CN1563362A - Extract of egg cell from human or animal and use for amplifying stem cell as well as induction and differentiation - Google Patents
Extract of egg cell from human or animal and use for amplifying stem cell as well as induction and differentiation Download PDFInfo
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- CN1563362A CN1563362A CN 200410033182 CN200410033182A CN1563362A CN 1563362 A CN1563362 A CN 1563362A CN 200410033182 CN200410033182 CN 200410033182 CN 200410033182 A CN200410033182 A CN 200410033182A CN 1563362 A CN1563362 A CN 1563362A
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- ovum
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Abstract
An extracted material from ovum of human being or animal is a new reagent for stem cells cloning and inducing technique. The preparing process of the material includes collecting raw material of ovum, washing, homogenating, freezing and melting, precipitating, picking top clear solution out, holding for impurity removal and loading.
Description
Invention field
The present invention relates to biomass cells extract and utilisation technology, be specifically related to human or animal's ovum extract and expansion of stem cells with induce application in the differentiation.
Background technology
To the research of ovum, for many years, relate to the fertility technical field more, still be not reported to the ovum extract and in expansion of stem cells and the research of inducing differentiation relevant technologies and application.
Since 1997, stem-cell research makes important progress, and discovers that the human body mescenchymal stem cell can be induced to differentiate into the mature cell of nerve, muscle, skin, cartilage, fat, tendon, liver, blood and types of organization.Amplification be transplanted to after inducing in the body can and respective organization cell good integration, bring into play specific biological function, can be used for that transplantation treatment is multiple organizes degeneration, histocyte degeneration necrosis, tissue injury or damaged etc., clinical good application prospects arranged.The research of embryonic stem cell has had gratifying achievements, has produced clone sheep " how sharp ".
Existing many about the expansion of stem cells of mescenchymal stem cell and the report of method of inducing differentiation and reagent, the technological method and the factor that are adopted about report have nothing in common with each other.Guangxi Medical College's journal the 18th the 2nd phase of volume of June in 2002 " human marrow-interstitial stem cell separates and cultivate the research of amplification method " is to obtain promising result with foetal calf serum (FCS) and the amplification of L-DMEM nutrient solution.Fudan Journal (medicine) 2003MAY, 30 (3) " vitro culture of human marrow mesenchymal stem cell and induce differentiation ", its amplification culture medium contains DMEM, foetal calf serum, endothelial cell growth factor (ECGF) (ECGF), dexamethasone, Regular Insulin, β-phospho-glycerol etc.Finding report method, reagent are different, do not form standard.
Summary of the invention
The invention provides a kind of ovum that directly obtains in the human or animal body through artificial the extraction and the ovum extract, the present invention also provides this ovum extract in external expansion of stem cells and the application of inducing in the differentiation.Ovum extract of the present invention expansion of stem cells with induce during differentiation uses, demonstrate the amplification efficiency of good expanding stem cells and the differentiation rate of inducing that induced dry-cell is divided into other cell, and effective, good reproducibility, stable performance.
Relevant noun note:
(1) stem 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): be the present more ASCs of research.Extensively be present in the multiple tissues such as adult's marrow, fat, 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: from human or animal's ovum, extract the complex mixture that molecular weight differs in size.Contain multiple promotion, regulation and control and inducing cell growth and differentiation factor.
(5) directional induction cell extract, respectively from human or animal's different tissues as histocyte extracts such as nerve, muscle, skin, cartilage, fat, tendon, liver, blood, the complex mixture that molecular weight differs in size.Contain multiple specificity and promote, regulate and control and induce, can break up to institute's committed cell by induced dry-cell to respective fine intracellular growth differentiation factor.Can be under the acting in conjunction of other factors as the neurocyte extract, the differentiation of induced dry-cell neuralward cell direction.
(6) expansion of stem cells: with natural compounds or cytokine etc. and stem cell under specified temp, humidity and the carbon dioxide conditions with after cultivating certain hour, stem cell population increases in a large number and keeps its original biological characteristics.
(7) 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.
The invention technical scheme
Human or animal's ovum extract comprises that mainly following method produces:
The ovum raw material collects → cleans → cell homogenates → freeze thawing → precipitate or filter → get supernatant liquor → separation and remove greater than the above Middle Molecular Substance → determination of protein concentration of 200,000 dalton (Da) → determination of activity → degerming → packing.
