CN1570088A - Neurocyte extract and its uses in mesenchyma stem cell differentiation induction - Google Patents
Neurocyte extract and its uses in mesenchyma stem cell differentiation induction Download PDFInfo
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- CN1570088A CN1570088A CN 200410044418 CN200410044418A CN1570088A CN 1570088 A CN1570088 A CN 1570088A CN 200410044418 CN200410044418 CN 200410044418 CN 200410044418 A CN200410044418 A CN 200410044418A CN 1570088 A CN1570088 A CN 1570088A
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Abstract
The invention relates to the human being or animal nerve cell extracts and the application thereof for inducing and differentiating the mesenchyme to nerve cell in vitro, which provides a new induction reagent for mesenchym induction technology. The preparation steps are: removing blood cell, connective tissue and capsule from nerve cell tissue, conducting cell homogenate, cell disrupting, precipitating, taking supernatant, separating and removing substance with the molecular weight over 200 thousand Da, subpackaging. It can be applied both in the organic combination of nerve cell extractive and the prior part and in the preparation of reagent box. The invention in the application for inducing and differentiating the mesenchym into nerve cell shows a favorable effect, with the induction differentiation rate being over 90%. The inducing differentiated nerve cell can be used for transplantation to treat multiple nerve diseases, having a favorable application prospect in clinical practice
Description
Invention field
The present invention relates to biomass cells extract and utilisation technology, be specifically related to human or animal's neurocyte extract and be induced to differentiate into utilisation technology in the neurocyte at mescenchymal stem cell.
Background technology
Since 1997, stem-cell research makes important progress, discover that human body mescenchymal stem cell (MSC) can be induced to differentiate into the mature cell of nerve, muscle, skin, cartilage, fat, tendon, liver, blood and types of organization, be transplanted in the body after amplification is induced and respective organization cell good integration, bring into play specific biological function, can be used for the multiple disease of transplantation treatment.The prompting of above-mentioned result of study, MSC amplification induce differentiation and neural like cell can be used for nervous system disorderss such as transplantation treatment senile dementia, Parkinson, encephalatrophy, peripheral nerve injury.At present this class disease does not have ideal treatment measure clinically, stem cell and induce differentiation after neural like cell transplant the optimal selection that will become above-mentioned disease treatment.
At present, the artificial neurocyte extract that extracts is a small molecular extract, mainly extracts from tissues such as the parotid gland, is applied to treat nervosa damage aspect with injection system.
In recent years, to inducing MSC to be divided into the used reagent of inducing of neurocyte more research has been arranged, MSC can be at beta-mercaptoethanol, be divided into neurocyte under the inducing of antioxidant such as dimethyl sulfoxide (DMSO) and vitamin A acid, China's microinvasion Neurological Surgery magazine 2004,9 (2) experimental studies report " vitro culture of human marrow mesenchymal stem cell and the research of neuralward cytodifferentiation " conclusion: Sodium Ferulate has the effect of the people MSC neuralward cytodifferentiation of the vitro culture of inducing.Prior art research method and induce reagent to have nothing in common with each other, the effect instability, and do not form standard.
Relevant noun note:
(1) in cell: be the cells of origin of various mature cells, according to the source and the difference of differentiation potential, be divided into embryonic stem cell (embryonic stemcells, ES) and adult stem cell (dult stem cells, ASCs) two classes.The potential amplification is arranged and induce the differentiation performance.
(2) mescenchymal stem cell (mesenchymal stem cells, MSCs is abbreviated as MSC): be that the more ASCs of research extensively is present in the multiple tissue of adult at present, and have multidirectional differentiation potential, can be induced to differentiate into the mature cell of broad variety, difference in functionality in vivo and in vitro.Can reach more than 50 times multiplication and keep the potential of its multidirectional differentiation external.
(3) DMEM and F12 substratum: cell cultures general basic substratum.
(4) ovum extract: extract from human or animal's ovum, the complex mixture that the molecular weight size does not normally wait contains multiple promotion, regulation and control and inducing cell growth and differentiation factor.It is in 200410033182.9 the patent application that the technical scheme of this technology is documented in number of patent application.
