CN1935987B - Method for differentiating osteoplastic cells by oriented inducing chick embryo primordial germ cell - Google Patents
Method for differentiating osteoplastic cells by oriented inducing chick embryo primordial germ cell Download PDFInfo
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- CN1935987B CN1935987B CN2006100966328A CN200610096632A CN1935987B CN 1935987 B CN1935987 B CN 1935987B CN 2006100966328 A CN2006100966328 A CN 2006100966328A CN 200610096632 A CN200610096632 A CN 200610096632A CN 1935987 B CN1935987 B CN 1935987B
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Abstract
The invention discloses the method used to directionally induce chick embryo primordial germ cell to differentiate into bone cell in vitro. It uses the inducer formed by 90% DMEM, 10% FBS, 5.5*10-5 beta-mercaptoethanol, 1*10-7mol/L dexamethasone, 0.01mol/L beta-sodium glycerophosphate, 0.05g/L vitamin C to process induction differentiating culturing in vitro for chick embryo primordial germ to differentiate into bone cell finally. It can build cell model for clinical medicine study, widely apply in tissue engineering field for providing reliable experience data, supply new source for cell substituting therapy and tissue therapy, even organ transplantation. The invention has the advantages of simple technology, convenient operation, strong repeatability, no need for special apparatus, onlyneed to master universal cell separating and culturing operation.
Description
Technical field
The present invention relates to a kind ofly the chick embryo primordial sexual cell is carried out the directional induction differentiation culture can obtain osteoblastic method, belong to therapeutic cloning, cell engineering, tissue engineering technique field external.
Background technology
(Embryonic Primordial germ cells is the cell of the primitive form of embryonic germ pedigree EPGCs) to embryo's archeocyte, is the ancester cell of sperm or ovum.EPGCs is as a kind of pluripotent stem cell, and is the same with the ES cell, can be divided into the cell of other types under external suitable condition, is one of the most promising seed cell in clinical medicine and the Tissue Engineering Study.Martin etc. with bone marrow stroma stem cell at the external scleroblast of successfully inducing into; Holtor etc. have studied the mouse bone marrow cells mescenchymal stem cell and have induced the effect of dexamethasone in the process of differentiation to scleroblast, prove that to adopt streaming perfusion culture method under the situation of dexamethasone also can directed differentiation be scleroblast lacking; Yarram etc. have analyzed Urogastron and the synergy of calcitriol in the induced osteogenesis cell processes.Ding Lianghua etc. adopt dexamethasone, sodium, vitamins C combined induction, and successfully will reach triple-substituted adult bone marrow stroma stem cell directed differentiation is scleroblast.At present, as a kind of important embryo's derived stem cells, aspects such as mesenchymal stem cells MSCs, mouse embryo stem cell and marrow stromal cell are more common in the research of its directed differentiation aspect greatly, and report seldom for the correlative study of chicken EPGCs.The differentiation research of embryo's derived stem cells will provide new source for cell replacement therapy and tissue treatment even organ transplantation from now on, repair or substitute because of the vital tissue that heredity or acquired disease cause, the defective and the damage of organ provide new approaches for human perfect, thus its differentiation research in whole stem-cell research in occupation of important position.
Summary of the invention
The purpose of this invention is to provide the method that a kind of strong osteoplastic cells by oriented inducing chick embryo primordial germ cell easy and simple to handle, repeatable breaks up.
Technical scheme of the present invention is: a kind of method of osteoplastic cells by oriented inducing chick embryo primordial germ cell differentiation, it is characterized in that utilizing the scleroblast inductor that the chick embryo primordial sexual cell is carried out external evoked differentiation culture, final directed differentiation is a scleroblast.
Described inductor is by 90%DMEM, 10%FBS, 5.5 * 10
-5Beta-mercaptoethanol, 1 * 10
-7Mol/L dexamethasone, 0.01mol/L sodium, 0.05g/L vitamins C are mixed with.
