CN103864900A - Induced osteogenesis short chain polypeptide as well as preparation method and application thereof - Google Patents
Induced osteogenesis short chain polypeptide as well as preparation method and application thereof Download PDFInfo
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- CN103864900A CN103864900A CN201410121725.6A CN201410121725A CN103864900A CN 103864900 A CN103864900 A CN 103864900A CN 201410121725 A CN201410121725 A CN 201410121725A CN 103864900 A CN103864900 A CN 103864900A
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- induced osteogenesis
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Abstract
The invention relates to an induced osteogenesis short chain polypeptide of which the structural formula is KIPKASSVPTEL. The induced osteogenesis short chain polypeptide has the advantages of relatively strong osteoinductive activity, capability of being synthesized in a large scale and low cost, and overcomes the defects that existing BMP-2 production equipment is very complex in preparation process, long in production cycle and low in yield, is difficult to be produced in a large scale, and is too expensive in price and the like, thereby having broad market prospects.
Description
Technical field
The invention belongs to clinical medicine domain, more particularly it relates to a kind of induced osteogenesis small peptide, its preparation method and the purposes in the medicine for the preparation of promotion osteanagenesis, bone defect repair.
Background technology
Chinese society progressively steps into aging society, and person in middle and old age's property orthopaedic disease incidence also will significantly rise, and therefore China also will face the very big demand of artificial bone graft's material.According to statistics, in the not free patient 1,500 ten thousand of state-owned limbs, owing to lacking the existing 3,000,000 people's amputation of reconstruction technique and material, damaged and the bone injury patient nearly 3,500,000 of the annual bone in the whole nation, 400,000 examples of performing the operation are per year calculated, the market scale of whole Chinese bone grafting material is also by the scale reaching more than 2,400,000,000 Renminbi, and this does not also comprise the bone grafting material needing in a large amount of dentistry orthomorphias.In the U.S., implement every year 450000 routine bone transplantations, bone grafting material market is up to 8,000,000,000 dollars.For European market, bone substitute products 35% are synthetic materials, and with annual 41.7% rate increase, bone transplantation 145775 examples in 2000, bone grafting material marketable value reaches 3,500,000,000 dollars.Above-mentioned data not yet comprise dental bone grafting material.Dental bone grafting material estimates at 1/4 of orthopaedics bone grafting material.As an important integral part in biomedical material, the market sales revenue of artificial bone graft's material is in the rate increase with annual 50%, and the market scale of artificial bone and potentiality will be very huge as can be seen here.
Artificial bone repair materials in the market mostly lacks induced osteogenesis active factor, and after these material implant into body, the T/A of skeletonization is limited, has limited to a certain extent the formation of new bone.Delicious peptide (bone morphogenetic proteins, BMPs) be to find at present unique somatomedin of induced osteogenesis separately, bone morphogenetic protein (bone morphogenetic protein BMP), as the most effective osteogenic induction active substance, has been widely used in the research of bone defect repair and union of fracture.Wherein the strongest with the osteogenic ability of BMP-2.But the natural BMP-2 transformation period is short, is diluted very soon and metabolism when topical application, not only limited amount, active unstable, can not meet clinical needs far away, and sneak into and in carrier, have many side effects.
Adopt at present the technology producer gene restructuring Delicious peptides such as molecular biology and genetic engineering both at home and abroad more, complicated process of preparation, the production cycle is long, and productive rate is low, and price is also very expensive, is difficult to scale operation.Use the gene recombination BMP-2 (rhBMP2) for preparing of transgenic technology also to have that transfection efficiency is low, expression time is shorter and the shortcoming such as the potential carinogenicity of virus vector.Although within 2002, U.S. FDA has been ratified rhBMP-2 gene engineering product, not yet enters domestic.These problems have greatly limited the clinical application of Delicious peptide.
Summary of the invention
The object of the invention is to overcome above-mentioned the deficiencies in the prior art part, particularly overcome existing BMP-2 production unit, preparation technology is very complicated, the production cycle is long, productive rate is low, be difficult to scale operation, the deficiency such as price is too expensive, and a kind of induced osteogenesis small peptide is provided.
