CN102584946A - Cam binding peptide and application thereof - Google Patents
Cam binding peptide and application thereof Download PDFInfo
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- CN102584946A CN102584946A CN2012100621120A CN201210062112A CN102584946A CN 102584946 A CN102584946 A CN 102584946A CN 2012100621120 A CN2012100621120 A CN 2012100621120A CN 201210062112 A CN201210062112 A CN 201210062112A CN 102584946 A CN102584946 A CN 102584946A
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Abstract
The invention discloses a CaM binding peptide, which belongs to an amino acid and is in the molecular type of peptide. The CaM binding peptide is a Sema4D calmodulin binding peptide synthesized in a laboratory, and contains a sequence of which the code corresponds to Sema4D binding CaM. The molecular structural formula of the Sema4D calmodulin binding peptide is NH2-GYLPRQCLKFRSALLIGKKKPKS-COOH. The CaM binding peptide is applied to tumor resistance and inhibition of in-vitro angiogenesis of an HUVEC (Human Umbilical Vein Endothelial Cell). The CaM binding peptide can possibly become a novel angiogenesis inhibitor, and a novel antitumor medicament is provided for clinical use.
Description
Technical field
The present invention relates to be used for antineoplastic polypeptide drugs field, be specifically related to a kind of CaM binding peptide and application thereof.
Background technology
At present, the wildness of cancer danger is being coerced the healthy of people. and to capturing of chronic disease is a great problem of pendulum in face of the mankind.In today of science prosperity, people's the objective still is placed on the little medicine of the searching strong and spinoff of antitumous effect own and tackles tumour.Great mass of data confirms that intracellular Ca2+ accent albumen (CaM) is closely related with formation, growth and the propagation of tumour.
20th century 70, the eighties, people just confirmed that calmodulin antagonist has sure restraining effect to tumor proliferation, transfer.CaM is as Ca in the born of the same parents
2+The acceptor egg from, participate in Ca in the born of the same parents
2+The adjusting of concentration, and normal cell propagation needs calcium, it is believed that therefore CaM is that normal cell propagation is necessary.The generation that has now found that certain this tumour is also closely related with CaM with development: late 1980s, people just have been found that CaM content is higher than normal cell in the tumour cell.The unordered propagation of CaM level and cell in many experiment proof cells, particularly malignant cell propagation is positive correlation.Be proportionate like CaM level and liver cancer growth speed, CaM content is normal 2-4 times in rodents and the birds tumour, and the normal zone of psoriatic's lesion region CaM concentration ratio is high 30 times.This shows that the variation of CaM content can cause the undesired propagation of cell and the variation of carefully roaring. secondly, the change of CaM molecular structure can make CaM and its part target body; Even the interaction between all target bodys changes; Thereby impel the growth of cancer cells, the for example discovery of cancer protein is exactly that of change of CaM molecule generation matter in the malignant cell falls card; It can promote the growth of cancer cells, makes tumour cell at scarce Ca
2+Still keep vigorous DNA synthesis capability under the condition.The change of conjugated protein recurring structure of CaM or conformation and CaM are to Ca in addition
2+Activated susceptibility changes, and cAMP content changes all closely related with the formation of tumour, so CaM becomes the novel targets of chemical anticancer therapeutic agent, thereby causes that people pay close attention to greatly.
Migration and invasion and attack are keys of malignant progression; Also be the difficult problem of clinical treatment, the migration and the invasion and attack of control tumour are important directions of antitumor drug research, and the migration of tumour is a multistage complex process; Tumor invasion can be divided into for three steps with shifting, and promptly sticked, degraded and move.Sticking is the initial of tumor cell invasion; Tumour cell is through the glutinous company of sticking mutually such as albumen, fibronectin familial combined hyperlipidemia collagen that connects of the layer of surperficial special receptor and basilar membrane; Then under the effect of proteolytic ferment and correlation factor; The invasion and attack basilar membrane, the degradation of cell epimatrix, in a single day tumour cell is broken through the promptly very fast in the substrate invasive growth of basilar membrane and is shifted.Therefore, blocking-up is any one process wherein, can suppress the migration and the invasion and attack of tumour cell.Have document to confirm: the migration invasion and attack of CaM and tumour are closely related.