The described differentiation step of inducing comprises:
1) separation of chick embryo primordial sexual cell, cultivate and go down to posterity:
Adopt independent enzymolysis to extract accumulative EPGCs in the sexual gland in the 19th phase and the 28th phase of chick embryo development from method.Cultivated 5～6 days, and the EPGCs clone was digested to individual cells, be inoculated in the new culturing bottle and cultivate with EDTA-trysinization liquid.Cell was reached for 4 generations;
2) the chick embryo primordial sexual cell breaks up to the scleroblast directional induction:
The chick embryo primordial sexual cell is replaced by the scleroblast inductor with the original base substratum after cultivating 48 hours, changed a not good liquor in per 3 days, to inducing after 21 days row dyeing to identify;
3) scleroblast dyeing is identified:
1. alkaline phosphatase (ALP) dyeing is identified;
2. calcium tubercle Von Kossa ' s dyeing is identified;
3. SABC immunohistochemical methods method detects the evaluation that type i collagen (Collagen I) is expressed.
Technology of the present invention is simple, and is easy and simple to handle, repeatable strong.Need not special instrument requirement, only need to be grasped the general cellular segregation and the operation of cultivation, simple and easy to do.Employing is by 90%DMEM, 10%FBS, 5.5 * 10
-5Beta-mercaptoethanol, 1 * 10
-7The inductor of mol/L dexamethasone, 0.01mol/L sodium, the preparation of 0.05g/L vitamins C, the chick embryo primordial sexual cell carries out directional induction in vitro and is divided into scleroblast, has proved that the chick embryo primordial sexual cell has the character of growing versatility.For clinic study is set up cell model, be widely used in field of tissue engineering technology reliable experimental is provided, for cell replacement therapy and tissue treatment even organ transplantation from now on provides new source, and then realize human perfect vital tissue, the defective of organ and the ideal of damage that causes because of heredity or acquired disease of repairing or substitute.The stem cell that the method applied in the present invention is suitable for other bird and mammal and other type equally carries out the directional induction in vitro differentiation.
Description of drawings
Fig. 1 be the EPGCs osteogenic induction after 21 days the row alkaline phosphatase (ALP) dyeing picture;
Fig. 2, Fig. 3 are EPGCs osteogenic induction row calcium tubercle Von Kossa ' s dyeing picture after 21 days;
Fig. 4 is EPGCs osteogenic induction row SABC immunohistochemical methods method dyeing picture after 21 days;
Embodiment
1, preparation inductor
With 90%DMEM, 10%FBS, 5.5 * 10
-5Beta-mercaptoethanol, 1 * 10
-7Mol/L dexamethasone, 0.01mol/L sodium, 0.05g/L vitamins C preparation inductor.
2, the separation of chick embryo primordial sexual cell, cultivate and go down to posterity
Adopt independent enzymolysis to extract accumulative EPGCs in the sexual gland in the 19th phase and the 28th phase of chick embryo development from method, with the cell inoculation that extracts to the feeder layer that has prepared, with adding 10% foetal calf serum, 2% chicken serum, 2mmol/L L-glutaminate, 1mmol/L Sodium.alpha.-ketopropionate, 5.5 * 10
-5Mol/L beta-mercaptoethanol, 10 μ l/ml non-essential amino acid, 10IU/ml murine leukemia supressor (mLIF), 5ng/ml human stem cell growth (hSCF), 10ng/ml Basic Fibroblast Growth Factor (bFGF), 0.04ng/ml human interleukin-11 (hIL-11), the high sugared DMEM nutrient solution of 10ng/ml rhIGF-1 (hIGF) is cultivated.EPGCs cultivated 5～6 days, with EDTA-trysinization liquid EPGCs clone and feeder layer cells are digested to individual cells together, be inoculated in the new culturing bottle and cultivate, together digest the feeder layer cells of getting off when going down to posterity and be removed, cell was reached for 4 generations by gelatin differential adherent method.
3, the chick embryo primordial sexual cell breaks up to the scleroblast directional induction:
The chick embryo primordial sexual cell is replaced by the scleroblast inductor with the original base substratum after cultivating 48 hours, changed a not good liquor in per 3 days, to inducing after 21 days row dyeing to identify.
4, scleroblast dyeing is identified:
1. alkaline phosphatase (ALP) dyeing is identified: induce that cell adopts improvement calcium cobalt method to measure osteoblastic ALP activity after 21 days, step is: induce the back cell with stationary liquid (cold acetone: dehydrated alcohol=3: 2) fix 1 hour, distilled water flushing 3 times, place matrix liquid, hatched 4～6 hours for 37 ℃.Drip Xiao Suangu, act on 5 minutes, the flowing water flushing.Drip the 20g/L ammonium sulfide, act on 5 minutes, the flowing water flushing, redyed 5 minutes in Yihong.