(5) neurocyte extract extracts from human or animal's neurocyte tissue, and the complex mixture that molecular weight differs in size contains multiple specificity and promotes, regulates and control and induce neuralward cell growth and differentiation factor.
(6) stem cell is induced differentiation: with natural compounds or cytokine and etc. and stem cell behind co-cultivation certain hour under specified temp, humidity and the carbon dioxide conditions, differentiation of stem cells is a mature cell.Different inducible factors induce back gained mature cell type different with condition.
Summary of the invention
The invention provides a kind of neurocyte tissue in the human or animal body that has drawn from, through artificial extraction, make the neurocyte extract of molecular weight less than 200,000 dalton (Da); The present invention also provides this neurocyte extract to be induced to differentiate in the neurocyte utilisation technology as inductor at external MSC, neurocyte extract of the present invention is induced to differentiate in the application of neurocyte at MSC, demonstrate the good differentiation rate of inducing, and effective, repeatability and good stability.
The invention technical scheme is as follows:
Human or animal's neurocyte extract mainly comprises following method extraction:
The neurocyte tissue is drawn materials, as sciatic nerve tissue or cerebral tissue, remove that red corpuscle, tunicle and reticular tissue etc. are non-to need composition → cell homogenates → cytoclasis → precipitation or filtration, get the material → determination of protein concentration of supernatant liquor → separation removal greater than the above molecular weight of 200,000 dalton (Da), determination of activity, degerming, frozen or be prepared into the reagent packing.
Concrete grammar is:
(1) gets human or animal's neurocyte tissue, remove internal memory red corpuscle and tunicle, reticular tissue, shred standby;
(2) below 4 ℃, homogenate 3-5 time;
(3) the capable cytoclasis of homogenate is broken fully to cell;
(4) under 1-4 ℃ of condition, place to go sediment and albumen floss are drawn supernatant liquor;
(5) supernatant liquor of Huo Deing is held back and separate to be removed molecular weight greater than the above macromolecular substance of 200,000 dalton (Da), and the molecular weight that makes albumen in the filtered liquid of acquisition and polypeptides matter is less than 200,000 dalton (Da);
(6) the filtered liquid determining the protein quantity of Huo Deing, determination of activity, degerming, frozen or aseptic subpackaged, this is a neurocyte extract of the present invention.
Carry out homogenate after the neurocyte tissue of step (1) shreds, also can add homogenate again after the dilution of 1-4 times of physiological saline; Step (3) cytoclasis can be used the method for chemistry or physics, and preferred freeze thawing is to place refrigerator frozen to freezing fully, under the condition that is not higher than 42 ℃ of water proofs, thoroughly melt then, as above the condition multigelation is 3-5 time, and smear microscopic examination routinely proves that cell is broken fully; The separation method of holding back of step (5) can be methods such as high speed centrifugation, electrophoresis, filtering membrane filtration, preferred filtering membrane filters under malleation or condition of negative pressure to be held back, its objective is macromolecular substance is separated, keep the composition of molecular weight less than 200,000 dalton (Da).The requirement of selecting for use of filtering membrane can be held back down the macromolecular substance of molecular weight greater than 200,000 dalton (Da).Neurocyte extract filtrate of the present invention is used the ordinary method generate a reagent, but pulvis or aqua.When making aqua, protein concn greater than 4 mg/ml for well, if protein concentration is low excessively in the aqua, when using in order to satisfy protein concentration, corresponding water capacity increases, and causes other interior composition consumption of nutrient solution of certain volume to reduce, and can not satisfy the nutrient solution ratio requirement.No matter be pulvis or aqua, during application, to the amount of asking for get final product according to activity unit.Reagent should be preserved the way cryopreservation by biotechnological formulation.
Described human or animal's neurocyte tissue is directly to obtain from human or animal body, animal again with Mammals for well.
