CN101868230A - Differentiation promoter and/or proliferation promoter for erythrocyte stem cells, use of methionine for preventing or treating senile anemia, and compositions containing methionine - Google Patents
Differentiation promoter and/or proliferation promoter for erythrocyte stem cells, use of methionine for preventing or treating senile anemia, and compositions containing methionine Download PDFInfo
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Abstract
It is a first object to develop a remedy efficacious in preventing senile anemia which is frequently observed due to the aging. It is a second object to provide an antiaging drug, which has little side effect and is inexpensive and efficacious, in anticipation of the coming aging society. It is a third object to provide a remedy which is efficacious against anemia caused by myelosuppression, more particularly speaking, anemia occurring as the side effect of the administration of an anticancer agent. It has been clarified for the first time that a drug containing, as the active ingredient, methionine (Met) alone, an amino acid composition containing methionine (Met) together with one or more members selected from among glycine (Gly), serine (Ser) and threonine (Thr) or, furthermore, an amino acid mixture selected from (1) Ile, Trp, Thr, Val, His, Phe,Lys and Leu, (2) Ile, Thr, Val, His, Lys and Leu, (3) Trp, Thr, His, Phe and Lys, and (4) Ile, Trp, Thr, Val, Phe and Leu is capable of ameliorating and healing senile anemia. Thus, preventives for senile anemia, remedies for senile anemia and drugs for preventing cell aging can be provided.
Description
Technical field
The present invention relates to differentiation accelerator and/or the enhancer of proliferation and the pre-age inhibiting technical field of erythrocyte stem cells.The invention particularly relates to the inhibition and the pre-age inhibiting technology of the erythrocyte stem cells hypofunctions such as hematopoietic stem cell in the erythrocyte propagation.
Background technology
Because the aging and age increases, wrinkle of skin not only appears, the variation that hair minimizing poliosis etc. occurs on the surface of health, the function of health also reduces gradually, various symptoms occur.For example, the synthetic reduction of illness such as hypertension, myocardial infarction, angina pectoris, insulin and the risk of diabetes that causes increases, sclerotin is loose, senile cataract, senile anemia etc.
As this aged countermeasure, all health food and the curative that comprise folkd therapy have been developed.
For example, along with the aging minimizing that melatonin can occur, thereby caused the research that the melatonin owing to the aging neurodegenerative disease that causes is given, reported that in recent years melatonin or its analog (No. the 5403851st, United States Patent (USP)) have pre-age inhibiting effect.In addition, the effect of this melatonin realizes by HGH, so also growth hormone is used (AnnuRev Med.2003 as aging preventive; 54:513-33.Epub 2001Dec 3).
In addition, the active oxygen of Sheng Chenging can cause the damage of cell and DNA, the peroxidating of lipid etc. in vivo, have all influences that cause with multiple disease association, so studied utilization with the antioxidant that is used to seal active oxygen, for example, utilize vitamin C, vitamin E etc., utilize (Japanese kokai publication hei 8-277282 numbers) such as alpha tocopherol and analog thereof, in addition, also exploitation has free radical scavenger (Japanese kokai publication hei 9-241637 number).
In addition, have report to claim: along with aging and in the various symptoms that take place, aspect the anemia relation, " active oxygen " that produces in the body can bring out anemia and immunodeficiency, and (Nature 431,997-1002).
With regard to senile anemia with regard to, as Chinese medicine, known by for example adding the flavor GUIPI TANG by what Radix Ginseng, Rhizoma Atractylodis or the Rhizoma Atractylodis Macrocephalae, Poria, Radix Glycyrrhizae, Rhizoma Zingiberis Recens, Fructus Jujubae, Semen Ziziphi Spinosae, Arillus Longan, Radix Polygalae, Radix Angelicae Sinensis, the Radix Astragali, the Radix Aucklandiae, Radix Bupleuri and Fructus Gardeniae were formed.
No. the 5403851st, [patent documentation 1] United States Patent (USP)
[patent documentation 2] Japanese kokai publication hei 8-277282 number
[patent documentation 2] Japanese kokai publication hei 9-241637 number
[non-patent literature 1] Annu Rev Med.2003,54,513-33.Epub 2001 Dec 3
[non-patent literature 2] Nature 431,997-1002
Summary of the invention
Problem to be solved by this invention
From being accompanied by aging and frequently existing anemia, be the 1st problem to the effective therapeutic agent of the prevention of senile anemia with exploitation.
And aging meeting from now on further develops sets out, and is the 2nd problem so that low, the inexpensive effective age resister of side effect to be provided.
Be not limited only to the old man with exploitation in addition, what can extensively be suitable for is the 3rd problem to the effective therapeutic agent of prevention anemia.
At last, for artificial blood manufacturing etc., be the 4th problem to prepare erythrocyte by cell culture effectively.
Solve the method for problem
The present inventor finds methionine (Met) and contains methionine in chance amino acid composition has keeping and promotes the effect of erythrocyte hematopoietic function, also produces effect very much improving on the senile anemia.Find that first the amino acid composition that contains methionine has the ability of replying senile anemia of improving, particularly, described compositions is for also containing glycine (Gly) and glutamic acid (Glu) amino acid composition as effective ingredient except that methionine.
In addition, the invention provides the external efficient erythrocyte propagation and/or the method for differentiation.
The invention effect
By of the present invention be the senile anemia preventive or the senile anemia therapeutic agent of effective ingredient with the methionine, for the senile anemia that may increase from now on, can carry out the inexpensive treatment of safety.In addition, by the differentiation accelerator and/or the enhancer of proliferation of erythrocyte stem cells of the present invention, can carry out necessary hemopoietic to the person's of strenuous exercise anemia, the side effect of anticarcinogen and the anemia that kidney disease causes etc., thereby internal therapy improves anemia on a large scale.And the present invention can prevent to follow aged cell function low, prevents catabiosis all sidedly.In addition, the erythrocyte can also pair cell cultivated of the present invention is effectively bred and is broken up.
This description comprises the content as the Japanese patent application 2007-300814 description of basis for priority of the present invention and/or accompanying drawing record.
The accompanying drawing summary
The hemoglobin content of respectively organizing the mice peripheral blood before [Fig. 1] ispol begins.Represent with meansigma methods ± standard deviation.*: P＜0.0001 (young mice is carried out student t check (student ' s t-test)).
The hemoglobin content of respectively organizing the mice peripheral blood after [Fig. 2] ispol gives.Represent with meansigma methods ± standard deviation.*: P＜0.01 (aged mouse (casein) is carried out student t check).
[Fig. 3] ispol is to the effect of 707fl. cell proliferation.Represent with meansigma methods ± standard deviation.*: P＜0.05 (condition of not adding ispol is carried out student t check)
[Fig. 4] is under removing specific amino acid whose situation, to the influence of 707fl. cell proliferation facilitation effect from the essential amino acids mixture.Represent with meansigma methods ± standard deviation.*: P＜0.005 (condition of not adding ispol is carried out student t check)
[Fig. 5] methionine and threonine are to the influence of 707fl. cell proliferation.Represent with meansigma methods ± standard deviation.*: P＜0.05 (condition of not adding ispol is carried out student t check)
[Fig. 6] ispol is to the propagation facilitation effect of the K562 cell that stems from the people.Represent with meansigma methods ± standard deviation.*: P＜0.05 (condition of not adding ispol is carried out student t check)
[Fig. 7] ispol is to the propagation facilitation effect of the HEL92.1.7 cell that stems from the people.Represent with meansigma methods ± standard deviation.*: P＜0.05 (condition of not adding ispol is carried out student t check)
[Fig. 8] methionine concentration is to the influence of 707fl. cell proliferation.Represent with meansigma methods ± standard deviation.