2. calcium tubercle Von Kossa ' s dyeing is identified: induce after 21 days cell to fix 1 hour with stationary liquid, PBS flushing 3 times, under ultraviolet lamp, contaminated 20～60 minutes with 2% Silver Nitrate, distillation washing 5 minutes, 5% sodium thiosulfate solution was handled 2 minutes, tap water flushing 5 minutes, haematoxylin redyeing, distillation washing 5～10 minutes.
3. SABC immunohistochemical methods method detects the evaluation that type i collagen (Collagen I) is expressed: induce after 21 days cell to fix 1 hour with stationary liquid, PBS washing 3 times, added peroxidase blocking-up liquid blocking-up endogenous peroxydase 10 minutes, added the 5%BSA confining liquid 20 minutes, add the COLI monoclonal antibody, 37 ℃ 1 hour, PBS washing 3 times, add biotin labeled two and resist, incubated at room 20 minutes, washing, add streptavidin-avidin-biotin complex (SABC) solution, incubated at room 20 minutes, washing, DAB colour developing.
1) the 4th generation EPGCs cultivated after 48 hours, under osteogenic induction agent effect, can induce to produce about 80% scleroblast (seeing Table 1).
Table 1 EPGCs is induced to differentiate into osteoblastic effect
Table?1?The?differereniation?rate?of?induction?of?osteoblast (n＝10)
Annotate: the identical person of same column letter represents difference not remarkable (P＞0.05), and the neighbor represents difference extremely significantly (P＜0.01).
2) induce the back cell after the dyeing of improvement calcium cobalt method, have black particle to exist in the experimental group most cells endochylema, chicken EPGCs is described to osteoblast differentiation, ALP dyeing is strong positive (Fig. 1).
3) induce the back cell dyeing after, visible brownish black deposit is brownish black mineralising tubercle in the experimental group extracellular matrix, showing has calcium deposition (Fig. 2,3).
4) induce the back cell after the dyeing of SABC immunohistochemical methods method, the experimental group cell is brown, is positive, and shows that chicken EPGCs directed differentiation is a scleroblast, and secretion type i collagen (Fig. 4).
Claims (1)
1. the method for osteoplastic cells by oriented inducing chick embryo primordial germ cell differentiation, utilize the scleroblast inductor that the chick embryo primordial sexual cell is carried out external evoked differentiation culture, final directed differentiation is a scleroblast, it is characterized in that the described differentiation step of inducing comprises:
1) be mixed with the osteocyte inductor:
With 90%DMEM, 10%FBS, 5.5 * 10
-5Beta-mercaptoethanol, 1 * 10
-7Mol/L dexamethasone, 0.01mol/L sodium, 0.05g/L vitamins C preparation inductor;
2) separation of chick embryo primordial sexual cell, cultivate and go down to posterity:
Adopt independent enzymolysis to extract accumulative chick embryo primordial sexual cell in the sexual gland in the 19th phase and the 28th phase of chick embryo development from method, with the cell inoculation that extracts to the feeder layer that has prepared, with adding 10% foetal calf serum, 2% chicken serum, 2mmol/LL-glutamine, 1mmol/L Sodium.alpha.-ketopropionate, 5.5 * 10
-5The mol/L beta-mercaptoethanol, 10 μ l/ml non-essential amino acid, 10IU/ml murine leukemia supressor, the 5ng/ml human stem cell growth, the 10ng/ml Basic Fibroblast Growth Factor, 0.04ng/ml human interleukin-11, the high sugared DMEM nutrient solution of 10ng/ml rhIGF-1 is cultivated, the chick embryo primordial sexual cell was cultivated 5～6 days, with EDTA-trysinization liquid chick embryo primordial sexual cell clone and feeder layer cells are digested to individual cells together, be inoculated in the new culturing bottle and cultivate, together digest the feeder layer cells of getting off when going down to posterity and be removed, cell was reached for 4 generations by gelatin differential adherent method;
3) the chick embryo primordial sexual cell breaks up to the scleroblast directional induction:
The chick embryo primordial sexual cell is replaced by the scleroblast inductor with the original base substratum after cultivating 48 hours, changed a not good liquor in per 3 days, to inducing after 21 days row dyeing to identify;
4) scleroblast dyeing is identified:
1. alkaline phosphatase staining is identified;
2. calcium tubercle Von Kossa ' s dyeing is identified;
3. SABC immunohistochemical methods method detects the evaluation that type i collagen is expressed.
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