CN1935986A - Method for differentiating bone cells by oriented inducing chick embryo stem spermatogonium - Google Patents
Method for differentiating bone cells by oriented inducing chick embryo stem spermatogonium Download PDFInfo
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- CN1935986A CN1935986A CNA2006100966313A CN200610096631A CN1935986A CN 1935986 A CN1935986 A CN 1935986A CN A2006100966313 A CNA2006100966313 A CN A2006100966313A CN 200610096631 A CN200610096631 A CN 200610096631A CN 1935986 A CN1935986 A CN 1935986A
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Abstract
The invention discloses the method used to directionally induce chicken embryo stem spermatogonium to differentiate into bone cell. Its features are that inoculating the chicken stem spermatogonium to culture bottle without feeder layer; using conditioned medium to induce culturing which is that the DMEM high glucose contains 10% calf blood serum, 2% chicken blood serum, 2mmol/L L-glutamine, 1mmol/L sodium pyruvate, 5.5*10-15ng/ml beta-mercaptoethanol, 10 mul/ml non-essential amino acid, 10-7mol/L dexamethasone, 0.01mol/L beta-phospho glycerol, 0.05g/L vitamin C. The invention has the advantages of advanced technology, simple operation, strong repeatability, providing possible for realizing tissue organ rebirthing and replacing. Thereby hopefully to find out the approach for build cell platform in large scale production, further a new study prospect can be exploited in developmental biology, medicine, genetics fields etc.
Description
Technical field
The present invention relates to chicken embryo stem spermatogonium and be divided into osteoblastic method, belong to cell engineering
Technical field.
Background technology
The former stem cell of chickens' extract (SSCs) has the versatility of differentiation, adds the suitable differentiation agent of inducing and can induce needed specific type cell and tissue in the vitro culture process, thereby caused people's very big concern.Present people are induced to differentiate into osteoblastic research to stem cell and are limited to mammal (rat, mouse) more, Gopalakrish.V etc. utilize mouse bone marrow stroma stem cell (BMSCs) to induce to be scleroblast, and Deliloglu-gurhan is from the inherent mechanism of molecular level research BMSCs induced osteogenesis cell; Zhang Weicheng, Ding Lianghua etc. utilize the thin induced osteogenesis cell of rat and people's marrow mesenchymal stem respectively, have all obtained success.
But the research to poultry SSCs also is in the starting stage at present, the research of chicken embryo SSCs is more common in separation method, is extracted purifying, obtain the former stem cell of chickens' extract, various kinds of cell factor isolated culture, aspects such as freezing preservation of SSCs and SSCs or testis tissue transplanting, and its external evoked Research on ability that is divided into various kinds of cell is not seen that as yet report is arranged.This experiment purpose is intended to study the ability of chicken embryo SSCs directional induction in vitro to osteoblast differentiation, for clinical value and the versatility of exploring stem spermatogonium (SSCs) provides material.
Summary of the invention
The purpose of this invention is to provide a kind of directional induction chicken embryo stem spermatogonium and be divided into osteoblastic method, with the extract former stem cell of chickens' extract that obtains, by research the former stem cell of chickens' extract is carried out external evoked cultivation, obtain the method for effective induced osteogenesis cell, for inducing the back that it is proceeded the amplification in vitro cultivation and is applied to clinical experiment, make it become a kind of proven technique.
Technical scheme of the present invention is: a kind of directional induction chicken embryo stem spermatogonium is divided into osteoblastic method, it is characterized in that the extract former stem cell of chickens' extract that obtains is inoculated in the no feeder layer culturing bottle, utilize conditioned medium to carry out inducing culture, described nutrient solution is: contain 10% calf serum, 2% chicken serum, 2mmol/L L-glutaminate, 1mmol/L Sodium.alpha.-ketopropionate, 5.5 * 10 in the DMEM high glucose medium
-5Mol/L beta-mercaptoethanol, 10 μ l/ml non-essential amino acid, 10
-7Mol/L dexamethasone, 0.01mol/L β-phospho-glycerol sodium, 0.05g/L vitamins C.
Technology of the present invention is advanced simple, need not special plant and instrument, and the elementary operation rule of a GPRS cell cultures is simple and easy to do.The versatility that has differentiation potential by the research stem cell provides material for clinical value and the versatility of exploring stem spermatogonium (SSCs), scleroblast after inducing is carried out external continuation amplification cultivation and is applied to clinical experiment, make it become a kind of proven technique, implement cell replacement therapy, realize that for final the regeneration and the replacement of histoorgan provide possibility, and wish whereby to find an approach of setting up the required cell platform of scale operation, and then be developmental biology, medical science, brand-new research prospect of genetics field developing.Induction method of the present invention also be applicable to other bird stem spermatogonium induce research, and workable.
Description of drawings
Fig. 1, Fig. 2 are the scleroblast picture after ALP dyeing;
Fig. 3, Fig. 4 are the scleroblast picture after Von kossa ' s dyeing;
Fig. 5, Fig. 6 are the scleroblast picture after immunohistochemical methods (type i collagen) dyeing.
Embodiment
Step 1: the isolation and purification of chicken embryo SSCs
Get 16-19 days Embryo Gallus domesticus of hatching, the aseptic testis Gallus domesticu that obtains.Digest through collagenase (1mg/ml), back trypsin 0.25%) digestion, to obtain SSCs, sustenticular cell and the mesenchymal cell etc. on the seminiferous epithelium, after the filtration of 350 order filter sieve, percoll gradient centrifugation, adherent velocity contrast, to obtain the stem spermatogonium of higher degree, use the trypan blue dyeing counting, the adjustment cell density is 1x10
5Individual/ml, be inoculated in 6 well culture plates, place 38 ℃, 5%CO
2Cultivate in the incubator.