The remission effect of the proliferation inhibition activity of the 707fl. cell that [Fig. 9] aminoacid [mixture of threonine (Thr) or glycine (Gly) and glutamic acid (Glu)] causes the high concentration methionine.Represent with meansigma methods ± standard deviation.*: P＜0.05 (matched group is carried out student t check)
The variation of [Figure 10] peripheral blood hemoglobin content in during the anticarcinogen actinomycin D is induced the efficiency assay of methionine in the anemia model.Represent with mean+/-standard error.
[Figure 11] induces peripheral blood hemoglobin content after the effectiveness actinomycin D of methionine in the anemia model begins 17 at the anticarcinogen actinomycin D.Represent with mean+/-standard error.There is marked difference between different literal, P＜0.05 (one factor analysis of variance and Newman-Cole's postmortem analysis (one-way ANOVA with Newman-Keuls ' s post hocanalysis))
The concentration of [Figure 12] methionine and the proliferative amount of 707fl. cell.Represent with meansigma methods ± standard deviation.Statistics is resolved and is used student t check to analyze.
[Figure 13] along with the concentration difference of methionine, the variation of erythrocyte Expression of Related Genes amount.Represent with meansigma methods ± standard deviation.Statistics is resolved and is used student t check to analyze.
[Figure 14] methionine through the time concentration change to the effect-1 of erythrocyte differentiation associated gene expression.In initial 3 days, subsequently 24 hours and again after cultivated the culture medium that the culture medium of using is respectively the methionine that contains following concentration etc. in 24 hours.Among the figure from left to right:
1：30μM→30μM→30μM
2：30μM→150μM→150μM
3：30μM→150μM→30μM
4：30μM→150μM→＞3μM
5：30μM→150μM+1％DMSO→150μM+1％DMSO
Represent with meansigma methods ± standard deviation.*: P＜0.05 (carrying out student t check) to 2
[Figure 15] methionine through the time concentration change to the effect-2 of erythrocyte differentiation associated gene expression.In initial 3 days, subsequently 24 hours and again after the culture medium of using of cultivating in 24 hours be respectively the culture medium that contains following concentration methionine etc.Among the figure from left to right:
1：30μM→30μM→30μM
2：30μM→150μM→150μM
3：30μM→150μM→30μM
4：30μM→150μM→＞3μM
5：30μM→150μM+1％DMSO→150μM+1％DMSO
Represent with meansigma methods ± standard deviation.*: P＜0.05 (carrying out student t check) to 2
The best mode that carries out an invention
1. preface
Anemia only is a kind of symptom, directly says, be because synthetic low, the erythrocytic destruction of erythrocytic generation minimizing, hemoglobin is hyperfunction etc. causes, but reason also has nothing in common with each other.
For example, erythrocytic generation reduces the generation that can enumerate the erythropoietin that (first) renal insufficiency etc. causes and reduces, the hematopoietic stem cell that (second) regeneration inhibition anemia etc. causes unusual, the reasons such as bone marrow of (third) leukemia or cancer.In addition, the synthetic minimizing of hemoglobin can have been enumerated reasons such as iron deficiency, is necessary to treat according to reason separately.
2. the propagation of senile anemia and erythrocyte stem cells promotes and promotes (differentiation and proliferation of erythrocyte stem cells promotes) from erythrocyte stem cells to erythrocytic differentiation
Early onset anemia majority is because the shortage of ferrum.The reason that anemia takes place because wear out is also not exclusively clear and definite, but compare with the early onset anemia, can enumerate being accompanied by the aged hematopoietic stem cell minimizing of bone marrow, cripetura red blood cell life span that is produced, the reasons such as alimentation amount minimizing that are accompanied by gastric mucosa atrophy.In the reason beyond the visceral hemorrhage of present stage and malnutrition etc., think because the age increase causes the low and erythrocytotropic induction of underactivity, especially autosynthesis ability of blood stem cell to be suppressed or is in the reduction state.
In addition, think that erythrocyte is divided into burst forming unit-erythroid (BFU-E) (BFU-E:burst-forming-unit-erythroid), colony forming unit-erythroid (CFU-E) (CFU-E:colony-forming-unit-erythroid), proerythroblast, protoerythrocyte, reticulocyte, erythrocyte from hematopoietic stem cell and generates.
So-called erythrocyte stem cells includes ancestral's erythrocyte (the red blood cell forerunner of erythroid progenitor Fine born of the same parents) such as hematopoietic stem cell, burst forming unit-erythroid (BFU-E) (BFU-E:burst-forming-unit-erythroid), colony forming unit-erythroid (CFU-E) (CFU-E:colony-forming-unit-erythroid), proerythroblast and protoerythrocyte among the present invention.
3. the prevention of the inhibition of the bone marrow function that the prevention of methionine (Met) and senile anemia or treatment, cell senescence suppress, the side effect of the reduction of the differentiation of erythrocyte stem cells and/or the promotion of propagation, the differentiation that is accompanied by aged erythrocyte stem cells and/or propagation function, anticarcinogen causes and the anemia that causes and improve and (hereinafter, use the code of 3 letters to represent aminoacid.)
3-1. the amino acid composition that the present inventor found through experiments Met and contains Met has keeping and promotes the effect of erythrocytic hematopoietic function, also produces effect improving on the senile anemia.
Find in addition, because Met and contain the giving of amino acid composition of Met, answer is confirmed as and is accompanied by the low or hyperfunction gene expression amount of aged expression, so can be used as, the amino acid composition of this specific composition suppresses to be accompanied by aged cell underactivity and uncomfortable medicament, in other words, it can be used as the cell senescence preventive.
3-2. need to prove that the amino acid composition of also finding Met and containing Met has the effect of promotion to the propagation as the erythroblastic 707fl. cell of class in mouse spleen source.Simultaneously, to using Met to promote resolving of propagation as the erythroblastic 707fl. gene expression of cells of class, the result shows, the same with the situation of aged mouse, the gene expression of haeme synthetase and erythrocyte tectine matter increases, the expression of gene of main interleukin (IL) of expressing in leukocyte etc. reduces, so infer that erythrocytotropic differentiation has obtained promotion.And at this moment; variation has appearred with the relevant gene expression of outer hereditary change (epigenetic change エ ピ ジ エ ネ テ イ Star Network な variation) of DNA (cytosine-5)-transmethylase 1 gene (Dnmt1) and histon deacetylase (HDAC) (Hdac) etc.; so infer in the cell proliferation of the 707fl. cell that Met causes promotes hereditary change outside in cell, being attended by.In fact, add after inhibitor Zebularine (ゼ Block ラ リ Application) as DNA (cytosine-5)-transmethylase 1 (DNMT1) acts on, Met promotes that to the propagation of 707fl. cell activity has been subjected to inhibition.