Growth of tumor has two different stages, and promptly changing into from avascular slow growth phase has the fast breeding of the blood vessel stage.If there is not vasculogenesis, the growth of primary tumo(u)r can not surpass 1-2mm
3Tumor vessel is that tumor tissues provides metabolic necessary oxygen and nutrition, thereby makes tumour be able to grow rapidly and for the far-end of tumour shifts transhipment is provided simultaneously.Therefore suppress the New Policy that vasculogenesis becomes antineoplaston in recent years.Recently have bibliographical information: CaM and neonate tumour blood vessel that confidential relation is also arranged, the CaM suppressor factor can angiogenesis inhibiting.Therefore shine new vitality again about the research of CaM and tumour.
But, the known calmodulin antagonist specificity of present known major part is not strong, and has untoward reaction.People attempt to seek the calmodulin antagonist that specificity is strong, untoward reaction is little, are used for oncotherapy.Polypeptide of the present invention possibly become effectively to be selected.
Summary of the invention
The purpose of this invention is to provide a kind of CaM binding peptide and application thereof, this invention not only can suppress the propagation of HeLa cell, and migration adheres to, and the extracorporeal blood vessel that can also suppress HUVEC is newborn, so it possibly developed and becomes a kind of novel antitumor drug.
For realizing above-mentioned purpose; CaM binding peptide provided by the invention, type are amino acid, and molecule type is a peptide; Said CaM binding peptide is a laboratory synthetic Sema4D calmodulin binding peptide; It contains the sequence of coding corresponding to Sema4D combination CaM, and the sequence of said Sema4D calmodulin binding peptide molecule is made up of an amino acid whose word brevity code, explains as follows: NH2-GYL PRQ CLK FRS ALL IGK KKP KS-COOH.
CaM is the acid small molecules solubility of an a kind of strand sphaeroprotein, is made up of 19 kinds, 148 amino acid, and molecular weight is 1.67 ten thousand, and iso-electric point is 4.3, is acidic protein.The calmodulin of different biogenetic derivations, its amino acid are formed and order or just the same, or a little difference is only arranged, and the popularity with distribution goes up the conservative property of height with evolving. and it is acidproof, and is heat-resisting, 90 ℃, heat and still can keep activity completely in 5 minutes.
The outer likeness in form dumbbell of CaM has two spheric ends, and middle continuous by a long and whippy spirane structure, each end has two Ca
2+Structural domain, each structural domain can combine a Ca
2+Thereby CaM intramolecularly and Ca
2+Four binding sites are arranged.The CaM molecule itself does not have the activity of enzyme, at no Ca
2+Situation under, do not show biological activity yet.Calmodulin and Ca in non-stimulated cells
2+Bonded avidity is very low; Yet, if because stimulation makes Ca in the cell
2+When concentration raises, Ca
2+Combine to form calcium-calmodulin mixture (Ca with calmodulin
2+/ CaM), will cause the variation of calmodulin configuration, strengthen calmodulin and a lot of target protein bonded avidity, thereby carried out its biological function, after calcium combined, the variation on the configuration took place in CaM, became the activator of some enzymes.When combining with enzyme again, cause the change of configuration of enzyme again, make by nonactive attitude to transfer activated state to, CaM-Ca
2+Having become these enzymes to do the time spent must obligato composition.
The biochemical reaction that CaM participates in is a lot, relates to many critical enzymes, as: during control information was transmitted, second messenger cAMP synthesized and the adenylate cyclase and the phosphodiesterase that decompose; Glycogen synthetic with decompose in phosphorylase kinase and the glycogen synthase kinase with storage power can be provided, relevant protein kinase with protein phosphorylation and dephosphorylation and protein phosphatase are separated enzyme, can regulate intracellular calcium concentration, play the Ca of calcium pump effect
2+-ATPase also has the MLCK relevant with smooth muscle contraction etc.
So CaM binding peptide of the present invention can be used for antitumor and application anti-angiogenic rebirth.
CaM binding peptide according to the invention can be used for suppressing the application of HeLa Cells in-vitro multiplication.
CaM binding peptide according to the invention can be used for suppressing HeLa Cells migration, adherent application.