The production process of neurocyte extract of the present invention is preferably under the above-mentioned cold condition and carries out, and to reduce loss of activity, prevents to pollute.Cytoclasis should be complete, can adopt chemistry or physical method, technology such as ultrasonic wave, and it is broken fully that frozen-thaw process need proceed to cell repeatedly.How many protein contents can because of changing that salt solution adds, protein concn is preferably more than 1 milligram/milliliter in the extracting solution, guarantee that other effective ingredient is able to abundant extraction, when being prepared into reagent, but reconcentration or be diluted to and require concentration, it is to contain the neurocyte extract with 100 milliliters of nutrient solutions to represent with protein concn in application that activity defines, contain protein more than 1 milligram promptly the performance activity is arranged, be the effective active scope.Frozen can be-20 ℃, can keep the activity of extract preferably, long-pending amount back preparation reagent.Degerming method with filtration method for well.
Neurocyte extract of the present invention is induced application in the differentiation at MSC, be with neurocyte extract of the present invention, basic medium and conditioned medium etc. and MSC co-cultivation, induce it to be divided into neural like cell, its application can be the application of neurocyte extract of the present invention in conjunction with prior art, also can be that neurocyte extract of the present invention becomes stem cell to induce the application of differentiation agents box with co-production such as other reagent such as basic mediums.The application of described test kit can be mainly by the application in the test kit of basic medium and ovum extract and/or the preparation of neurocyte extract.Described basic medium with the mixed culture medium that is selected from DMEM and F12 for well.
Using significant quantity is reactive conditions: represent with protein concn to contain the neurocyte extract in the nutrient solution cumulative volume, be shown as that i.e. performance has activity more than the protein 1% (mg/ml), be the effective active scope, be effective active scope more at 1%-100%.In this field of activity, induce differentiation rate to reach more than 60%.
Through repetitious experiment, be example with people MSC, amplification cultivation 15 days, go down to posterity four times, the MSC after the amplification is induced to differentiate into neurocyte, cultivated again 15 days, go down to posterity four times, the differentiation rate of inducing that is induced to differentiate into neurocyte reaches effect of the present invention, and repeatability and good stability.The inducing cell qualification result: morphology, the evaluation of neurocyte specific antigens sign and neurotransmitter analysis etc. all meet the neurocyte feature.
Neurocyte extract of the present invention is induced to differentiate in the application of neurocyte at MSC, demonstrates the good differentiation rate of inducing, and more than 60%, suitable application induces differentiation rate to reach more than 95%.
Described contained composition of human or animal's neurocyte extract and content thereof are unclear fully as yet at present, existing report confirms, contain the potential adjusting cell growth and the factor of breaking up in the neurocyte slurry, also contain rich in protein and correlation factor, for inducing differentiation, MSC provides ideal comprehensive nutrient environment jointly with other substratum and correlative factor, show as when keeping the MSC growth, induce its neuralward cytodifferentiation.
Now the present invention is further elaborated in conjunction with the embodiments:
Embodiment 1, the preparation of neurocyte extract
(1) get human or animal's sciatic nerve cell tissue, irritate physiological saline by artery and vein immediately, remove the red corpuscle that retains in the blood vessel to greatest extent, shave out the non-compositions that need such as tunicle, reticular tissue, blood vessel, shred, adding equivalent physiological saline is standby;
(2) tissue homogenate or stamp mill be under 4 ℃ of conditions, ten thousand rev/mins of homogenate of 1-3 3-5 time, each 1 minute; Should notice that refiner is unsuitable overheated, otherwise portion of tissue liquid loses activity;
(3) homogenate is placed-20 ℃ of refrigerators frozen to freezing fully, general more than 24 hours, water proof thoroughly melts under 42 ℃ the condition not being higher than then, and as above the condition multigelation is 3-5 time, and smear microscopic examination routinely proves cell fragmentation fully;
(4) under 1-4 ℃ condition natural subsidence 2-10 hour, draw supernatant liquor, filter with 3-4 layer sterile gauze, place to go sediment and albumen floss, under 4 ℃ of conditions, centrifugal 30 minutes of 400-600g gets supernatant liquor;
(5) filters the supernatant liquor that obtains with filtering membrane under malleation or condition of negative pressure, molecular weight cut-off is greater than the macromolecular substance more than 200,000 dalton (Da), and the molecular weight that makes albumen in the filtered liquid and polypeptides matter is less than 200,000 dalton (Da);
(6) measuring protein concentration is 20 mg/ml, and application of active is measured and met, and the filtration method degerming is diluted to protein concentration with stroke-physiological saline solution and is 10 mg/ml, and is aseptic subpackaged, is aqua reagent.