The prevention and the improvement of the anemia that 3-3. the propagation of erythrocyte stem cells promotes and/or differentiation promotes, following reason is caused: be accompanied by the marrow function that the side effect of the reduction of the propagation of aged erythrocyte stem cells and/or differentiation function or anticarcinogen causes and suppress
Now clear and definite Met can promote the propagation of erythrocyte stem cells, also can promote the erythrocytotropic differentiation of erythrocyte stem cells.
That is to say, the differentiation and/or the facilitation of the erythrocyte stem cells by Met, the anemia that can cause in the disease of the side effect of the normal person's who comprises the old people anemia, the person's of strenuous exercise anemia, anticarcinogen or kidney etc. must be carried out among the crowd on a large scale of hemopoietic, can be by giving Met or containing the amino acid composition prevention of Met and improve the low of hematopoietic function such as anemia.
In addition, as causing the anticarcinogen of anemia,, enumerate the anticarcinogen that suppresses bone marrow function and cause anemia especially though be that any anticarcinogen of anemia is an object with caused side effect as side effect.For example can enumerate taxanes anticarcinogen such as anthracene nucleus class, paclitaxel and Docetaxel such as platinum class, amycin and epirubicin such as cisplatin and carboplatin.
Present inventors etc. after the effect of having found aforesaid Met erythroblastic propagation and differentiation to class, have studied the valid density scope of Met and the influence of concentration in the system that has used cultured cell.At first, the erythroblastic propagation of class is promoted that the concentration range of effective Met is 25 μ M～50000 μ M, preferred 30 μ M～5000 μ M, more preferably 50 μ M～3500 μ M, the most preferably scope of 50 μ M～200 μ M.Need to prove, the concentration of Met within the specific limits, the scope below the 2mM of high concentration, near about 200 μ M, the effect of on cell proliferation weakens, and does not know which kind of mechanism this is.Perhaps also might be owing to there is the different proliferation function of cell proliferation mechanism in methionine concentration.
Find in addition, during the concentration less than 25 μ M of Met, the erythroblastic proliferation function of class is died down, but the erythroblastic differentiation facilitation of class is advantage.
Find also that simultaneously the differentiation facilitation that can cause Met to be advantage under the state of low concentration further strengthens by further reduce the concentration of Met once more after the Met concentration rising of a property crossed.That is to say,, can regulate and control the propagation and the differentiation of erythrocyte stem cells more efficiently if give Met violent concentration change.So think, a rising of crossing the Met concentration in the hemopoietic tissue of property as takes place in humans and animals when picked-up Met, then can promote the propagation of erythrocyte stem cells.Think: if further reduce next differentiation of occasion of the concentration of Met once more, therefore originally only make the amount of former erythrocyte stem cells propagation can cause erythrocytic a large amount of generation, consequently erythrocyte number increases.From in the past document as can be known, the picked-up of too much Met can cause hemolytic anemia, thinks that this is that differentiation being suppressed when the erythrocytic propagation of ancestral obtained continuing to promote because when the picked-up of the Met of surplus, consequently undifferentiated erythrocyte produces in a large number, thereby causes hemolytic anemia.
4. the amino acid composition (amino acid preparation) that contains Met
The amino acid composition that the present invention uses comprises that the aminoacid constituent that contains Met is as the differentiation accelerator of the erythrocyte stem cells of effective ingredient and/or rise in value promoter, anemia preventive, treatment for anemia agent, senile anemia preventive, senile anemia therapeutic agent or cell senescence preventive.
The present invention preferably includes and contains differentiation accelerator and/or enhancer of proliferation, senile anemia preventive, treatment for anemia agent or the cell senescence preventive of following amino acid composition as the erythrocyte stem cells of effective ingredient, and described compositions contains has also added Gly and Glu except that Met.
The present invention includes and contain differentiation accelerator and/or enhancer of proliferation, anemia preventive, treatment for anemia agent, senile anemia preventive, senile anemia therapeutic agent or the cell senescence preventive of following amino acid composition as the erythrocyte stem cells of effective ingredient, described compositions further contains isoleucine (Ile), tryptophan (Trp), valine (Val), histidine (His), phenylalanine (Phe), lysine (Lys) and/or leucine (Leu) as any adding ingredient in the aforementioned amino acid composition.
Particularly, comprise and contain differentiation accelerator and/or enhancer of proliferation, anemia preventive, treatment for anemia agent, senile anemia preventive, senile anemia therapeutic agent or the cell senescence preventive of following amino acid composition as the erythrocyte stem cells of effective ingredient, described amino acid composition comprises Met, Gly, serine (Ser), threonine (Thr), Ile, Trp, Val, His, Phe, Lys and/or Leu.
Each aminoacid that constitutes amino acid composition of the present invention be D-body or L-body all can, preferred L-body.In addition, free amino acid also can, the salt that allows pharmaceutically or on the food to use also can.For example, each amino acid salts, hydrochlorate, lactate all can.
As amino acid composition of the present invention, but example is as follows.
4-1. contain differentiation accelerator and/or enhancer of proliferation, anemia preventive, treatment for anemia agent, senile anemia preventive, senile anemia therapeutic agent or the cell senescence preventive of following amino acid composition as the erythrocyte stem cells of effective ingredient, described compositions has also been added be selected from Gly, Glu, Ser and Thr a kind with upper amino acid except that Met.
The present invention includes and contain differentiation accelerator and/or enhancer of proliferation, anemia preventive, treatment for anemia agent, senile anemia preventive, senile anemia therapeutic agent or the cell senescence preventive of following amino acid composition as the erythrocyte stem cells of effective ingredient, described compositions has also been added be selected from Gly, Glu, Ser and Thr a kind with upper amino acid except that Met.When share Gly and Glu, demonstrated inhibition effect to the hemolytic anemia that causes under the too much picked-up situation of Met.
Need to prove, the present invention includes differentiation accelerator and/or enhancer of proliferation, anemia preventive, treatment for anemia agent, senile anemia preventive, senile anemia therapeutic agent or the cell senescence preventive of the erythrocyte stem cells that comprises following amino acid composition, described compositions is added with Gly and Glu in Met.Share Gly and Glu and be added among the Met and add Gly separately and compare, can relax the inhibitory action of the Met pair cell of high concentration.
4-2. with differentiation accelerator and/or enhancer of proliferation, senile anemia preventive, senile anemia therapeutic agent or the cell senescence preventive of following amino acid composition as the erythrocyte stem cells of effective ingredient, described compositions has also been added the aminoacid more than a kind that is selected from Ile, Trp, Thr, Val, His, Phe, Lys and Leu except that Met.
Preference is as differentiation accelerator and/or enhancer of proliferation, anemia preventive, treatment for anemia agent, senile anemia preventive or the cell senescence preventive of the erythrocyte stem cells that comprises following ispol, described mixture is selected from the ispol that (1) contains Ile, Thr, Val, His, Met, Lys and Leu, (2) contain the ispol of Trp, Thr, His, Phe, Met and Lys, (3) contain the ispol of Ile, Trp, Thr, Val, Phe, Met and Leu.
5. the use form of compositions of the present invention
The use form of relevant amino acid composition of the present invention is not particularly limited, and can be used as diet usefulness, the auxiliary diet usefulness of nutrition, also can be used as medical.Under as edible situation, this amino acid composition can directly eat, and also can use as additive in various food.In addition, drink as beverage after compositions of the present invention also can be dissolved in water, in this case, also can add other nutritional labeling again, for example water soluble vitamins, taurine etc.In addition, in order to improve palatability, drink as beverage after can adding acids such as salt, citric acid such as sodium chloride and/or other suitable taste.For the reason of stability, also can add pH regulator agent, chelating agen etc.