Compared with prior art, the present invention has the following advantages:
CaM binding peptide of the present invention is that Sema4D calmodulin binding peptide not only has the effect of angiogenesis inhibitors; Also has the inhibition tumor cell proliferation; Adhere to; The specific effect of migration, being expected to develop becomes a kind of novel angiogenesis inhibitors, for clinical a kind of new antitumor drug is provided.
Above-mentioned explanation only is the general introduction of technical scheme of the present invention, understands technique means of the present invention in order can more to know, and can implement according to the content of specification sheets, below with preferred embodiment of the present invention and conjunction with figs. specify as after.
Embodiment
To combine embodiment below, specify the present invention.
One, Sema4D calmodulin binding peptide suppresses the propagation of HeLa cell
This experimental selection HeLa Cells has tentatively been inquired into the effect of Sema4D calmodulin binding peptide to HeLa cells in vitro propagation.The HeLa cell inoculation places in 37 ° of C, the 5%CO2 incubator and cultivates in the RPMI-1640 nutrient solution that contains 10% foetal calf serum, penicillium mould 100U/ml, Streptomycin sulphate 100ug/ml.Every 48h changes liquid, goes down to posterity 1 time, and the logarithmic phase cell is chosen in experiment.
Step:
1) inoculating cell: with 0.25% trysinization logarithmic phase cell, cell is made into single cell suspension, is inoculated in 96 orifice plates, every hole 100ul with suitable density with perfect medium;
2) culturing cell: culture plate is put into incubator, and at 37 ℃, 5%CO2 cultivates administration behind 24 h;
3) experiment is provided with drug-treated group and blank group: the every hole of drug-treated group adds the RPMI-1640 complete culture solution that contains different concns (0.2,1,5,25uM) Sema4D calmodulin binding peptide, and each concentration is all established 3 and answered holes.The every hole of blank group adds the RPMI-1640 nutrient solution that 100ul does not contain polypeptide.
4) colour generation: after cultivating 48 h, every hole adds MTT solution (5mg/ml) 20uL, and 37 degree continue to hatch 4h, stops cultivating, and carefully discards substratum and MTT in the hole, and every hole adds 100ul DMSO, concussion 10min, and Shi Jia Za fully dissolves;
5) colorimetric: select the 490nm wavelength, on enzyme-linked immunosorbent assay instrument, measure each hole light absorption value, the record result.
The result shows: Sema4D calmodulin binding peptide treatment group and blank group relatively, except 0.2uM organized, each concentration drug group OD value all was starkly lower than control group (P < 0.01), the 1uM polypeptide promptly can significantly suppress the propagation of HeLa cell.And with the rising of administration concentration, each concentration drug group OD value reduces gradually.Explain that Sema4D calmodulin binding peptide has significant inhibitory effect to the propagation of HeLa cell.
Two, Sema4D calmodulin binding peptide suppresses the adhesion of HeLa cell
Tumour cell attaches to (like fiber link albumen, Laminin ELISA and IV collagen type) on basilar membrane and the extracellular matrix components through film surface receptor (integrating element, cadherin and laminin receptor etc.).Sticking is the moving step of beginning of cancer cells invasion and attack, and the tumour cell of high invasion and attack and the heterogeneity ability of sticking of basement membrane components increase usually, and the homogeneity between tumour cell is sticked ability and then can be descended.Above-mentioned characteristic helps tumour cell and separates with mother cell, and invades healthy tissues such as basilar membrane.
This experiment is main adopts MTT colorimetric measuring HeLa cell the sticking of (Matrigel) on extracellular matrix.Matrigel extracts the solubility basilar membrane extract that obtains from the EHS murine sarcoma; Be known as " artificial Matrigel " again; Its staple is type and the glutinous albumen that connects of layer, and is very similar with basilar membrane, thereby in experiment, is used as the substitute of basilar membrane.
Step:
1) aqua sterilisa is prepared 2 kinds of solution respectively: 10g/L BSA (1%), 50mg/L Matrigel, 1:8 diluent;
2) encapsulate basilar membrane: Matrigel adds 96 well culture plates respectively with the 50ul/ hole, and 4 ℃ are spent the night, and BSA is control substrate (negative control);
3) aquation basilar membrane: residual liquid in the sucking-off culture plate, every hole adds the serum-free medium that 50ul contains 10g/L BSA, 37 ℃, 30min;
4) inoculating cell: with 0.25% trypsin digestion and cell (normal cultured or dosing 24h), using the 1% substratum adjustment cell density that contains 10g/L BSA is 10
5Individual/ml, respectively with the 100ul cell suspension inoculation in 96 well culture plates that encapsulate Matrigel, every group of parallel 3 samples;
5) culturing cell: 37 ℃ of conventional 1h that cultivate, observe and photograph;
6) the MTT colourimetry detects: after cultivating 1h, and the ELIASA reading.
