CN105456293A - Stem cell-based medicinal product for treating diabetes and preparing method thereof - Google Patents
Stem cell-based medicinal product for treating diabetes and preparing method thereof Download PDFInfo
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- CN105456293A CN105456293A CN201410451828.9A CN201410451828A CN105456293A CN 105456293 A CN105456293 A CN 105456293A CN 201410451828 A CN201410451828 A CN 201410451828A CN 105456293 A CN105456293 A CN 105456293A
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Abstract
The invention discloses a stem cell-based medicinal product for treating diabetes. The stem cell-based medicinal product contains mesenchymal stem cells separated and extracted from the human umbilical cord, the expression of stem cell surface markers CD73, CD90 and CD105 is higher than 95%, and the expression of cell surface markers CD14, CD34, CD45, CD19 and HLA-DR is lower than 3%. The stem cell-based medicinal product can be differentiated into pancreatic beta cells under induction, so that insulin is secreted and blood sugar concentration is effectively reduced, and then a new method is provided for overcoming the defect that diabetes treatment depends on insulin and radically curing diabetes. The invention further provides a method for preparing the stem cell-based medicinal product. The method is easy to operate, raw material sources are wide, ethic limitation is avoided, and large-scale production can be conducted.
Description
Technical field
The invention belongs to stem-cell research field, specifically a kind of stem cell medicine being used for the treatment of diabetes and preparation method thereof.
Background technology
Diabetes are a kind of because of a series of clinical syndromes that insulin in body is absolute or relative deficiency causes, and mainly comprise I type and type Ⅱdiabetes mellitus.In recent years, onset diabetes rate is in rising trend, and the rejuvenation that more becomes.Expect 2025,3.34 hundred million person-times will be had to suffer from diabetes in world wide, diabetes have become the large persistent ailment threatening human health.Dissimilar diabetes all can cause the β cell in pancreas can not produce enough insulins to fall hypoglycemic concentration, cause the generation of hyperglycemia.In current Therapeutic Method, although Drug therapy effectively can reduce blood glucose, effectively can not repair islet function; Though insulin injection can control symptom, delay and reduce the generation of complication, but diabetes can not be made thoroughly to cure, and life-time service insulin can produce the serious complication such as such as blind, renal failure, obesity and produce insulin resistant, every day, insulin injection itself also brought great misery to patient in addition, brought heavy financial burden to patient, family and society; Although islet cell transplantation is an effective therapeutic strategy, but problems faced is still medical science and the ethnics Problem such as donor shortage and immunologic rejection.
In recent years, stem-cell research is that the treatment of various disease provides a kind of new means.Stem cell is original and the cell of not fully differentiation, has the potential function regenerating various histoorgan, thus for the treatment of reproducing transplanting and major disease of histoorgan provides hope.Mescenchymal stem cell (MesenchymalStemCells, MSCs) mesoderm is originated from, all separable acquisition in the Various Tissues such as bone marrow, umbilical cord, umbilical blood, fat and organ, possess oneself's amplification and the Multidirectional Differentiation of stem cell, under the different phase of growing and certain environmental conditions, the histiocyte of multiple mesenchymal origin can be divided into, as skeletonization, cartilage and adipose cell, can transdifferentiationof be also other histiocytes multiple such as myocyte, neurocyte, vascular endothelial cell, liver pancreatic cell.MSCs has many-sided effects such as regeneration, reparation and immunomodulating concurrently.Mescenchymal stem cell (the Umbilical-DerivedMesenchymalStemCells in umbilical cord source, UMSCs) be present in the mescenchymal stem cell in umbilical cord jelly of Wharton and tissues surrounding vascular, compared with the mescenchymal stem cell deriving from bone marrow, UMSCs has more significant advantage, comprising: gathering convenient is non-invasive operation; The mescenchymal stem cell deriving from fetal cord is inmatureer than mesenchymal stem cells in adult human with combined density gradient centrifugation, propagation and differentiation potential larger; Umbilical cord abundance, can carry out large-scale production.These advantages make it in treatment diabetes, show more wide application prospect.
