14 simultaneously with flow cytometry.In 24 hours that cultivate, 48 hours, 72 hours collecting cells; Add phycoerythrin (phycoerythrin, PE) mouse anti human CD11b monoclonal antibody of labelling (BD company, the U.S.) or CD14 monoclonal antibody (BD company respectively after the PBS washing; The U.S.) and corresponding PE-mice IgG1 homotype control antibodies (BD company; The U.S.) 5 μ l, after 4 ° of C lucifuges are hatched 40min, the positive rate of Flow cytometry CD11b and CD14.Shown in Fig. 2-left side, UC-MSC and ATRA raise the expression of cell surface CD11b respectively, and use time effect associating enhancing, this rise effect presentative time dependency simultaneously.Matched group NB4 cell positive rate basic value is 4.35 ± 2.29%, and UC-MSC, ATRA and 24 hours positive rates of UC-MSC/ATRA synergy are respectively 18.37 ± 2.53%, 26.6 ± 5.37% and 46.6 ± 6.09%; Be respectively 30.7 ± 1.97%, 38.0 ± 8.67% and 65.5 ± 9.02% in 48 hours; Be respectively 39.83 ± 2.53%, 63.33 ± 14.59% and 88.7 ± 7.27% in 72 hours.And for the detection of CD14 shown in Fig. 2-right side, it is lasting low expression level, and does not have significant difference between each group.We think in view of the above, and it is differentiation and maturation to grain that UC-MSC induces the NB4 cell, but not breaks up to mononuclear cell.
(4) (propidium iodide, PI) (sigma company, the U.S.) mixes method Flow cytometry cell cycle distribution respectively to organize the cell propidium iodide in the collections in 48 hours of cultivating.Be specially and respectively organize the NB4 cell fixing 24 hours, after the PBS washing, add 5ul final concentration 50ug/ml RNAase (Sigma company, the U.S.) with 70% cold ethanol, 37 ℃ hatch 30 minutes after, PBS flush away RNAase.Add 50ug/ml PI 500ul again, after the room temperature lucifuge was hatched 5 minutes, flow cytometer detected.As shown in Figure 3, UC-MSC and ATRA produce G0/G1 phase Cycle Arrest to the NB4 cell respectively, both couplings, and effect is more obvious.The G0/G1 phase, with the corresponding downward modulation of S phase cell proportion, and G2/M phase ratio did not have significant change trend when blocking.
2. after informed consent, gather APL patient's bone marrow fluid 5ml.The patient meets the FAB diagnostic criteria, and morphology confirms that leukaemia's ratio is more than 95%.Adopt the lymphocyte separation medium separating bone marrow single nuclear cell.In 6 orifice plates, set up leukaemia and UC-MSC co-culture system, set up 4 experimental grouies, be i.e. leukaemia's matched group, UC-MSC processed group, ATRA processed group and UC-MSC/ATRA Combined Treatment group.UC-MSC is inoculated in 6 orifice plates in advance, and 5 * 10
5/ hole places 37 ℃, 5%CO
2Cultivate in the saturated humidity incubator, reach 80%～90% merge after, 30Gy irradiation in caesium source stops cell proliferation.Leukaemia's initial concentration 10
5/ ml, the 4ml/ hole is inoculated in 6 orifice plates, is incubated at RPMI 1640 culture medium that contain 10% hyclone, 100 μ g/ml streptomycins and 100U/ml penicillin.Cultivate after 48 hours, blow and beat repeatedly gently when collecting the leukaemia, avoid destroying the UC-MSC cellular layer of lower floor.Through detecting leukocyte surface differentiation antigen CD11b; We prove that UC-MSC can promote acute promyelocytic leukemia patient's leukaemia to break up at the end eventually to grain system equally; And when uniting use with low dose of ATRA (10nM), short differentiation has additive effect.As shown in Figure 4, leukaemia CD11b basal expression rate is 13.1%, and UC-MSC and ATRA can cause respectively and transfer to 61.4% and 88.8% on the CD11b positive rate, and UC-MSC/ATRA unites use, and positive rate transfers to 95.1% on further.The detection that CD14 expresses shows that the CD14 positive rate is lower than 3% all the time.In view of the above, we can conclude that UC-MSC can induce acute promyelocytic leukemia cell to the differentiation of grain system.
In sum, content of the present invention is not confined in the above embodiments, and the knowledgeable people in the same area can propose other embodiment easily within technological guidance's thought of the present invention, but this embodiment is included within the scope of the present invention.
Claims (8)
1. the application of human mesenchymal stem cell in preparation treatment M3 type leukemia medicament.
2. the application of human mesenchymal stem cell according to claim 1 in preparation treatment M3 type leukemia medicament is characterized in that the medication combined application of human mesenchymal stem cell and inducing leukemia cell differentiation.
3. the application of human mesenchymal stem cell according to claim 2 in preparation treatment M3 type leukemia medicament is characterized in that the medicine of said inducing leukemia cell differentiation is an all-trans-retinoic acid.
4. according to claim 1, the application of 2 or 3 described human mesenchymal stem cells in preparation treatment M3 type leukemia medicament, it is characterized in that said human mesenchymal stem cell is to obtain in the middle of depleted neonatal umbilical cord placenta tissue with the histaminase digestion method.
5. according to claim 1, the application of 2 or 3 described human mesenchymal stem cells in preparation treatment M3 type leukemia medicament, it is characterized in that said human mesenchymal stem cell separates acquisition from the human bone marrow cell.
6. according to claim 1, the application of 2 or 3 described human mesenchymal stem cells in preparation treatment M3 type leukemia medicament, it is characterized in that said human mesenchymal stem cell separates acquisition from people's fat or endometrial tissue.
7. according to claim 1, the application of 2 or 3 described human mesenchymal stem cells in preparation treatment M3 type leukemia medicament, it is characterized in that said human mesenchymal stem cell medicine adopts the intravenous injection mode.
8. the application of human mesenchymal stem cell according to claim 7 in preparation treatment M3 type leukemia medicament is characterized in that said intravenous consumption is (0.5 ~ 5) * 10
6/ kg.
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| CN105395570A (en) * | 2015-09-23 | 2016-03-16 | 广东颐养抗衰老研究院 | Composition for treating senile leukemia |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105395570A (en) * | 2015-09-23 | 2016-03-16 | 广东颐养抗衰老研究院 | Composition for treating senile leukemia |
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Application publication date: 20121128 |