CN104861076B - A kind of fused polypeptide and its application in skeletonization and vascularization is promoted - Google Patents
A kind of fused polypeptide and its application in skeletonization and vascularization is promoted Download PDFInfo
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- CN104861076B CN104861076B CN201510317325.7A CN201510317325A CN104861076B CN 104861076 B CN104861076 B CN 104861076B CN 201510317325 A CN201510317325 A CN 201510317325A CN 104861076 B CN104861076 B CN 104861076B
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Abstract
Application the invention discloses a kind of fused polypeptide for belonging to Biochemistry and Molecular Biology technical field and its in skeletonization and vascularization is promoted.It is formed by the peptide fusion with the polypeptide for promoting bone uptake effect and with promotion angiogenesis function, amino acid sequence such as sequence table SEQ ID NO：Shown in 3.The fused polypeptide of the present invention promotes the peptide fusion of bone uptake and vascularization to form by having, have both the effect of promoting bone uptake and vascularization, the mutant fusion polypeptides of the present invention have effects that stronger promotion bone uptake and vascularization, for the research and development of drug of bone uptake and vascularization is promoted to provide basis.
Description
Technical field
The invention belongs to Biochemistry and Molecular Biology technical fields, and in particular to a kind of fused polypeptide and its promote
Application in skeletonization and vascularization.
Background technology
Bone morphogenetic protein (BMPs) is a kind of one group of cell factor for being capable of peculiar smell skeletonization, is found that 20 so far
It is a variety of, wherein, BMP-2 is most important growth factor in BMP families, and BMP-2 inducing bone mesenchymals stem cell is to skeletonization side
Most strong to the ability of differentiation, still, the protein half-life is short, it is difficult to persistently play a role.It is prepared and its purifying the step of ratio
It is more complicated, it is not easy to obtain, therefore, study the polypeptide segment of its active site, synthesized using chemical synthesis process, become one kind and grind
Study carefully trend.At present, the active site research of BMP-2 is ripe, still, the active and native protein phase of the segment in vivo
Than activity reduction is more, and is not easy targeting and fixes.Natural B MP-2 bioactive peptide molecules are easily degraded in vivo, internal activity
It is very low.
Vascularization polypeptide is a kind of polypeptide factor that can promote angiogenesis, organizational project, regeneration medium vessels
Formation is crucial, and current technology is to form deficiency, and no any regeneration of blood supply cannot all succeed, and native blood vessels form polypeptide point
Son is small, is easily degraded in vivo, and internal activity is very low.
Invention content
The purpose of the present invention is to provide a kind of fused polypeptides that can promote bone uptake and vascularization simultaneously.
The present invention also aims to provide the preparation side of fused polypeptide that is above-mentioned while promoting bone uptake and vascularization
Method and its application.
It is a kind of that there is the fused polypeptide for promoting bone uptake and vascularization, by having the polypeptide and tool that promote bone uptake effect
There is the peptide fusion for promoting angiogenesis function to form；The amino acid sequence such as sequence that there is the polypeptide for promoting bone uptake effect
List SEQ ID NO：Shown in 1；It is described that there is the polypeptide such as sequence table SEQ ID NO for promoting angiogenesis function：Shown in 2.
The polypeptide with promotion bone uptake effect is sequence table SEQ ID NO：Amino acid residue sequence warp shown in 1
Cross substitution, missing or the addition of 1 to 3 amino acid residue and with the polypeptide for promoting bone uptake effect.
The polypeptide with promotion angiogenesis function is sequence table SEQ ID NO：Amino acid residue sequence shown in 2
By the substitution of 1 to 3 amino acid residue, missing or addition and with the polypeptide for promoting angiogenesis function.
Application of the above-mentioned fused polypeptide in the drug for promoting bone uptake and vascularization is prepared.
A kind of pharmaceutical composition for including above-mentioned fused polypeptide.
Described pharmaceutical composition further includes the carrier of pharmaceutical acceptable, excipient or adjuvant in addition to containing fused polypeptide.
The carrier of the pharmaceutical acceptable is keyhole limpet hemocyanin, bovine serum albumin(BSA), ovalbumin or hemoglobin.