Embodiment 2, and MSC is induced to differentiate into the application of neurocyte
The application of test kit, ovum extract aqua contains the protein 10 mg/ml, and above-mentioned neurocyte extract aqua contains the protein 10 mg/ml, prepares per 100 milliliters of test kits, each component independent packaging consumption:
(1) DMEM and F12 mixed culture medium are 80 milliliters
(2) human body ovum extract aqua is 10 milliliters
(3) neurocyte extract aqua is 10 milliliters
The conventional promoting growth of cell factor, antioxidant and microbiotic etc. with significant quantity can be arranged in the test kit.
Method for inducing and cultivating:
1, mescenchymal stem cell separation, purifying: from tissues such as marrow, fat, separate obtaining mononuclearcell according to a conventional method, carry out earlier that the surperficial special sign antigen with mescenchymal stem cell carries out positive and negative immunoscreening after the cell adherent culture of former generation, obtain pure mescenchymal stem cell.If cell quantity is enough, also can directly carry out separation and purification with separating the mononuclearcell that obtains.According to experiment purpose and cell quantity, carry out cell amplification earlier and cultivate desired number or directly induce with this reagent.
2, mescenchymal stem cell adherent culture: is 1 * 10 with mescenchymal stem cell with the basic medium dilution
5Cell/ml is by * 10
4Cell/cm2 density is inoculated in the culturing bottle (ware), carries out vitro culture to cell by the mescenchymal stem cell amplification in vitro method and covers with a bottle wall, carries out inducing culture after the 2/3 above cytogamy.Mescenchymal stem cell criterion: form: fusiformis is the regular aligned growth of flamboyancy.Surface molecular sign: CD34
-, CD45
-, CD103
+, CD106
+, SH2
+, SH4
+
3, inducing culture: shift out the nutrient solution behind the adherent culture cell, wash 1-2 time with physiological saline, the cell of above-mentioned amount is induced, needing test kit nutrient solution cumulative volume is 10ML, sets active protein content and is respectively 50%, then gets DMEM/F12 substratum 9ml, 0.5 milliliter of ovum extract, 0.5 milliliter of neurocyte extract contains two kinds of each 5mg of cell extract protein in this 10ML nutrient solution, active protein concentration is respectively 50%.Place 37 ℃, 5%CO2,95% humidity condition to cultivate down in the mixture of test kit nutrient solution and cell, continuously the observation of cell upgrowth situation.
4, inducing cell is identified:
(1) form: cell is spindle shape before inducing, the flamboyancy growth, and regular arrangement, nucleus is big, and the endochylema composition is few relatively.Cultivated about 3 days, it is round that cell becomes gradually, stretches out projection then, forms many projections neurocyte like cell.
(2) neuronal cell specific antigens sign is identified: adopt the immunocytochemistry pair cell to identify.The result is: neuronic special mark neuron specific enolase (NSE), nerve-specific nucleoprotein (NeuN), intermediate nerve silk (NFOm), Protein tau and some tubulins (tubulin-β, MAP-2, TuJ-1) are positive.
(3) neurotransmitter analysis: adopt gas/mass spectroscopy, neurotransmitter generations such as Dopamine HCL are arranged in the culture supernatant.
(4) inductivity: more than 90%.
Through a plurality of embodiment, get the different protein concentrations of neurocyte extract in the nutrient solution respectively and make inducing culture as 5%, 10%, 20%, 35%, 50%, 75%, 100% and 150%, all obtain satisfied effect, demonstrate good repeatability and stable.Technical scheme of the present invention is not subjected to the restriction of embodiment.
Claims (10)
1. neurocyte extract comprises that mainly following method produces:
(1) gets human or animal's neurocyte tissue, remove red corpuscle, tunicle and reticular tissue, shred standby;
(2) cell homogenates;
(3) the capable cytoclasis of homogenate is broken fully to cell;
(4) supernatant liquor is got in precipitation or filtration;
(5) separate the removal molecular weight greater than the above material of 200,000 dalton (Da);
(6) determination of protein concentration, determination of activity, degerming, frozen or the preparation reagent aseptic subpackaged, this is a neurocyte extract of the present invention.