Summary of the invention
For solving the deficiency of existing treatment diabetes method, and the source related in acquisition stem cell process is limited and ethical issues, the invention provides a kind of stem cell medicine being used for the treatment of diabetes and preparation method thereof.
The present invention for the technical scheme realizing its object and adopt is:
Be used for the treatment of a stem cell medicine for diabetes, this stem cell medicine comprises the mescenchymal stem cell of separation and Extraction from people's umbilical cord, and this mescenchymal stem cell can be divided into beta Cell of islet after induction.
Further, the expression of cell surface marker CD73, CD90, CD105 of described mescenchymal stem cell is all higher than 95%, and the expression of cell surface marker CD14, CD34, CD45, CD19 and HLA-DR is all lower than 3%.
Further, described mescenchymal stem cell is further through the stem cell of subculture in vitro separately cultivation after separation and Extraction.
Further, described mescenchymal stem cell is under the microscope in spindle shape, and bag slurry is bright, and cell space is little, has third dimension.
Further, described mescenchymal stem cell have to skeletonization direction differentiation with to the multi-lineage potential becoming fat direction to break up.
Prepare the described method being used for the treatment of the stem cell medicine of diabetes, comprise the following steps:
(1) separation and Extraction mescenchymal stem cell from umbilical cord;
(2) original cuiture of mescenchymal stem cell;
(3) Secondary Culture of mescenchymal stem cell;
(4) mescenchymal stem cell that step (3) obtains is made stem cell medicine;
(5) quality testing of stem cell medicine.
Further, the separation and Extraction of described mescenchymal stem cell comprises the following steps: operate in superclean bench, is carried out disinfection by the umbilical cord ethanol of fresh acquisition, then cleans umbilical cord with normal saline, is separated and divests cord vessels, obtain magnificent Tong Shi glue part.
Further, the original cuiture of described mescenchymal stem cell comprises the following steps: shredded by magnificent Tong Shi glue, bottle is planted after dilution, adopting in CO2 gas incubator organizes adherent method to cultivate, change liquid by full dose and remove non-attached cell gradually, treat that cell fusion degree reaches 70-80% and can gather in the crops primary cell.
Further, the quality testing of described stem cell medicine comprises antibacterial, fungus, mycoplasma, virus, endotoxin, thermal source detects and cytoactive detects.
The invention has the beneficial effects as follows: the present invention take umbilical cord as the source of stem cell, through extraction and isolation, original cuiture, Secondary Culture make stem cell medicine, this is the stem cell of a class reduced immunogenicity to utilize the surface markers of flow cytomery cell to prove, show that gained mescenchymal stem cell can breed, have multi-lineage potential by experiment, the most important thing is, it can differentiation-inducing one-tenth beta Cell of islet, thus is used for the treatment of diabetes.Stem cell in invention formulation is from umbilical cord, and it is convenient to gather, and is non-invasive operation; Gained mescenchymal stem cell is inmatureer than other mescenchymal stem cell, propagation and differentiation potential larger; Umbilical cord abundance, can carry out large-scale production; Do not limit by ethics, immunogenicity is low, has boundless application prospect in treatment diabetes field.
Accompanying drawing explanation
Fig. 1 is the primary cell of umbilical cord derived mesenchymal stem cell.
Fig. 2 is second filial generation umbilical cord derived mesenchymal stem cell.
Fig. 3 is the testing result of the cell surface marker of umbilical cord derived mesenchymal stem cell.
Fig. 4 is the growth curve of the cell of umbilical cord derived mesenchymal stem cell.
Fig. 5 is that umbilical cord derived mesenchymal stem cell is to the dyeing qualification becoming the differentiation of fat direction.
Fig. 6 is the dyeing qualification that umbilical cord derived mesenchymal stem cell breaks up to skeletonization direction.
Fig. 7 is in animal model, and four groups of mices are in the testing result of the fasting glucose of the 3rd day.
Fig. 8 is in animal model, the blood glucose level monitor result of four groups of mices.
Fig. 9 is in animal model, the insulin level monitoring result of four groups of mices.