Concrete grammar is:
(1) gets human or animal's ovum, with physiological saline washing 2-3 time;
(2) below 4 ℃, homogenate 3-5 time;
(3) homogenate is placed refrigerator frozen to freezing fully, thoroughly melt under the condition that is not higher than 42 ℃ of water proofs then, as above the condition multigelation is 3-5 time, and smear microscopic examination routinely proves cell fragmentation fully;
(4) under 1-4 ℃ of condition, place to go sediment and albumen floss are drawn supernatant liquor;
(5) supernatant liquor of Huo Deing separates the macromolecular substance promptly remove greater than the above molecular weight of 200,000 dalton (Da), makes the albumen in the filtered liquid of acquisition and the molecular weight of polypeptides matter be lower than 200,000 dalton (Da);
(6) with the filtered liquid degerming that obtains, determining the protein quantity, pulvis or aqua are made in determination of activity, in-20 ℃ frozen or aseptic subpackaged, this is an ovum extract of the present invention.
The ovum of step (1) can directly carry out homogenate, also can add homogenate again after the dilution of 1-4 times of physiological saline again; The separation method of step (5) can be methods such as high speed centrifugation, electrophoresis, filtering membrane filtration, and preferred filtering membrane filters under malleation or condition of negative pressure to be held back.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).Product ordinary method generate a reagent, but pulvis or aqua.When making aqua, protein concn,,, will the amount of asking for get final product according to activity unit during application no matter be pulvis or aqua for well greater than 4 mg/ml.
Described human or animal's ovum is directly to obtain from human or animal body, animal with Mammals for well.
The production process of ovum extract of the present invention is preferably under the cold condition and carries out, and reduces loss of activity, prevents to pollute.It is broken fully that frozen-thaw process need proceed to cell repeatedly.Protein content can extract by making method of the present invention because of how many differences of salt solution adding, and protein concn is more than 1 mg/ml in the extracting solution.Degerming method is to be filtered into.
Human or animal's ovum extract is in expansion of stem cells and the application of inducing in the differentiation, particularly in the amplification of mescenchymal stem cell and induce the application of differentiation, show good amplification efficiency and induce differentiation rate, more than expansion of stem cells 500-1000 times, induce more than the differentiation rate 60%-90%.Ovum extract reagent of the present invention is in expansion of stem cells and the application of inducing in the differentiation, can be the application of ovum extract reagent of the present invention in conjunction with prior art, also can be that ovum extract reagent is being prepared into expansion of stem cells and is inducing the application of differentiation agents box.The application of described test kit can be the application in the test kit of mainly being made by basic medium and ovum extract and/or directional induction cell extract, and what described directional induction cell extract reagent can be by prior art induces the differentiation agents replacement.Described basic medium with the mixed culture medium that is selected from DMEM and F12 for well.Described directional induction cell extract series technique is the separate case patent application simultaneously.
Of the present inventionly induce differentiation to use not limited by amplification technique of the present invention, can be applicable to other technology amplification mescenchymal stem cell induce differentiation.
Application test and effect:
Stem cell is example amplification cultivation 15 days with the mescenchymal stem cell, goes down to posterity four times, and the mescenchymal stem cell after the amplification is induced to differentiate into neurocyte, cultivates 15 days again, goes down to posterity four times.The result shows: expanding stem cells reaches more than 1000 times, and the differentiation rate of inducing that is induced to differentiate into neurocyte reaches more than 90%.
Amplifying cells is identified:
Mescenchymal stem cell criterion: form: fusiformis is the regular aligned growth of flamboyancy.Surface molecular sign: CD34
-, CD45
-, CD103
+, CD106
+, SH2
+, SH4
+Qualification result meets.
Inducing cell is identified:
(1) morphology: meet the neurocyte morphological feature;
(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 (tubulinO β, MAPO2, TuJO1) are positive;
(3) neurotransmitter analysis: adopt gas/mass spectroscopy, neurotransmitter generations such as Dopamine HCL are arranged in the culture supernatant.The electricity Physiological Analysis has neural sample discharge.
Qualification result meets.