Another object of the present invention is to the synthetic method of the induced osteogenesis small peptide that provides such.
The induced osteogenesis small peptide that another object of the present invention is to provide such is in the purposes for the preparation of promoting in the medicine of osteanagenesis, bone defect repair.
The invention provides a kind of induced osteogenesis small peptide, it is characterized in that structural formula is KIPKASSVPTEL.
The present invention also provides the synthetic method of such induced osteogenesis small peptide, it is characterized in that adopting Peptide synthesizer to pass through FMOC/tBu solid-phase polypeptide synthesis method synthetic.The thick peptide of gained is through gel chromatography preliminary purification, finally by crossing its purity of high-efficient liquid phase chromatogram technique analysis, freezing and obtain.
Induced osteogenesis small peptide of the present invention has advantages of strong bone-inducting active, can synthesize on a large scale and price lower, its having overcome existing BMP-2 production unit, preparation technology is very complicated, production cycle is long, productive rate is low, be difficult to scale operation, price is too expensive etc. not enough time, has kept high bone-inducting active, therefore, market outlook are very wide.
Embodiment
Introduce in detail the test cell line of induced osteogenesis small peptide of the present invention below.
1. induced osteogenesis polypeptide of the present invention is synthetic
Obtain induced osteogenesis small peptide KIPKASSVPTEL of the present invention by FMOC/tBu solid-phase polypeptide synthesis method synthesis method as known in the art.
2. test cell line
(1) test pertinent instruments equipment
| Instrument title | Model | Manufacturer |
| Clean bench | NU-126-006 | Nuaire |
| Research grade inverted microscope | IX41 | OLYMPUS |
| Refrigerated centrifuge | 5810R | EPPENDORF |
| Carbonic acid gas incubator | NU4750E | Nuaire |
(2) test associated materials
SD MSC mono-strain (RASMX-01001), source: Cyagen
Cell state: cell state is good, form homogeneous.
The aseptic detected result of cell: feminine gender
(3) test related reagent
SD Rat Mesenchymal Stem Cells perfect medium (RASMX-90011-500), 1 × PBS(PBS-10001-100),
SD rat interstital stem cell Osteoinductive differentiation perfect medium (RASMX-90021-200), 0.25%Trypsin-0.04%EDTA (TEDTA-10001-100),
(above reagent is all from Cyagen)
BMP-2 (Beijing Bioisystech Co., Ltd of Yi Qiao divine boat)
Induced osteogenesis small peptide of the present invention
Alkaline phosphatase assay test kit (Science and Technology Ltd. is built up in Nanjing)
(4) testing sequence
I, cell are prepared: in the time that SD MSC cytogamy reaches 80%, cell is gone down to posterity, be inoculated in 24 orifice plates that were coated with through 0.1% gelatin, for osteogenic induction.
A. cell dissociation, inoculation:
A. supernatant discarded, cleans cell 2 times with 1 × PBS, adds 1mlPBS and 1ml0.25%Trypsin-0.04%EDTA peptic cell;
B. digest 1-2 minute, under microscope, visible cell gap increases, and cell rounding is clapped the wall of culture vessel with have gentle hands, adds immediately the SDMSC perfect medium of 2mL to stop digestion.
C. use suction pipe imbitition, blow and beat gently culture vessel surface, 3-5 time repeatedly, make cell thoroughly depart from a bottle ware diapire, cell is moved in centrifuge tube, then add 1 × PBS to wash 1-2 time in culturing bottle, and washing lotion is transferred in centrifuge tube in the lump.
D. 1100rpm carries out centrifugal 4 minutes;
E. supernatant discarded adds complete culture solution, fully mixes, and is on average inoculated in 3 24 orifice plates that were coated with gelatin, shakes up, and is positioned in the CO2gas incubator of 37 ℃ and cultivates.
The preparation of II, induced osteogenesis small peptide: induced osteogenesis small peptide is weighed and is distributed into 3 parts (1mg/ parts), get a copy of it and dissolve, other are stored in-20 ℃ of refrigerators for for subsequent use.Add the aseptic water dissolution 1mg induced osteogenesis small peptide of using of 1ml in centrifuge tube, filter, be sub-packed in EP pipe, be stored in-80 ℃ of refrigerator-freezers for use.