Detailed description of the invention
Embodiment 1
The preparation of the mescenchymal stem cell preparation in umbilical cord source
Operate in superclean bench, the umbilical cord ethanol of fresh acquisition is carried out disinfection, umbilical cord is cleaned again with normal saline, separation divests cord vessels, obtain magnificent Tong Shi glue part, magnificent Tong Shi glue is shredded, after dilution, plants bottle, adopting in CO2 gas incubator organizes adherent method to cultivate, basis of microscopic observation cell growth status, can see that cell attachment grows, in spindle shape, bag slurry is bright, cell space is less, has third dimension, changes liquid remove non-attached cell gradually by full dose, treat that cell fusion degree reaches 70-80% and can gather in the crops primary cell, the results are shown in Figure 1; Gained primary cell is carried out Secondary Culture in DMEM culture medium, to reach the object that purification and amplification cultivates mescenchymal stem cell, the results are shown in Figure 2; Collect 3rd ~ 8 generation mescenchymal stem cell, make stem cell medicine, cryopreservation.
Embodiment 2
The surface markers research of the mescenchymal stem cell in umbilical cord source
Utilize flow cytomery cell surface marker, the results are shown in Figure 3.Cell surface marker CD73, CD90, the CD105 relevant with MSCs are high expressed, and the expression of each generation is all stable, all higher than 95%; Cell surface marker CD14, CD34, CD45, CD19 of being correlated with hematopoietic cell and express low, all lower than 3% with the HLA-DR that immunologic rejection is relevant.Prove repeatedly go down to posterity posterior umbilicus band source MSCs contain can stablize breed stem cell, the labelling that cell high expressed MSCs is relevant, do not express or low expression hematopoietic cell and the cell surface marker relevant to transplant rejection, this type of MSCs cell is pointed out to be the stem cell of a class reduced immunogenicity, may in anti-autoimmune disease, treatment is transplanted rear repulsion aspect and is played a role.
Embodiment 3
The proliferation research of the mescenchymal stem cell in umbilical cord source
Planted by passage cell in 96 orifice plates, divide 12 groups, often organize 7 holes, every hole adds cell suspension 200 μ l.Get cultured cell line at random, according to mtt assay microplate reader, OD value is carried out to each experimental group and blank group and detect, get mean, draw stem cell growth curve, the results are shown in Figure 4.Experimental result shows that mescenchymal stem cell can normal proliferative.
Embodiment 4
The multi-lineage potential research of the mescenchymal stem cell in umbilical cord source
Be seeded in by cell to be detected in 12 orifice plates, use control medium and Osteoinductive differentiation respectively, adipogenic induction division culture medium, afterwards continuous observation, treat that cellular morphology changes, and carries out dyeing qualification.Adipogenic induction, to about the 21st day, carries out oil red O stain, the results are shown in Figure 5.Osteogenic induction, to about the 28th day, carries out Alizarin red staining, the results are shown in Figure 6.Experimental result show mescenchymal stem cell have to skeletonization direction differentiation with to the multi-lineage potential becoming fat direction to break up.
Embodiment 5
Mice is transplanted to the experiment of the mescenchymal stem cell in umbilical cord source
By the C57 mice that 60 body weight are 20-30g, be equally divided into 4 groups, wherein 45 are carried out modeling, and 15 as Normal group.Be set as following processing mode:
Modeling situation:
Divide cage labelling to each group of mice, weigh after overnight fasting, afterwards normal drinking water, duty changes bedding and padding.
When the 3rd day, fasting 4 hours, survey fasting glucose, correlated results is shown in Fig. 7, and three modeling group compared with normal matched group blood glucose significantly raise as seen from Figure 7, there was no significant difference between group, modeling success.After modeling first week, carry out cellular transplantation therapy.Normal group conveniently flow process is raised.
Umbilical cord mesenchymal stem cells being changed into the method transforming β cell is: recovered by the umbilical cord mesenchymal stem cells of freezer storage, cultivate in Tissue Culture Dish after washing, medium component is DMEM high glucose medium, obtains the umbilical cord mesenchymal stem cells of adherent growth; Add hyclone FBS and basic fibroblast growth factor bFGF; After 3 days, cell is all adherent, cleans 3 times with PBS, and replaced medium composition is serum-free DMEM/F12; And add four kinds of adenoviruss according to following concentration: PdX1:2.96 × 10
8iU/ml, Mafa:8.52 × 10
9iU/ml, Ngn3:5.24 × 10
9iU/ml; FOXA2:4.35 × 10
9iU/ml, at 5%CO
2incubator in cultivate 3 days, Induction Process terminates, and obtains the mesenchyma stem cell differentiation induction beta Cell of islet that is.