Significant quantity: contain ovum extract active ingredient with per 100 milliliters of nutrient solutions and represent with protein concn, be shown as contain protein more than 1 milligram i.e. performance activity is arranged, be the effective active scope, the 1-100 milligram is effective active scope more.In this field of activity, expanding stem cells reaches more than 500 times, induces differentiation rate to reach more than 60%.
The significant quantity field of activity of the above protein content of described ovum extract, the representative application that covers mescenchymal stem cell and embryonic stem cell should be effective.Verified, vitamin A acid and mercaptoethanol are to the amplification of mescenchymal stem cell and to induce differentiation be effective, be applied to embryonic stem cell and also demonstrate identical effect, illustrate embryonic stem cell and mescenchymal stem cell have with amplification potential, it is feasible that ovum extract of the present invention is used the amplification of embryonic stem cell.
Composition and content in described human or animal's ovum extract are not clear and definite as yet, but the ovum slurry has regulating and controlling effect to fetal development and stem cell growth.The existing report confirms to contain in the ovum slurry the potential adjusting cell growth and the factor of breaking up, also contain rich in protein and correlation factor in the extract, for expansion of stem cells and induce differentiation that ideal comprehensive nutrient environment is provided, can make a large amount of amplifications of stem cell population and keep its original characteristics of cell biology, this point is confirmed in above-mentioned mescenchymal stem cell amplification is used.Ovum extract of the present invention and other directional induction factor, as the combination of directional induction cell extract, inducing in the differentiation application of stem cell, show as and induce it to break up to committed cell when keeping stem cell growth, this point is induced to differentiate in the neurocyte application at above-mentioned mescenchymal stem cell and is confirmed.
Through the experiment of repeated multiple times, product of the present invention and utilisation technology, repeatability and good stability.
Now in conjunction with the embodiments to inventing further elaboration:
Embodiment 1, the preparation of ovum extract
(1) get human or animal's ovum 10 grams, with physiological saline washing 2-3 time, the physiological saline that adds equivalent 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;
(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.Smear microscopic examination routinely proves that cell is broken fully;
(4) under 1-4 ℃ condition natural subsidence 2-10 hour, draw supernatant liquor;
(5) filter place to go sediment and albumen floss with 3-4 layer sterile gauze;
(6) under 4 ℃ of conditions, centrifugal 30 minutes of 400-6008, get supernatant liquor:
(7) under malleation or condition of negative pressure, filters the supernatant liquor of acquisition with filtering membrane, hold back macromolecular substance, make the albumen in the filtered liquid and the molecular weight of polypeptides matter be lower than 200,000 dalton (Da) greater than 200,000 above molecular weight;
(8) measuring protein concn is 20 mg/ml, and filtration sterilization is diluted to 10 mg/ml concentration with physiological saline, and-20 ℃ frozen or aseptic subpackaged.
Embodiment 2, and mesenchyme is in cell amplification
Test kit, ovum extract protein 10 mg/ml
Per 100 milliliters of test kit each component amounts of taking:
(1) DMEM and F12 mixed culture medium are 90 milliliters;
(2) human body ovum extract is 10 milliliters;
The each component independent packaging.
The conventional promoting growth of cell factor, antioxidant and microbiotic etc. with significant quantity can be arranged in the test kit.
Amplification method:
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.
2, as getting the 10ml nutrient solution, then get (1) 9.8ml in the test kit, (2) 0.2ml, this is an active protein content 20%, with the mixture of test kit nutrient solution and cell place go down to posterity repeatedly under 37 ℃, 5%CO2,95% humidity condition four be commissioned to train foster, observation of cell upgrowth situation continuously.Show that amplification efficiency is 1100 times.
3, inducing cell is identified: mescenchymal stem cell criterion: form: fusiformis is the regular aligned growth of flamboyancy.Surface molecular sign: CD34
-, CD45
-, CD103
+, CD106
+, SH2
+, SH4
+Qualification result meets.
Embodiment 3, and mescenchymal stem cell is induced to differentiate into neurocyte
Test kit, ovum extract protein 10 mg/ml neurocyte extract protein 10 mg/ml
Per 100 milliliters of test kit each component amounts of taking:
(1) DMEM and F12 mixed culture medium are 80 milliliters;
(2) human body ovum extract is 10 milliliters;
(3) the neurocyte extract is 10 milliliters;
The each component independent packaging.