2. neurocyte extract according to claim 1 is characterized in that, comprises that mainly following method produces:
(1) gets human or animal's neurocyte tissue, remove internal memory red corpuscle and tunicle and reticular tissue, shred standby;
(2) below 4 ℃, homogenate 3-5 time;
(3) method of cell disruption is freeze thawing, places refrigerator frozen to freezing fully homogenate, thoroughly melts not being higher than under 42 ℃ of conditions of water proof then, and as above the condition multigelation is 3-5 time, smear microscopic examination routinely, and cell is fragmentation fully;
(4) under 1-4 ℃ of condition, remove sediment and albumen floss, draw supernatant liquor;
(5) supernatant liquor of Huo Deing separate to be removed molecular weight greater than the above macromolecular substance of 200,000 dalton (Da), and the molecular weight that makes albumen in the filtered liquid of acquisition and polypeptides matter is less than 200,000 dalton (Da);
(6) the filtered liquid determining the protein quantity of Huo Deing, protein concn be more than 1 milligram/every milliliter of filtrate, determination of activity, and degerming, frozen or preparation reagent is aseptic subpackaged.
3, neurocyte extract according to claim 1 and 2 is characterized in that, adds 1-4 times of physiological saline in the neurocyte tissue of step (1) and dilutes homogenate again.
4, neurocyte extract according to claim 1 and 2 is characterized in that, the separation method of step (5) is to filter under malleation or condition of negative pressure with filtering membrane.
5, neurocyte extract according to claim 3 is characterized in that, the separation method of step (5) is to filter under malleation or condition of negative pressure with filtering membrane.
6, according to claim 1,2 or 5 described neurocyte extracts, it is characterized in that described animal is a Mammals.
7, the neurocyte extract of claim 1 is induced application in the differentiation at mescenchymal stem cell, is the application that external mescenchymal stem cell is induced to differentiate into neurocyte.
8, application according to claim 7 is characterized in that, described application is the application in the preparation test kit.
9, application according to claim 8 is characterized in that, the application of described test kit is the application of the test kit of the basic medium mixed culture medium that is selected from DMEM and F12.
10, according to Claim 8 or 9 described application, it is characterized in that the application of described test kit is the application in the test kit of basic medium and ovum extract and/or the preparation of neurocyte extract.
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| CN1274815C CN1274815C (en) | 2006-09-13 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1948466B (en) * | 2006-11-10 | 2010-05-12 | 中国人民解放军军事医学科学院野战输血研究所 | Reagent box used for preparing bone marrow interstitial stem cell |
| CN103937739A (en) * | 2010-06-01 | 2014-07-23 | 碧雅诗株式会社 | Mesenchymal stem cell attractant and method for attracting mesenchymal stem cell |
| CN104004713A (en) * | 2014-05-14 | 2014-08-27 | 安沂华 | Method for inducing and culturing umbilical cord mesenchymal stem cells into nerve cells |
| CN108542877A (en) * | 2017-03-23 | 2018-09-18 | 广州资生生物科技有限公司 | A kind of preparation method of micromicron grade cell extract |
-
2004
- 2004-05-10 CN CN 200410044418 patent/CN1274815C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1948466B (en) * | 2006-11-10 | 2010-05-12 | 中国人民解放军军事医学科学院野战输血研究所 | Reagent box used for preparing bone marrow interstitial stem cell |
| CN103937739A (en) * | 2010-06-01 | 2014-07-23 | 碧雅诗株式会社 | Mesenchymal stem cell attractant and method for attracting mesenchymal stem cell |
| CN103937739B (en) * | 2010-06-01 | 2017-12-08 | 碧雅诗株式会社 | The diverting agent of mesenchyma lineage stem cells and the guidance method of mesenchyma lineage stem cells |
| CN104004713A (en) * | 2014-05-14 | 2014-08-27 | 安沂华 | Method for inducing and culturing umbilical cord mesenchymal stem cells into nerve cells |
| CN108542877A (en) * | 2017-03-23 | 2018-09-18 | 广州资生生物科技有限公司 | A kind of preparation method of micromicron grade cell extract |
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| CN1274815C (en) | 2006-09-13 |
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