The processing method of umbilical cord mesenchymal stem cells: recovered by umbilical cord mesenchymal stem cells, washing, is resuspended in and preserves in injection, count, according to about 10
5the dosage an of/Mus carries out subpackage, respectively with 10 μ l long-neck electrophoresis suction nozzles suck Cell saps, then make cell natural subsidence in suction nozzle cervical region, from suck surplus liquid.
The results of beta Cell of islet, centrifugal, counting, prepares after the same method.
Transplant operation mode: mice is carried out back and shaves hair and disinfection, ensures operating room environment sterilization.Cut off the left renal tract skin of mice dorsal part, after exposing kidney, pressing, extrudes kidney around, and physiological saline solution keeps moistening, kidney peplos cuts off osculum, by there being in ready the plastic suction pipe of cell to insert kidney peplos, importing with liquid phase pin, returning after transplanting and receiving kidney, skin suture, carries out surface sterilization.
Transplant operation same day, and postoperatively carry out weekly a fasting glucose and detect, application blood glucose meter and reagent paper, tail vein blood is monitored, and records result, the results are shown in Figure 8.
According to bimestrial periodic monitoring, no significant difference after the blood glucose value of sham-operation transplantation group and UMSCs transplantation treatment group and modeling can be seen, remain on higher level, illustrate that islet function does not recover, cannot blood glucose regulation be carried out, to type 1 diabetes without therapeutical effect.
Transplantation group in beta Cell of islet kidney peplos, after modeling, this treated animal blood glucose raises, but after transplantation treatment, blood glucose steadily declines gradually, compare basic coincidence with Normal group, after describing differentiation, beta Cell of islet is implanted in type 1 diabetes animal model experiment in vivo and has good therapeutic effect.
Insulin level need extract serum, considers that mice blood volume is few, monthly monitors once, and mice on an empty stomach rear feeding is about 1h, the blood sampling of application capillary pipette, every eyeground vein clump gets blood 50 μ about l, can obtain the about 20 μ l of serum, through dilution, use ELISA mode to detect, the results are shown in Figure 9.
As can be seen from serum insulin levels, after experiment mice feed, just there is difference in each group of serum insulin levels, the transplanted cells same day (1w) Normal group mice serum insulin obviously rises, and three modeling groups are all without obviously rising, after showing modeling, mouse islets is without normally producing insulin function.During 4w, can see that sham operated rats serum insulin levels is without obvious change, the serum insulin levels difference of beta Cell of islet transplanting and Normal group is little, and UMSCs transplantation group has certain insulin secretion.During 8w third time detect, can see that beta Cell of islet transplantation group serum insulin levels has certain reduction, and levels found in normal controls raise, these may and food-intake, the factors such as beta Cell of islet time-to-live be correlated with.
The above; be only the specific embodiment of the present invention, but protection scope of the present invention is not limited thereto, anyly belongs to those skilled in the art in the technical scope that the present invention discloses; the change that can expect easily or replacement, all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain of claim.
Claims (9)
1. be used for the treatment of a stem cell medicine for diabetes, it is characterized in that, this stem cell medicine comprises the mescenchymal stem cell of separation and Extraction from people's umbilical cord, and this mescenchymal stem cell can be divided into beta Cell of islet after induction.
2. a kind of stem cell medicine being used for the treatment of diabetes according to claim 1, it is characterized in that, the expression of cell surface marker CD73, CD90, CD105 of described mescenchymal stem cell is all higher than 95%, and the expression of cell surface marker CD14, CD34, CD45, CD19 and HLA-DR is all lower than 3%.
3. a kind of stem cell medicine being used for the treatment of diabetes according to claim 1, is characterized in that, described mescenchymal stem cell is further through the stem cell of subculture in vitro separately cultivation after separation and Extraction.