Under therapeutic medical situation, can give that (Longitude pipe throw with), rectum give, inject, infusing the general approach that gives such as gives and use by orally give, through pipe.As orally give, can be for above-mentioned composition self or for the powder, granule, tablet, the capsule that mix with the carrier that pharmaceutically allows, excipient, diluent etc., contain tablet, syrup etc.In addition, as injection, can use to be added with suitable buffer agent, isotonic agent etc. and dissolved injection in sterile purified water.Preferred oral gives.
Because Met of the present invention and to contain the amino acid composition of Met as safe as a house so set its dosage in very wide scope, need to prove, give method and need not also have nothing in common with each other according to application target.For example, Met content is that 0.1%～1% solution can orally give in the scope of 1ml～600ml on the 1st.In addition, for example, when adding Gly and Glu among the Met and using as compositions, for example, the ratio of Met: Gly: Glu can use for 10: 1～10: 1～10.And compositions of the present invention and ferrum, folic acid, vitamin B12 etc. share to expect better effect.
In addition, particularly, can enumerate for example scope about 10mg/50kg individuality/sky～100g/50kg individuality/sky, the scope about preferred 100mg/50kg individuality/sky～10g/50kg individuality/sky, the more preferably dosage about 130mg/50kg individuality/sky.Promote the concentration range of effective Met can enumerate 25 μ M～50000 μ M, preferred 30 μ M～5000 μ M, more preferably 50 μ M～3500 μ M, the scope of 50 μ M～200 μ M most preferably to the propagation of erythrocyte stem cells.Can enumerate less than 25 μ M, preferred 0.01 μ M～20 μ M, more preferably 0.1 μ M～10 μ M, the scope of 0.1 μ M～5 μ M most preferably as being divided into effective Met concentration range under the main situation to erythrocyte with protoerythrocyte.
6. the cultivation of erythrocyte stem cells
6-1. cell culture additive
The present invention comprises that also containing Met promotes or/and the cell culture medium additive that differentiation promotes as the propagation that is used for erythrocyte stem cells of effective ingredient.
Among the present invention is that the enhancer of proliferation and/or the differentiation accelerator of the erythrocyte stem cells of effective ingredient can be added into erythrocyte stem cells with Met, promptly uses in the erythrocytic cell culture medium of ancestral such as burst forming unit-erythroid (BFU-E) (BFU-E:burst-forming-unit-erythroid), colony forming unit-erythroid (CFU-E) (CFU-E:colony-forming-unit-erythroid), proerythroblast and protoerythrocyte.The cell culture medium that these erythrocyte stem cells are used can be any culture medium, and for example, the unipotency ancestral erythrocyte that can buy on the market (the red blood cell forerunner of single differentiation potency Fine born of the same parents) is with culture medium etc.
As the concentration of in aforementioned culture medium, adding, at first, promote the concentration range of effective Met as propagation, can enumerate 25 μ M～50000 μ M, preferred 30 μ M～5000 μ M, more preferably 50 μ M～3500 μ M, the scope of 50 μ M～200 μ M most preferably erythrocyte stem cells.In addition, promote effective Met concentration range, can enumerate less than 25 μ M, preferred 0.01 μ M～20 μ M, more preferably 0.1 μ M～10 μ M, the scope of 0.1 μ M～5 μ M most preferably as differentiation to erythrocyte stem cells.
6-2. the cultural method of erythrocyte stem cells
The present invention contains the cultural method of the erythrocyte stem cells that comprises following operation: (1) uses the culture medium that contains high concentration Met to make the operation of erythrocyte stem cells propagation, and (2) use the culture medium that contains low concentration Met to make the operation of erythrocyte stem cells differentiation.And, the present invention contains the cultural method of the erythrocyte stem cells that comprises following operation: (1) uses the Met culture medium that contains high concentration to make the operation of erythrocyte stem cells propagation, and (2) by reusing the culture medium that contains low concentration Met makes the erythrocyte stem cells differentiation of breeding in aforementioned (1) operation operation, thereby further strengthen the operation of differentiation facilitation.Or the present invention includes the cultural method of the erythrocyte stem cells that contains following operation: described operation is controlled the propagation and the differentiation of erythrocyte stem cells efficiently by being used in combination the culture medium that contains low concentration and high concentration Met.
As aforementioned erythrocyte stem cells, as previously mentioned, comprise ancestral's erythrocyte such as hematopoietic stem cell, burst forming unit-erythroid (BFU-E) (BFU-E:burst-forming-unit-erythroid), colony forming unit-erythroid (CFU-E) (CFU-E:colony-forming-unit-erythroid), proerythroblast and protoerythrocyte.
As the concentration of the high concentration Met that contains in aforementioned (1) operation, can enumerate 25 μ M～50000 μ M, preferred 30 μ M～5000 μ M, more preferably 50 μ M～3500 μ M, the scope of 50 μ M～200 μ M most preferably.In addition, as the concentration of the low concentration Met that contains in aforementioned (2) operation, can enumerate less than 25 μ M, preferred 0.01 μ M～20 μ M, more preferably は 0.1 μ M～10 μ M, the scope of 0.1 μ M～5 μ M most preferably.
Amino acid whose picked-up causes the increase of the peripheral blood hemoglobin content of aged mouse
The ICR male mice is raised until 20 monthly ages under the condition of MF feedstuff (Oriental yeast) of freely drinking water, freely ingest, it is divided into 3 groups (aged mouses) according to body weight.In addition, the ICR male mice (young mice) that used for 5 ages in week in contrast.Test feed is as shown in table 1, uses following feedstuff: with the refining feedstuff (casein content 14%) of AIN-93M is basic feedstuff, and 14% caseic 2% the amount that will be equivalent to that this basic feedstuff contains is replaced into amino acid composition.The duration of test feedstuff of freely ingesting.
In aminoacid is organized, 2 groups have been designed, as shown in table 2, the aminoacid that has mixed 16 seed amino acids mixes 1 (AAM1) group (aged mouse (AAM1)) and gives that to give essential amino acids be that main aminoacid mixes 2 (AAM2) groups (aged mouse (AAM2)).Organize (casein is organized) at the refining feedstuff of in contrast AIN-93M and be provided with each 1 group of young mice and aged mouse (casein).