The excipient is Arabic gum, and lanolin, cocounut oil, potato starch, sweet potato starch, sodium carboxymethyl starch is low to take
For hydroxypropyl cellulose, crosslinked polyvinylpyrrolidone, the one or more in gas-producing disintegrant.
The adjuvant is Freund's complete adjuvant, and incomplete Freund's adjuvant, aluminum hydroxide adjuvant, corynebacterium, fat is more
Sugar, cell factor.
Beneficial effects of the present invention：The fused polypeptide of the present invention is by having the peptide fusion for promoting bone uptake and vascularization
Form, have both and promote bone uptake and the effect of vascularization, mutant fusion polypeptides of the invention have stronger promotion bone uptake and
The effect of vascularization, for the research and development of drug of bone uptake and vascularization is promoted to provide basis.
Specific embodiment
With reference to specific embodiment, the present invention will be further described.
Embodiment 1
1st, the preparation of fusogenic peptide
The fused polypeptide of the present embodiment is obtained by FMOC/tBu solid phase polypeptide synthesis as known in the art：
Skeletonization polypeptide SEQ ID NO：1(KIPKASSVPTELSAISTLYL)；
Angiogenesis polypeptide SEQ ID NO：2(KLTWQELYQLKYKGI)；
Fused polypeptide SEQ ID NO：3(KIPKASSVPTELSAISTLYLKLTWQELYQLKYKGI).
2nd, it is tested into Osteogenic Cells
Test associated materials：
SD MSC mono- plant (RASMX-01001), source：Cyagen
Cell state：Cell state is good, and form is uniform.
Cell Sterility testing result：It is negative
Test related reagent：
Human embryonic stem cell medium mTeSR1 (Canadian Stemcell companies), 1 × PBS (PBS-10001-100),
Human mesenchymal stem cell Osteoinductive differentiation culture medium C P1202 (Wei Tong biotech firms of Shenzhen), 0.25%
Trypsin-0.0,4%EDTA (TEDTA-10001-100), BMP-2 (Beijing Bioisystech Co., Ltd of Yi Qiao divine boats)
The induced osteogenesis polypeptide alkaline phosphatase assay kit of the present invention (Science and Technology Ltd. is built up in Nanjing).
Test procedure：
I, cell prepares：When SD MSC cell fusions are up to 80%, cell is passed on, is inoculated in through 0.1% gelatin
In 24 orifice plates being coated with, for osteogenic induction.
A. cell dissociation, inoculation：
A. it discards supernatant, cell is cleaned 2 times with 1 × PBS, add in 1mlPBS and 1ml 0.25%Trypsin-0.04%
EDTA vitellophags；
B. it digesting 1-2 minutes, visible cell gap increases under microscope, cell rounding, and the wall of culture vessel is patted with hand,
The SDMSC complete mediums for adding in 2mL immediately terminate digestion.
C. liquid is drawn with suction pipe, gently blows and beats culture vessel surface, 3-5 times repeatedly, cell is made thoroughly to be detached from a bottle ware bottom
Wall moves into cell in centrifuge tube, then adds in 1 × PBS into culture bottle and wash 1-2 times, and washing lotion is transferred to centrifuge tube together
In.
D.1100rpm centrifugation 4 minutes is carried out；
E. addition complete culture solution is discarded supernatant, abundant mixing is averagely inoculated in 3 and was coated in 12 orifice plates of gelatin,
It shakes up, is positioned in 37 DEG C of carbon dioxide incubator and cultivates.
The preparation of II, induced osteogenesis polypeptide：The weighing of induced osteogenesis polypeptide is distributed into 3 parts (1mg/ parts), takes a copy of it
It is dissolved, other are stored in -20 DEG C of refrigerators for spare.Addition 1ml is sterile to use water dissolution 1mg osteogenins in centrifuge tube,
Filtering, is sub-packed in EP pipes, is stored in -80 DEG C of refrigerator-freezers to use.