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, DMEM/F12 substratum 9ml then, 0.5 milliliter of ovum extract, 0.5 milliliter of neurocyte extract, contain two kinds of each 5mg of cell extract protein in this 10ml nutrient solution, active protein content is respectively 50%.Place 37 ℃, 5%CO2,85% 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 (tubulinO β, MAPO2, TuJO1) are positive.
(3) neurotransmitter analysis: adopt gas/mass spectroscopy, neurotransmitter generations such as Dopamine HCL are arranged in the culture supernatant.
(4) inductivity: 95%.
Through the amplification of a plurality of embodiment and induce differentiation culture, all obtain satisfied effect, demonstrate good repeatability and stable.Being induced to differentiate in the application of insulin secretory cell, liver cell etc. stem cell is feasible equally.Technical scheme of the present invention is not subjected to the restriction of embodiment.
Claims (9)
1, a kind of human or animal's ovum extract comprises that mainly following method produces:
The ovum raw material collects → cleans → cell homogenates → freeze thawing → precipitate or filter → get supernatant liquor → separation and remove greater than the above Middle Molecular Substance → determination of protein concentration of 200,000 dalton (Da) → determination of activity → degerming → packing.
2, human or animal's ovum extract according to claim 1 is characterized in that, comprises that mainly following method produces:
(1) gets human or animal's ovum, with physiological saline washing 2-3 time;
(2) below 4 ℃, homogenate 3-5 time;
(3) homogenate is placed refrigerator frozen to freezing fully, thoroughly melt under the condition that is not higher than 42 ℃ of water proofs then, as above the condition multigelation is 3-5 time, and smear microscopic examination routinely proves cell fragmentation fully;
(4) under 1-4 ℃ of condition, place to go sediment and albumen floss are drawn supernatant liquor;
(5) supernatant liquor of Huo Deing separates the macromolecular substance remove greater than the above molecular weight of 200,000 dalton (Da), makes the albumen in the filtered liquid of acquisition and the molecular weight of polypeptides matter be lower than 200,000 dalton (Da);
(6) with the filtered liquid degerming that obtains, determining the protein quantity, determination of activity, in-20 ℃ frozen or aseptic subpackaged, this is an extract of the present invention.
3, human or animal's ovum extract according to claim 1 and 2 is characterized in that, adds 1-4 times of physiological saline diluted for use in the ovum of step (1).
According to claim 2 or 3 described human or animal's ovum extracts, it is characterized in that 4, the separation method of step (5) is to filter under malleation or condition of negative pressure with filtering membrane to hold back.
5, human or animal's ovum extract according to claim 1 and 2 is characterized in that described animal is a Mammals.
6, human or animal's ovum extract of claim 1 is used with inducing in the differentiation at external expansion of stem cells.
7, application according to claim 6 is characterized in that, described application is in mescenchymal stem cell amplification and the application or the application in the embryonic stem cell amplification of inducing in the differentiation.
According to claim 6 or 7 described application, it is characterized in that 8, described application is the application in the preparation test kit.
9, application according to claim 8 is characterized in that, basic medium is the application of the mixed culture medium of DMEM and F12 in the described test kit.
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|---|---|---|---|
| CNB2004100331829A CN100519741C (en) | 2004-04-03 | 2004-04-03 | Extract of egg celsl from animals |
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|---|---|---|---|
| CNB2004100331829A CN100519741C (en) | 2004-04-03 | 2004-04-03 | Extract of egg celsl from animals |
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| CN1563362A true CN1563362A (en) | 2005-01-12 |
| CN100519741C CN100519741C (en) | 2009-07-29 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110292178A (en) * | 2019-07-26 | 2019-10-01 | 中山大学 | A kind of preparation method and its preparation for extracting youth element from animal egg cell |
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| US5480772A (en) * | 1993-02-03 | 1996-01-02 | Brandeis University | In vitro activation of a nucleus |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110292178A (en) * | 2019-07-26 | 2019-10-01 | 中山大学 | A kind of preparation method and its preparation for extracting youth element from animal egg cell |
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