III, induced osteogenesis:
A. just induction: in the time that cytogamy reaches 80% left and right, carry out osteogenic induction.Every group two multiple hole, wherein 2 hole negative controls: use mescenchymal stem cell complete culture solution to cultivate; 2 hole positive controls: use interstital stem cell Osteoinductive differentiation complete culture solution to cultivate; Cultivate containing the BMP-2 mescenchymal stem cell complete culture solution of 10ug/ml in 2 holes; Cultivate containing the BMP-2 mescenchymal stem cell complete culture solution of 30ug/ml in 2 holes; Cultivate containing the induced osteogenesis polypeptide mescenchymal stem cell complete culture solution of the present invention of 10ug/ml in 2 holes; Cultivate containing the induced osteogenesis polypeptide mescenchymal stem cell complete culture solution of the present invention of 30ug/ml in 2 holes.2 plates repeat.
B. within every 3 days, induce and change liquid.
IV. alkaline phosphatase detects:
Get and put cell different detection times, measure test kit (Nanjing is built up) according to alkaline phosphatase (AKP) each group of AKP content is detected.Concrete operations are as follows:
A. digest each porocyte, centrifugal after, carry out cell counting, adjust cell quantity, guarantee that 50ul cell resuspended liquid contains and be greater than 5 × 10
5cell.With 0.05ml damping fluid re-suspended cell, according to the form below adds corresponding solution successively
Note: the phenol standard stock solution of the preparation of 0.1mg/ml phenol Standard Applying Solution: 1.1mg/ml: distilled water=1:10 dilution
B. mix immediately, 520nm, 0.5cm or 1cm optical path colorimetric, blank tube zeroing, surveys each pipe absorbancy.
Calculation formula:
V. experimental result
A. two weeks alkaline phosphatase assay:
B. three weeks alkaline phosphatase assay:
Can be found out by the above results, the result of induced osteogenesis small peptide of the present invention and positive control sample is contrasted, can find that induced osteogenesis small peptide of the present invention completely can inducing cell growth.And the result of the sample of induced osteogenesis small peptide of the present invention and BMP-2 is contrasted, can find that induced osteogenesis small peptide of the present invention can realize the effect of the inducing cell suitable with BMP-2 growth, and induced osteogenesis small peptide of the present invention owing to thering is stronger bone-inducting active, can synthesize on a large scale and price lower, there are wide market outlook thereby compare BMP-2.
The present invention relates to a kind of induced osteogenesis small peptide, its structural formula is KIPKASSVPTEL.
Claims (3)
1. induced osteogenesis small peptide, is characterized in that its structural formula is KIPKASSVPTEL.
2. the method for the induced osteogenesis small peptide of preparation claim 1, is characterized in that adopting Peptide synthesizer to pass through FMOC/tBu solid-phase polypeptide synthesis method synthetic.
3. the induced osteogenesis small peptide of claim 1 is in the purposes for the preparation of promoting osteanagenesis medicine.
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| CN201410121725.6A CN103864900A (en) | 2014-03-28 | 2014-03-28 | Induced osteogenesis short chain polypeptide as well as preparation method and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2017513474A (en) * | 2014-04-10 | 2017-06-01 | ウィスコンシン・アルムナイ・リサーチ・ファウンデーションＷｉｓｃｏｎｓｉｎ Ａｌｕｍｎｉ Ｒｅｓｅａｒｃｈ Ｆｏｕｎｄａｔｉｏｎ | Hydrogel compositions for use in cell elongation and differentiation |
-
2014
- 2014-03-28 CN CN201410121725.6A patent/CN103864900A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2017513474A (en) * | 2014-04-10 | 2017-06-01 | ウィスコンシン・アルムナイ・リサーチ・ファウンデーションＷｉｓｃｏｎｓｉｎ Ａｌｕｍｎｉ Ｒｅｓｅａｒｃｈ Ｆｏｕｎｄａｔｉｏｎ | Hydrogel compositions for use in cell elongation and differentiation |
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Application publication date: 20140618 |