The result shows: compare with the blank group; Sema4D calmodulin binding peptide treatment group can obviously suppress the adhesion of HeLa cell on Matrigel; 20uMSema4D calmodulin binding peptide can be reduced to 72% with cell adhesion; Explain Sema4D calmodulin binding peptide can anticancer the moving step of beginning of invasion and attack, thereby transfer that maybe anticancer.
Three, Sema4D calmodulin binding peptide suppresses the migration of HeLa cell
Migration is one of requisite link in the tumour cell transfer process.Tumour cell separates with the parent knurl, passes through vessel wall, when attacking peripheral healthy tissues, needs certain motor capacity.We have taked two kinds of experimental techniques to verify the influence of Sema4D calmodulin binding peptide to the HeLa cell movement.
The scratch experiment step:
1) aqua sterilisa is prepared 2 kinds of solution respectively: 10g/L BSA (1%), 50mg/L Matrigel, 1:8 diluent;
2) encapsulate basilar membrane: Matrigel adds 96 well culture plates respectively with the 50ul/ hole, and 4 ℃ are spent the night, and BSA is control substrate (negative control)
3) aquation basilar membrane: residual liquid in the sucking-off culture plate, every hole adds the serum-free medium that 50ul contains 10g/L BSA, 37 ℃, 30min;
4) monolayer is cultivated in preparation: TCD is adjusted into 5 * 10
5Individual/ml, be inoculated in 96 well culture plates that encapsulate Matrigel, every group of parallel 3 samples, the perfect medium with 10% are cultivated until forming cell monolayer;
5) artificial cut: on single-layer culturing cell, mark " one " font cut with the rifle head, mirror is record cut district relative distance down, washs with serum-free medium then;
6) change 1% substratum that contains 10g/L BSA, the experimental group dosing is cultivated 24h and is changed 10% perfect medium, and continuation is cultivated 24h and taken a picture.
The Migration experimental procedure:
1) test previous day, the Transwell cell is positioned in 24 orifice plates, following chamber adds the RPMI-1640 nutrient solution of 600ul, and last chamber adds the RPMI-1640 nutrient solution of 100ul, and incubator spends the night for use;
2) test the same day, the chamber adds the RPMI-1640 nutrient solution that contains 10 %FBS of 600ul at first downwards, inserts the Transwell cell then, again the HeLa cell is adjusted to 5 * 10 with the RPMI-1640 substratum
5The concentration of cells/ ml (contains 5 * 10 to the last indoor adding 100ul HeLa cell of Transwell cell
4Individual cell).In 37 ℃, the incubator of 5 % CO2;
3) cultivate 24 h after, wipe the HeLa cell on surface, film top with cotton swab, be close to permeable membrane under the edge shearing of chamber, place fixedly 20min of 4% Paraformaldehyde 96;
4) after PBS gave a baby a bath on the third day after its birth time, 1% violet staining 30min, microscopically be 6 cell count that visuals field counting passes cell at random.
The cell scratch experiment is the result show, Sema4D calmodulin binding peptide also can reduce the motion of HeLa cell, and 20uMSema4D calmodulin binding peptide treatment group cell healing rate is starkly lower than control group; Transwell cell result shows that further Sema4D calmodulin binding peptide can reduce the transfer ability of HeLa cell.Hint Sema4D calmodulin binding peptide can suppress the migration of tumour cell, possibly be developed as the medicine of anti metastasis.
Four, Sema4D calmodulin binding peptide suppresses HUVEC cells in vitro angiogenesis
As aforementioned, angiogenesis inhibiting becomes the New Policy of antineoplaston.In the experiment, endotheliocyte can form the fenestral fabric that is similar to tube chamber on Matrigel, and therefore, Matrigel rubber moulding pattern commonly used is intended intravital angiogenesis in the experiment in vitro.