[table 1]
The composition of feedstuff
[table 2]
Ispol is formed
| AAM1 (weight %) | AAM2 (weight %) | AAM3 (weight %) | |
| ??Asn | ??- | ??- | ??- |
| ??Asp | ??0.2 | ??- | ??- |
| ??Ala | ??4.3 | ??- | ??- |
| ??Arg | ??4.9 | ??16 | ??- |
| ??Ile | ??4.7 | ??11.5 | ??11.9 |
| ??Gly | ??11.4 | ??- | ??- |
| ??Gln | ??- | ??- | ??- |
| ??Glu | ??3.7 | ??17 | ??- |
| ??Cys | ??- | ??- | ??- |
| AAM1 (weight %) | AAM2 (weight %) | AAM3 (weight %) | |
| ??Ser | ??2.1 | ??- | ??- |
| ??Tyr | ??8.6 | ??- | ??- |
| ??Trp | ??3.6 | ??3.8 | ??18.5 |
| ??Thr | ??6.8 | ??9.4 | ??10.8 |
| ??Val | ??5.5 | ??9.5 | ??10.6 |
| ??His | ??3.2 | ??3.8 | ??4.2 |
| ??Phe | ??5.0 | ??3.8 | ??15 |
| ??Pro | ??16.5 | ??5.0 | ??- |
| ??Met | ??0.6 | ??2.5 | ??3.7 |
| ??Lys | ??12.5 | ??3.8 | ??13.3 |
| ??Leu | ??6.4 | ??14 | ??11.9 |
From each tail vein of organizing of aged mouse and young mice, gather blood, use hemoglobin B-test and light (with the pure medicine of light) to measure the hemoglobin content of peripheral blood.At this moment, young mice was 5 ages in week, and each is organized aged mouse and was for 20 monthly ages.And the n=9 of young mice, the n=15 of aged mouse.Statistics is resolved and is used student t check, and young mice is carried out significance test.
The result as shown in Figure 1.AMM1 in the bracket of aged mouse represents to prepare to comprise the group of the mixture of 17 seed amino acids, and AMM2 represents to prepare to give the group (amino acid whose kind reference table 1) of the mixture of 12 seed amino acids.Before giving and young mice compare, significant reduction has appearred in the hemoglobin content of aged mouse, hematopoietic function also has significant reduction.
Subsequently, allow the aged mouse aminoacid mixing AAM1 or the AAM2 shown in the table 1 that in 2 months, ingest of each group.Subsequently, the tail vein is taken a blood sample, making the hemoglobin content in the mensuration peripheral blood that uses the same method.This moment with aforementioned identical, the n=9 of young mice, each organizes aged mouse all is n=15.Statistics is resolved and is used student t check, and the aged mouse that casein is organized carries out significance test.
The result as shown in Figure 2.Do not ingest and do not find the answer of hemoglobin content in the amino acid whose aged mouse, but the peripheral blood hemoglobin content of the amino acid whose aged mouse of ingesting then returns back to the level identical with young mice.This demonstration, the Moriamin Forte mixture can promote the erythrocyte hematopoietic function, and can improve effectively and follow aging and peripheral blood hemoglobin content that reduce.
Ispol is to the facilitation effect of the cell proliferation of mice class protoerythrocyte (707fl. cell)
For the increase of determining in embodiment 1 to increase owing to the age peripheral blood hemoglobin content that reduces is amino acid whose effect, use 707fl. cell (cell of purchase ECACC (European cell strain, microbial preservation center European Collection of Cell Cultures) type strain) to study.707fl. cell is the class protoerythrocyte that can be divided into erythrocytic mouse spleen source.This 707fl. cell uses interpolation hyclone in RPMI1640 culture medium (SIGMA) so that its ultimate density is 5% culture medium that obtains (containing 5% hyclone RPMI1640 culture medium) cultivates.When measuring beginning, in 96 hole microtest plates with about 1 * 10
4Cells/well is sowed cell, and adds the ispol of various concentration (about 0.5%～0.001%) therein.At this moment, the pH that adjusts ispol adds near the neutrality.After 3 days, use the WST-1 algoscopy to measure the propagation of cell cell culture in this state.That is, add the Premix WST-1 reagent (Takarabio) of 10 μ l in each hole (well), hatched in CO2 gas incubator 1～4 hour, then the WST-1 that causes of the dehydrogenase in the living cells mitochondrion decomposes the first that generates
Amount can be measured under A450nm (reference A655nm).
At first to as studying (amino acid whose kind etc. are with reference to table 2) based on the AAM2 of the mixture of 12 seed amino acids of essential amino acids and as the AAM3 of the mixture of 9 kinds of essential amino acids.
The result as shown in Figure 3.Both have the effect that promotes the 707fl. cell proliferation to have observed AAM2 and AAM3.The result who uses aged mouse to obtain among above-mentioned result and the embodiment 1 is consistent, shows that the supposition that the evaluation system of this uses 707fl. cell is bred facilitation effect for aminoacid to erythrocyte stem cells is effective.
Determine as the aminoacid that the propagation of erythrocyte stem cells is promoted active main body
Will be from confirming that among embodiment 2 having propagation to promote to remove among the active essential amino acids mixture AAM3 mixture that 2 to 3 seed amino acids obtain to the class protoerythrocyte was dissolved into as cell culture containing in the 5% hyclone RPMI1640 culture medium with culture medium., remove amino acid whose being combined as herein, branched-chain amino acid (Val, Leu, Ile), basic amino acid (His, Lys), aromatic amino acid (Trp, Phe), other aminoacid (Met, Thr).And, the concentration that contains in the concentration of every seed amino acid and 1% solution as the AAM3 on basis is equated.Each Freamine is injected 96 hole microtest plates respectively with the series of 2 times of dilutions, every hole 50 μ l.To wherein adding the suitably cell (about 1 * 10 of dilution
4Cell) 50 μ l cultivated 3 days in CO2 gas incubator.In each hole (well), add the PremixWST-1 reagent of 10 μ l subsequently, in CO2 gas incubator, hatched 1～4 hour, and under A450nm (reference A655nm), measure and confirm cell proliferation.
The result as shown in Figure 4.From AAM3, remove in each amino acid whose mixture at AAM3 and great majority, in concentration is 0.004%～0.008% scope, all confirmed to promote active the propagation of 707fl. cell.Yet, when removing Met and Thr, but not observe cell proliferation and promote activity, this demonstration promotes that to the erythroblastic propagation of class active main body probably is Met or Thr.
At this, use and above-mentioned same method have further studied Met and Thr promotes active to the erythroblastic propagation of class.
The result as shown in Figure 5.It is active to have confirmed that Met promotes the erythroblastic propagation of class, but the propagation that does not but confirm Thr promotes activity, and this shows that propagation promotes that active major part depends on Met.In addition, infer that Met promotes that to the erythroblastic propagation of class active optimum concentration is about 1～5 μ g/ml.On the one hand, observed AAM3 near 0.005% (W/V) the erythroblastic propagation of class is promoted activity, the Met amount that contains this moment is whole 3.3% (W/V)=1.65 μ g/ml and current basically identical as a result.
At this, also have report to claim if the Met hyperphagia then can cause the side effect of hemolytic anemia.In addition, also there is report to claim that this phenomenon can be suppressed by aminoacid such as Gly, Ser, Thr.Can infer the erythroblastic cell proliferation facilitation effect of class from above-mentioned phenomenon and Met: under the situation that Met excessively absorbs, exceedingly promote the propagation of erythrocyte stem cells in the hemopoietic tissue, promote haemolysis and induce anemia thereby produce incomplete erythrocyte.
In addition, think and to suppress because the reason of the inductive hemolytic anemia of Met hyperphagia is: add as the auxiliary element in hemopoietic and effectively as material necessity of biological component amino acid whose by aminoacid such as Gly.That is to say, think that the interpolation of following material in the cell proliferation of Met promotes to induce is the auxiliary important document of hemopoietic: essential glutathion in as erythrocyte, as erythrocyte in the biological component Gly of material of haemachrome and succinyl CoA and as the biological component branched-chain amino acid (BCAA) of the material of succinyl CoA with as the Ser of the synthetic material of Met, Gly, as the aminoacid that relates to the material of citric acid circulation biological component (this is supply from the citric acid circulation owing to succinyl CoA).Think that amino acid whose participation is not only as being used for the material of the synthetic biological component of erythrocyte, and for example Gly and succinyl CoA on amount evenly picked-up also have effect.In addition, Gly also is synthetic, the glutathion of necessary nucleic acid in the cell division and the biosynthetic material of creatine.