Step 2: the in-vitro inducing of chicken embryo SSCs is cultivated
SSCs cultivated after 3-4 days, with condition induced liquid (basic medium+10 of preheating
-7Mol/L dexamethasone+0.01mol/L β-phospho-glycerol sodium+0.05g/L vitamins C) carries out half amount and change liquid.Observed in per 24 hours later on, full dose was changed liquid in per 72 hours.Randomly draw 10 non-overlapped visuals field (* 100) during observation, be calculated to be the ratio that osteocyte accounts for the SSCs cell count.(table 1)
Step 3: alkaline phosphatase (ALP) improvement calcium cobalt method
Chicken SSCs is cultivated about 3 weeks at the conditions in vitro inductor, discard inductor, put in the stationary liquid distilled water flushing 3 times fixedly 8-10 minute.Put in the matrix liquid, hatched 4-6 hour for 37 ℃.Take out and drip several Xiao Suangus, act on 5-7 minute, the PBS flushing.Drip 20g/l ammonium sulfide number droplet, act on 5-8 minute, PBS washes.Redyed 5 minutes in Yihong, and microscopy is dried in flushing.
Alkaline phosphatase (ALP) improvement calcium cobalt method qualification result: examine under a microscope the scleroblast after ALP dyeing, chocolate particle or block precipitation are arranged in its cell cytosol, i.e. ALP dyeing is positive.Observe and to send out existing cytogamy and become big sheet, the intercellular substance is difficult to differentiation, also has manyly to exist with the individual cells form, and nucleus clearly (is seen Fig. 1,2).
Step 4: calcium tubercle Von Kossa ' s method
Chicken SSCs is cultivated about 3 weeks at the conditions in vitro inductor, discard inductor, the PBS flushing, in 95% ethanol fixedly 8-10 minute, PBS flushing, 5% Silver Nitrate normal temperature lucifuge 15 minutes, uviolizing 15-20 minute, distilled water flushing, in 5% Sulfothiorine 2-3 minute, distilled water flushing, eosin stain was redyed 3-5 minute, microscopy is dried in flushing.
Calcium tubercle Von Kossa ' s method is identified the scleroblast result: examine under a microscope the scleroblast after Vonkossa ' s dyeing, can clearly see that the calcium deposition zone is that tubercle is black, its black precipitate is distributed in the formation matrix of cell (seeing Fig. 3,4).
Step 5: immunohistochemical methods type i collagen (Collagen I) is identified
To induce the scleroblast in 4 weeks to discard induced liquid, with stationary liquid (cold acetone: dehydrated alcohol=3: 2) fix 1 hour, the PBS flushing, added peroxidase blocking-up liquid blocking-up endogenous peroxydase 8-10 minute, added the 5%BSA confining liquid 15-20 minute, and added Collagen I monoclonal antibody, 37 ℃ of water-baths 1 hour, the PBS flushing, add two anti-(biotin labelings), incubated at room 15-20 minute, distilled water flushing, add streptavidin-avidin-biotin complex (SABC) solution, incubated at room 15-20 minute, flushing, DAB colour developing, dry microscopy.
Immunohistochemical methods type i collagen (Collagen I) is identified the reef knot fruit: the cell after the directional induction is after the dyeing of SABC method, and cell is brown, shows that chicken embryo SSCs directed differentiation is a scleroblast, and the secretion type i collagen, and (seeing Fig. 5,6) is positive.
Table 1 SSCs is induced to differentiate into the scleroblast result
Table 1 The differentiation rate of induction of osteoblasts (n＝10)
| Experiment group Groups | Developmental stage Stages | SSCs cell count Number of SSCs | Scleroblast is counted Number of osteoblasts | Differentiation rate Differentiation rate |
| Experimental group Test Group control group Control Group | 16-19d 16-19d | 32.30±2.71 31.25±1.86 | 26.51±3.02 0.55±0.63 | 0.82±0.04 a 0.011±0.02 b |
Annotate: alphabetical d represents to hatch fate, represents difference extremely significantly (P＜0.01) between the alphabetical ab.
Claims (1)
1, a kind of directional induction chicken embryo stem spermatogonium is divided into osteoblastic method, it is characterized in that the extract former stem cell of chickens' extract that obtains is inoculated in the no feeder layer culturing bottle, utilize conditioned medium to carry out inducing culture, described nutrient solution is: contain 10% calf serum, 2% chicken serum, 2mmol/L L-glutaminate, 1mmol/L Sodium.alpha.-ketopropionate, 5.5 * 10 in the DMEM high glucose medium
-5Mol/L beta-mercaptoethanol, 10 μ l/ml non-essential amino acid, 10
-7Mol/L dexamethasone, 0.01mol/L β-phospho-glycerol sodium, 0.05g/L vitamins C.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182269A (en) * | 2018-07-26 | 2019-01-11 | 佛山科学技术学院 | A kind of cultivating system and method making the efficient differentiating into nerve cells of stem spermatogonium |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182269A (en) * | 2018-07-26 | 2019-01-11 | 佛山科学技术学院 | A kind of cultivating system and method making the efficient differentiating into nerve cells of stem spermatogonium |
| CN109182269B (en) * | 2018-07-26 | 2022-07-05 | 佛山科学技术学院 | Culture system and method for efficiently differentiating spermatogonial stem cells to nerve cells |
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Open date: 20070328 |