Step:
1) experiment is taken out Matrigel previous day from-20 degree, places with the required rifle of experiment is first-class that to be positioned over 4 degree refrigerators subsequent use;
2) experiment same day, Matrigel with after serum free medium 1:1 mixes, to be got 50ul and places 96 orifice plates, cell culture incubator is hatched 1h;
3) get experiment and use endotheliocyte, perfect medium is diluted to 3 * 10
5Individual/ml, every hole 100ul adds in 96 orifice plates of hatching in advance, establishes control group, the experimental group dosing;
4) microscopically is observed the vasculogenesis situation behind the 24h, and meter vascularization number.
The result shows: compare with the blank group, after Sema4D calmodulin binding peptide was handled, the cell multiform became incomplete reticulated structure.Explain that treatment group HUVEC cell forms the fenestral fabric that is similar to tube chamber on Matrigel ability obviously descends.Sema4D calmodulin binding peptide can be developed as the active drug of anti-angiogenic rebirth.
The above is merely the preferred embodiments of the present invention, is not limited to the present invention, and for a person skilled in the art, the present invention can have various changes and variation.All within spirit of the present invention and principle, any modification of being done, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1.CaM binding peptide; Type is an amino acid, and molecule type is a peptide, it is characterized in that; Said CaM binding peptide is a laboratory synthetic Sema4D calmodulin binding peptide; It contains the sequence of coding corresponding to Sema4D combination CaM, and the sequence of said Sema4D calmodulin binding peptide molecule is made up of an amino acid whose word brevity code, explains as follows: NH2-GYL PRQ CLK FRS ALL IGK KKP KS-COOH.
2. CaM binding peptide according to claim 1 is characterized in that: said CaM binding peptide is being used for antineoplastic application.
3. CaM binding peptide according to claim 1 is characterized in that: said CaM binding peptide is in the newborn application of extracorporeal blood vessel that is used to suppress HUVEC.
4. CaM binding peptide according to claim 1 and 2 is characterized in that: said CaM binding peptide is in the application that is used to suppress the HeLa Cells in-vitro multiplication.
5. CaM binding peptide according to claim 1 and 2 is characterized in that: said CaM binding peptide is in the migration that is used to suppress HeLa Cells.
6. CaM binding peptide according to claim 1 and 2 is characterized in that: said CaM is combined in the adhesion that peptide is used to suppress HeLa Cells.
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| CN2012100621120A CN102584946A (en) | 2012-03-12 | 2012-03-12 | Cam binding peptide and application thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2006235271A1 (en) * | 2005-04-07 | 2006-10-19 | Novartis Vaccines And Diagnostics Inc. | SEMA4D in cancer diagnosis, detection and treatment |
| US20080255244A1 (en) * | 2007-04-11 | 2008-10-16 | Fred Hutchinson Cancer Research Center | CaM Kinase II Inhibitor Improves Retinoic Acid Therapy and Inhibits the Proliferation of Myeloid Leukemia Cells |
-
2012
- 2012-03-12 CN CN2012100621120A patent/CN102584946A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2006235271A1 (en) * | 2005-04-07 | 2006-10-19 | Novartis Vaccines And Diagnostics Inc. | SEMA4D in cancer diagnosis, detection and treatment |
| US20080255244A1 (en) * | 2007-04-11 | 2008-10-16 | Fred Hutchinson Cancer Research Center | CaM Kinase II Inhibitor Improves Retinoic Acid Therapy and Inhibits the Proliferation of Myeloid Leukemia Cells |
Non-Patent Citations (3)
| Title |
|---|
| PAOLO CONROTTO ET AL: "Sema4D induces angiogenesis through Met recruitment by Plexin B1", 《BLOOD》, vol. 105, no. 11, 4 January 2005 (2005-01-04) * |
| PEIPEI MOU ET AL: "Identification of a Region within the Cytoplasmic Domain of Sema4D That Binds Calmodulin and Regulates Shedding of the Sema4D Exodomain in Platelets", 《53RD ASH ANNUAL MEETING AND EXPOSITION》, 13 December 2011 (2011-12-13) * |
| 吴亚平: "钙调蛋白与肿瘤", 《国外医学肿瘤学分册》, no. 5, 31 December 1985 (1985-12-31) * |
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