Aminoacid is to the propagation facilitation of the blood cell line cell that stems from the people
People's cell has been studied similarly the effect of the cell proliferation promotion of Met.People's cell is selected the K562 cell that stems from chronic lymphocytic leukemia (erythroleukemia) for use, and (two kinds all is cells of buying ECACC (EuropeanCollection of Cell Cultures) type strain with stemming from the leukemic HEL92.1.7 cell of protoerythrocyte property.)。No matter which kind of cell all has report to claim to be divided into erythrocyte.When measuring beginning, in 96 hole microtest plates with about 1 * 10
4Cells/well is sowed cell, and adds the ispol of various concentration (0.5%～0.008% scope) therein.At this moment, the pH that adjusts ispol adds near neutral afterwards.After 3～4 days, use the WST-1 algoscopy to confirm the propagation of cell cell culture in this state.
The result as shown in Figure 6 and Figure 7.Same with the 707fl. cell, confirmed that ispol also has the propagation facilitation effect to K562 cell and HEL92.1.7 cell.And, compare with AAM2 as the AAM3 of essential amino acids mixture, confirmed the effect that on cell proliferation is higher.From above-mentioned research as can be known, the people is the same with mice, and hemopoietic has demonstrated the effect that promotes to essential amino acids to erythrocyte.
The Met amount essential to the 707fl. cell proliferation
The influence of the concentration of the necessary methionine of research 707fl. cell proliferation.
Add hyclone (contain RPMI1640 culture medium that 5% hyclone do not contain Met) with 5% final concentration in the culture medium of from RPMI1640, removing methionine (the RPMI1640 culture medium that does not contain Met) in (Funakoshi) as minimal medium.In addition, also with containing the RPMI1640 culture medium preparation 100mM Met solution that 5% hyclone does not contain Met.2 times of dilution series of the above-mentioned Met solution of preparation are injected 96 hole microtest plates respectively with 50 μ l/ holes in containing the RPMI1640 culture medium that 5% hyclone do not contain Met.And with the 707fl. cell with contain RPMI1640 culture medium that 5% hyclone do not contain Met carry out muddy outstanding so that its concentration is 2 * 10
5Cell/ml, and injection has added the hole of the Met dilution series in 50 μ l/ holes.In CO2 gas incubator, cultivate after 3 days, use the WST-1 algoscopy to determine the situation of cell proliferation.
The result as shown in Figure 8.Confirmed cell proliferation facilitation effect when the concentration of Met is 25 μ M～50000 μ M, and in the concentration of 50 μ M～200 μ M, confirmed the highest cell proliferation facilitation effect the 707fl. cell.
Embodiment 6
The amino acid whose research of the cell inhibitory effect effect that mitigation high concentration Met causes
Shown in embodiment 3, high concentration Met compares with the Met concentration that is suitable for most cell proliferation, suppresses the propagation of 707fl. cell.Therefore, relax this inhibitory action, carried out relaxing the amino acid whose research of the proliferation inhibition activity that high concentration Met causes in order to identify.Each seed amino acid as supplying to test uses (first) Thr or (second) Gly and Glu, mixes with identical weight with Met respectively to prepare.Each ispol is dissolved in as the RPMI1640 culture medium that contain 5% hyclone of cell culture with culture medium, and its dilution series is injected 96 hole microtest plates with 50 μ l respectively.Add the cell (about 5 * 10 that 50 μ l have suitably diluted therein
4Cell/50 μ l), in CO2 gas incubator, cultivated 3 days.Subsequently, in each hole, add the WST-1 reagent of 10 μ l, continued to hatch 2～4 hours, under A450nm (reference A655nm), measure, and compare cell proliferation.
The result as shown in Figure 9.Met detects the cell inhibitory effect effect when surpassing the concentration of 12.5 μ g/ml.When Thr and Met coexist with same concentrations, can be more than 12.5 μ g/ml, delay inhibitory action in the scope of less than 25 μ g/ml.Relative therewith, when Gly and Glu coexist with same concentrations with Met respectively, confirm inhibition to the proliferation inhibiting effect that Met produced more than the 50 μ g/ml, shown better effect than Thr.
Embodiment 7
The anticarcinogen actinomycin D is induced the research of the effectiveness of Met in the anemia model
[method]
The mice of buying was tamed 10.From tail vein blood 10 μ l, measure hemoglobin (Hb) amount the 7th day of domestication.And the mensuration body weight, divide into groups according to Hb amount and body weight.After the grouping, the intraperitoneal that mice is carried out 2 continuous actinomycin D of 5 days gives, each between 2 days at interval.Dosage is half amount that gives 5 days LD50 in the mouse peritoneal continuously, i.e. 0.07mg/kg body weight/day.And actinomycin D with physiological saline solution after, give with 100 μ l/ individualities.And the normal saline that will not contain actinomycin D carries out intraperitoneal according to the dosage of 100 μ l/ individualities and gives, as negative control group.
When experiment finishes, force orally give amino acid solution or aquesterilisa when beginning to give actinomycin D.The concentration of Met aqueous solution is 0.2%, 1%, 5% aqueous solution.With aquesterilisa as negative control.In addition, with the negative control of 1%Thr aqueous solution as the amino acid solution relevant with the hemopoietic effect.No matter be amino acid solution or aquesterilisa, dosage is 200 μ l/ individuality/skies.
Duration of test carries out the blood sampling of 10 μ l with suitable frequency to the tail vein, and measures the Hb amount.
After the above-mentioned off-test, mice executions of taking a blood sample entirely under anesthesia, and collection spleen and myeloid tissue are carried out gene analysis.
It should be noted that the formation of group (respectively organizing 10) is as described below.
The 1st group: the normal saline intraperitoneal gives, aquesterilisa orally give group.
The 2nd group: intraperitoneal gives actinomycin D (0.07mg/kg body weight/day), aquesterilisa orally give group.
The 3rd group: intraperitoneal gives actinomycin D (0.07mg/kg body weight/day), 1%Thr aqueous solution orally give group.
The 4th group: intraperitoneal gives actinomycin D (0.07mg/kg body weight/day), 0.2%Met aqueous solution orally give group.
The 5th group: intraperitoneal gives actinomycin D (0.07mg/kg body weight/day), 1.0%Met aqueous solution orally give group.
The 6th group: intraperitoneal gives actinomycin D (0.07mg/kg body weight/day), 5.0%Met aqueous solution orally give group.
[result]
The change result of the peripheral blood Hb amount of measuring in the duration of test as shown in figure 10.Begin the maximum that the Hb amount reduces after 17 days.The peripheral blood Hb that begins after 17 days measures as shown in figure 11.At this moment, confirmed that the 1%Thr aqueous solution organizes and give not have significant difference between the aquesterilisa group.Relative therewith, confirm the Hb amount rising trend in the 0.2%Met aqueous solution is organized, and 1% and the 5%Met aqueous solution have significantly rising in organize.