4. a kind of stem cell medicine being used for the treatment of diabetes according to claim 1, is characterized in that, described mescenchymal stem cell is under the microscope in spindle shape, and bag slurry is bright, and cell space is little, has third dimension.
5. a kind of stem cell medicine being used for the treatment of diabetes according to claim 1, is characterized in that, described mescenchymal stem cell have to skeletonization direction differentiation with to the multi-lineage potential becoming fat direction to break up.
6. prepare the method being used for the treatment of the stem cell medicine of diabetes according to claim 1, it is characterized in that, comprise the following steps:
(1) separation and Extraction mescenchymal stem cell from umbilical cord;
(2) original cuiture of mescenchymal stem cell;
(3) Secondary Culture of mescenchymal stem cell;
(4) mescenchymal stem cell that step (3) obtains is made stem cell medicine;
(5) quality testing of stem cell medicine.
7. the method preparing stem cell medicine according to claim 6, it is characterized in that, the separation and Extraction of described mescenchymal stem cell comprises the following steps: operate in superclean bench, the umbilical cord ethanol of fresh acquisition is carried out disinfection, umbilical cord is cleaned again with normal saline, separation divests cord vessels, obtains magnificent Tong Shi glue part.
8. the method preparing stem cell medicine according to claim 6, it is characterized in that, the original cuiture of described mescenchymal stem cell comprises the following steps: shredded by magnificent Tong Shi glue, bottle is planted after dilution, adopting in CO2 gas incubator organizes adherent method to cultivate, change liquid by full dose and remove non-attached cell gradually, treat that cell fusion degree reaches 70-80% and can gather in the crops primary cell.
9. the method preparing stem cell medicine according to claim 6, is characterized in that, the quality testing of described stem cell medicine comprises antibacterial, fungus, mycoplasma, virus, endotoxin, thermal source detection and cytoactive and detects.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106620704A (en) * | 2016-11-04 | 2017-05-10 | 广州赛莱拉干细胞科技股份有限公司 | Preparation for treating diabetes and preparation method and application of preparation for treating diabetes |
| CN107460158A (en) * | 2017-08-10 | 2017-12-12 | 河南省银丰生物工程技术有限公司 | A kind of inducing umbilical cord mesenchymal stem is divided into the method to form insulin |
| CN114939157A (en) * | 2022-06-15 | 2022-08-26 | 玺瑞生命科学(深圳)有限公司 | Stem cell medicine for treating diabetes and preparation method thereof |
| JP2023002657A (en) * | 2015-01-08 | 2023-01-10 | ジュンクツセル バイオメド マニュファクチャリング ゲーエムベーハー | Genetically modified mesenchymal stem cells expressing α1-antitrypsin (AAT) |
| WO2023038161A1 (en) * | 2021-09-07 | 2023-03-16 | 주식회사 티스템 | Composition of membrane-free stem cell extract for preventing and treating diabetes |
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2014
- 2014-09-05 CN CN201410451828.9A patent/CN105456293A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2023002657A (en) * | 2015-01-08 | 2023-01-10 | ジュンクツセル バイオメド マニュファクチャリング ゲーエムベーハー | Genetically modified mesenchymal stem cells expressing α1-antitrypsin (AAT) |
| JP7374527B2 (en) | 2015-01-08 | 2023-11-07 | ジュンクツセル バイオメド マニュファクチャリング ゲーエムベーハー | Genetically modified mesenchymal stem cells expressing α1-antitrypsin (AAT) |
| CN106620704A (en) * | 2016-11-04 | 2017-05-10 | 广州赛莱拉干细胞科技股份有限公司 | Preparation for treating diabetes and preparation method and application of preparation for treating diabetes |
| CN107460158A (en) * | 2017-08-10 | 2017-12-12 | 河南省银丰生物工程技术有限公司 | A kind of inducing umbilical cord mesenchymal stem is divided into the method to form insulin |
| WO2023038161A1 (en) * | 2021-09-07 | 2023-03-16 | 주식회사 티스템 | Composition of membrane-free stem cell extract for preventing and treating diabetes |
| CN114939157A (en) * | 2022-06-15 | 2022-08-26 | 玺瑞生命科学(深圳)有限公司 | Stem cell medicine for treating diabetes and preparation method thereof |
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