Can reaffirm that by this test Met has inhibition or improves effect the anemia of following bone marrow function to suppress.In order to suppress or to improve the anemia of following bone marrow function to suppress, now clear and definite Met concentration is that 1% orally give has effect of sufficient, but Met concentration is also can expect effect at 0.2% o'clock.The dosage of 1%Met is about 130mg/50kg individuality/sky converting under people's the situation according to the body surface area conversion.This is the amount that can give fully for the people.Now confirmed the erythrocyte hemopoietic facilitation effect of Met, the peripheral blood Hb that not only in aged mouse, can recover to reduce amount, the anemia that the side effect of anticarcinogen is caused also produces effect.
Embodiment 8
The actinomycin D that gives Met is induced the gene expression quantitative analysis of the spleen and the bone marrow of anemia mice
[method]
Collection supplies in mouse spleen and the myeloid tissue of embodiment 7, and the RNA Later reagent that the Ambion commercial firm of about 10 times of volumes of using-system produces soaks, and leaves standstill a night under 4 ℃.Tissue after handling is stored under-80 ℃ until extracting RNA.From preserve tissue, extract and purifying RNA is to use the RNeasy test kit of qiagen company and carries out according to incidental use rules.The quality of the RNA of the biological analyser that uses Agilent company after to purification is confirmed.And adopt GeneChip, the mice 430_2.0 array of Affymetrix commercial firm and use purified RNA to carry out the little array analysis of DNA.Following research is to by giving the effect (in the mice of the 5th group of embodiment 7) that mice that actinomycin D induces anemia gives 1%Met: the gene expression amount of more above-mentioned mice and induce anemia and give the gene expression amount of mice (confession is in the mice of the 2nd group of embodiment 7) of the contrast of organizing as aminoacid of aquesterilisa by actinomycin D.
[result]
The erythrocyte related gene that the expression that is caused by 1%Met is risen is as shown in table 3.It should be noted that, in table 3, use numeric representation with respect to the relative expression quantity of each gene expression amount that actinomycin D is induced anemia and given the mice of aquesterilisa.
Compare with the mice that gives aquesterilisa, rise as the spleen of the erythrocyte hemopoietic tissue of mice and most number average of the erythrocyte related gene in the bone marrow.This result shows that the erythrocytic differentiation that causes of Met obtains promoting.In addition, (another name CD117 transmembrane tyrosine kinase is expressed in the hemopoietic progenitor cell of colony forming cell etc., but is not expressed in the CFU-GM of B cell line the c-kit that the gene that express to rise is expressed involving from bone marrow stem cell to CFU-E.The interaction partners hemopoietic of CD117 and part is very important) and part (stem cell factor: also claim SCF) and even expression increases in the differential period after CFU-E TfR (CD71), haeme synthetase and erythrocyte membrane protein matter.Therefore, the effect of Met be present in decision erythrocyte differentiation from the unipotent stem cell to the erythrocyte in this broad range.
[table 3]
The actinomycin D that Met gives is induced the erythrocyte related gene that rises in the mice of anemia
| The gene bank searching number | Describe | Spleen | Bone marrow | |
| ??NM_011638 | TfR | ??3.5 | ??2.6 | |
| ??NM_010369 | Alpha-Glycophorins | ??1.9 | ??1.7 | |
| ??BE307351 | ??CD36 | ??2.0 | ??1.9 | |
| ??BB333334 | The Kit proto-oncogene | ??1.0 | ??1.5 | |
| ??BB815530 | The Kit part | ??3.0 | ??1.7 | |
| ??AY033898 | The eIF2 alpha kinase that haemachrome is regulated, the synthetic translation of hemoglobin is regulated | ??2.3 | ??1.5 | |
| ??NM_007998 | Ferrochelatase | ??2.8 | ??1.5 | |
| ??NM_013631 | Pyruvate kinase, liver and erythrocyte | ??1.0 | ??1.6 | |
| ??NM_007563 | 2, the 3-diphosphoglycerate mutase | ??0.7 | ??1.3 | |
| ?? | Carbonic anhydrase | 1 | ??1.0 | ??1.6 |
| ?? | Carbonic anhydrase | 2 | ??1.0 | ??1.1 |
| ?? | Carbonic anhydrase | 3 | ??3.2 | ??2.5 |
| ??U03487 | Erythrocyte membrane protein 4.2 | ??3.2 | ??2.6 | |
| ??BB533969 | Erythrocyte membrane protein 4.1 | ??1.0 | ??1.9 | |
| ??U87455 | Erythrocyte α spectrin | ??1.3 | ??1.2 | |
| ??AW912678 | Spectrin SH3 territory conjugated |
??3.2 | ??1.5 |
Embodiment 9
Relation between erythroblastic differentiation of the concentration of Met and class and the propagation facilitation effect
Met concentration in mice or the human plasma is about about 30 μ M.In addition, in the bone marrow as main hemopoietic tissue, the Met concentration in its tissue fluid is (＜3 μ M) under detectability.So think, and compare under the external cell culture condition, the extracellular Met concentration in the hemopoietic tissue of organism is much smaller usually.And can be clear and definite from Fig. 8 of front, Met demonstrates the propagation facilitation effect to the class protoerythrocyte in 25 μ M～50000 μ M scopes.
Therefore, the 707fl. gene expression of cells under following two kinds of situations is compared research: exist to the class protoerythrocyte show the propagation facilitation effect 150 μ M concentration Met situation and with the bone marrow fluid non-existent substantially situation of Met similarly.
[method]
The hyclone that adds ultimate density and be 5% concentration in not containing the RPMI1640 culture medium of Met is prepared culture medium (containing the RPMI1640 culture medium that 5% hyclone does not contain Met).And be formulated in the culture medium of having added Met in the above-mentioned culture medium, so that Met is various concentration (containing the RPMI1640 culture medium that each concentration Met contains hyclone).
The 707fl. cell was cultivated 3 in containing the RPMI1640 culture medium that 30 μ M Met contain 5% hyclone.To cultivate the back cell and be divided into 2 parts and collect, a use contains the RPMI1640 culture medium that 5% hyclone do not contain Met and carries out suspendible again, and making its concentration is about 5 * 10
5Cell/ml.Added 5% hyclone because contain the RPMI1640 culture medium that hyclone do not contain Met, so contain the Met that stems from serum that has an appointment about 1～3 μ M herein.The Met that another part usefulness contains 150 μ M contains the RPMI1640 culture medium of 5% hyclone and carries out suspendible again, and making its concentration is about 5 * 10
5Cell/ml.Then the cell under each condition is divided into 3 parts, cultivate 24 hours respectively after, a part of getting cell culture fluid is determined the degree of cell proliferation with the WST-1 algoscopy.
[result]
With contain in the RPMI1640 culture medium that 5% hyclone do not contain Met cultured cells (Met of＞3 μ M) and compare, the cell number that contains cultured cells (150 μ M Met) in the RPMI1640 culture medium that 150 μ M Met contain 5% hyclone presents significant increase (Figure 12).Then from these cells, extract RNA, use PCR in real time (polymerase chain reaction) to come erythrocyte correlating markings expression of gene amount is compared research.As erythrocyte differentiation sign, measure the gene expression amount of alpha-Glycophorins and ferrochelatase, described alpha-Glycophorins is abundant and a protein that specificity exists in erythrocytic cell is membranaceous, and described ferrochelatase is that the haemachrome biosynthesis is a kind of of rate-limiting enzyme in the enzyme.And, use β actin gene in contrast this moment.Consequently, with contain in the RPMI1640 culture medium that 150 μ M Met contain 5% hyclone cultured cells (150 μ M Met) and compare, two expression of gene that contain cultured cells (＞3 μ M Met) in the RPMI1640 culture medium that 5% hyclone do not contain Met significantly raise (Figure 13).So the result shows, under the situation that the Met higher than common concentration exists, than the differentiation of erythrocyte stem cells, more promotes the propagation of erythrocyte stem cells; And in contrast, under the situation that the low Met of concentration exists,, more promote differentiation than propagation.According to The above results as can be known, there is the function of experiencing of experiencing Met concentration in the erythrocyte stem cells, can controls propagation and differentiation dexterously according to extracellular Met concentration.
Along with the relation between erythroblastic differentiation of the class of Met concentration change and the propagation facilitation effect
[method]
In containing the RPMI1640 culture medium that 5% hyclone do not contain Met, add Met, making its concentration is 150 μ M, and to continue to wherein adding final concentration be that 1% dimethyl sulfoxide (DMSO) prepares culture medium (contain 150 μ M Met and contain the RPMI1640 culture medium that 5% hyclone contains 1%DMSO).
Use contains RPMI1640 culture medium (30 μ M) that 30 μ M Met contain 5% hyclone the 707fl. cell is divided into the 1st～5 5 groups, cultivates 3 days.Subsequently, exchange to the RPMI1640 culture medium (30 μ M) that contains 30 μ M Met and contain 5% hyclone once more with the 1st group, respectively the 2-4 group is exchanged to the RPMI1640 culture medium (150 μ M) that contains 150 μ M Met and contain 5% hyclone respectively, the 5th group exchanged to contain 50 μ M Met and contain the RPMI1640 culture medium (150 μ M+1%DMSO) that 5% hyclone contains 1%DMSO, and cultivated 24 hours.
Exchange to the RPMI1640 culture medium (30 μ M) that contains 30 μ M Met and contain 5% hyclone with the 1st and the 3rd group respectively subsequently, exchange to the RPMI1640 culture medium (150 μ M) that contains 150 μ M Met and contain 5% hyclone with the 2nd group, exchange to the RPMI1640 culture medium (＞3 μ M) that contains 5% hyclone and do not contain Met with the 4th group, exchange to the Met that contains 150 μ M with the 5th group and contain the RPMI1640 culture medium (150 μ M+1%DMSO) that 5% hyclone contains 1%DMSO, and cultivated 24 hours.Herein, 1%DMSO adds as the relevant positive control of differentiation.After cultivating end, from each group cell, extract RNA, use PCR in real time comparing research as the alpha-Glycophorins of erythrocyte correlating markings gene and the gene expression amount of ferrochelatase.At this moment, gene uses the β actin in contrast.
[result]
Be to continue cultured cells under the 30 μ M to compare in Met concentration, be the expression decreased of erythrocyte differentiation associated gene in the cultured cells under the 150 μ M in Met concentration, so think:, more promote to breed than differentiation.Relative therewith, compare with continuing to use 30 μ M Met culture medium or exchange to the situation that contains 30 μ M Met culture medium by the culture medium that contains 150 μ M Met, if make the concentration of Met be reduced to less than 3 μ M, then the expression of differentiation associated gene is risen; Compare with the situation of cultivating in the presence of 1%DMSO, if make the concentration of Met be reduced to less than 3 μ M, then the expression of differentiation associated gene reaches essentially identical level (Figure 14, Figure 15).These results show, because acute variation has taken place the concentration of Met, in the erythroblastic 707fl. cell as class, have carried out from cell proliferation to induced differentiation efficiently.Therefore, Met can regulate and control the propagation and the differentiation of erythrocyte stem cells efficiently.
Utilize possibility on the industry
The invention provides senile anemia prophylactic and senile anemia therapeutic agent, the possibility of use is arranged at pharmaceuticals manufacturing industry and food manufacturing.
This description is directly introduced in whole publications, patent or the patent application of quoting in this description as a reference.
Claims (10)
1. the differentiation accelerator of erythrocyte stem cells and/or enhancer of proliferation, it contains Met as effective ingredient.
2. the differentiation accelerator of the erythrocyte stem cells of claim 1 and/or enhancer of proliferation, it further contains Glu and Gly.
3. the differentiation accelerator of the erythrocyte stem cells of claim 1 and/or enhancer of proliferation, it further contains the ispol that is selected from following (1)～(3)
(1) Ile, Thr, Val, His, Lys and Leu
(2) Trp, Thr, His, Phe and Lys
(3) Ile, Trp, Thr, Val, Phe and Leu.
4. diet product, it contains the differentiation accelerator and/or the enhancer of proliferation of each erythrocyte stem cells among the claim 1-3.
5. anemia preventive or treatment for anemia agent, it contains the differentiation accelerator and/or the enhancer of proliferation of each erythrocyte stem cells among the claim 1-3.
6. senile anemia preventive or senile anemia therapeutic agent, it contains the differentiation accelerator and/or the enhancer of proliferation of each erythrocyte stem cells among the claim 1-3.
7. cell senescence preventive, it contains Met as effective ingredient.
8. the cell senescence preventive of claim 7, it further contains Glu and Gly.
9. the cell senescence preventive of claim 7, it further contains the ispol that is selected from following (1)～(3)
(1) Ile, Thr, Val, His, Lys and Leu
(2) Trp, Thr, His, Phe and Lys
(3) Ile, Trp, Thr, Val, Phe and Leu.
10. diet product, it contains among the claim 7-9 each cell senescence preventive.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2007300814 | 2007-11-20 | ||
| JP2007-300814 | 2007-11-20 | ||
| PCT/JP2008/070042 WO2009066562A1 (en) | 2007-11-20 | 2008-11-04 | Differentiation promoter and/or proliferation promoter for erythrocyte stem cells, use of methionine for preventing or treating senile anemia, and compositions containing methionine |
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| CN101868230A true CN101868230A (en) | 2010-10-20 |
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| KR (1) | KR20100098400A (en) |
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| JPWO2012147901A1 (en) * | 2011-04-28 | 2014-07-28 | 味の素株式会社 | Anticancer drug side effect improving composition |
| JP6851060B2 (en) * | 2016-09-29 | 2021-03-31 | 日本メナード化粧品株式会社 | Hematopoietic stem cell differentiation promoter |
| EP4349344A1 (en) * | 2021-05-25 | 2024-04-10 | Ajinomoto Co., Inc. | Composition for improving or preventing iron deficiency anaemia |
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| EP0891719A1 (en) * | 1997-07-14 | 1999-01-20 | N.V. Nutricia | Nutritional composition containing methionine |
| IL157367A0 (en) * | 2001-03-09 | 2004-02-19 | Nestle Sa | Composition improving age-related physiological deficits and increasing longevity |
| JP2003155234A (en) * | 2001-11-20 | 2003-05-27 | Fancl Corp | Antiaging composition |
| JP2005198642A (en) * | 2003-12-15 | 2005-07-28 | Yutaka Miyauchi | Liquid drinking product using fruit skin of citrus fruit as raw material |
| WO2007029730A1 (en) * | 2005-09-06 | 2007-03-15 | Meiji Dairies Corporation | Amino acid composition for prevention or treatment of senile anemia |
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