III, induced osteogenesis：
A. it just induces：When cell fusion is up to 80% or so, osteogenic induction is carried out.Every group of two multiple holes, wherein 2 holes are negative
Control：Use mescenchymal stem cell complete culture solution culture；2 hole positive controls：It is complete using interstital stem cell Osteoinductive differentiation
Full nutrient solution culture；BMP-2 mescenchymal stem cell complete culture solution culture of 2 holes containing 10ug/ml；2 holes are containing 30ug/ml's
BMP-2 mescenchymal stem cell complete culture solution cultures；Between the induced osteogenesis polypeptide of the present invention of 2 holes containing 10ug/ml
Mesenchymal stem cells complete culture solution culture；The induced osteogenesis polypeptide mescenchymal stem cell of the present invention of 2 holes containing 30ug/ml is trained completely
Nutrient solution culture.2 plates repeat.
B. it carries out within every 3 days induction and changes liquid.
IV. alkaline phosphatase detects：
Different detection time point cells are taken, according to alkaline phosphatase (AKP) assay kit (Nanjing is built up) to each group AKP
Content is detected.Concrete operations are as follows：
A. each hole cell is digested, after centrifugation, cell count is carried out, adjusts cell quantity, ensures that 50ul cell re-suspension liquids contain
Have more than 5X105Cell.Cell is resuspended with 0.05ml buffer solutions, sequentially adds corresponding solution：It measures in pipe and adds in 0.05mL
Cell suspension, 0.05mL buffer solutions, 0.05mL matrix liquids；0.05mL 0.1mg/ml phenol Standard Applying Solutions are added in standard pipe,
0.05mL buffer solutions, 0.05mL matrix liquids；0.05mL distilled waters, 0.05mL buffer solutions, 0.05mL matrix liquids are added in blank tube.
Abundant 37 DEG C of mixing water-bath 15 minutes, adds in 1.5mL developing solutions, mixing, 520nm, 0.5cm 1cm optical path colorimetrics, blank tube
Zeroing, surveys each pipe absorbance.
V. experimental result
Two weeks alkaline phosphatase assay：
The result of the fused polypeptide of the present embodiment and positive control sample is compared it can be seen from the above results,
It can be found that the fused polypeptide of the present invention is fully able to induced cell growth.And better than the effect of single skeletonization polypeptide.And it incite somebody to action this
The fused polypeptide of invention and the result of the sample of BMP-2 are compared, it can be found that the present invention fused polypeptide can realize and
The effect of the comparable induced cell growths of BMP-2, and the fused polypeptide of the present invention and BMP-2 compound use effects are more preferable.
The gene order of the fused polypeptide of the present invention and BMP-2 active peptides is transferred to animal table by the method for genetic engineering
It up to carrier, is transferred in Mice Body, obtains the cartilaginous tissue of mouse, crack protein matter passes through Western Blot detection expression
The amount of fused polypeptide and BMP-2 active peptides finds fused polypeptide, and the degradation speed of cell is slow in vivo, lives than natural BMP-2
Property stabilized peptide improve 10 times or more, active duration is long.
2nd, angiogenesis is tested
The process of angiogenesis is complicated process, and angiogenesis is carried out with the ishemic part of mouse lower limb ischemia model
Treatment, after operation 1 day, in the lower section at ligation position, i.e. gastrocnemius position carries out angiogenesis polypeptide and fused polypeptide respectively
Injection annotates the polypeptide 0.1mL of 0.1mg/mL, and injection is primary daily, 8 days after surgery, 16 days, 32 days, 8 days blood vessels after operation
It is 0.22 to generate the mouse of polypeptide therapeutic or so leg blood flow velocity ratio, and mouse of fused polypeptide treatment or so leg velocity ratio is
0.30, mouse of control group treatment or so leg velocity ratio is 0.10, mouse of angiogenesis polypeptide treatment in 32 days or so after operation
Leg blood flow velocity ratio is that the mouse of 0.28 fused polypeptide treatment or so leg velocity ratio is 0.39, mouse of control group treatment or so leg
Velocity ratio is 0.11.
Vascular wall generation could promote blood flow velocity, and described above, angiogenesis polypeptide and fused polypeptide can play thorn
Sharp capilary is formed, and improves the ability of blood flow velocity.And fused polypeptide effect is stronger.
During injection, the keyhole limpet hemocyanin 0.01ml of 0.1mg/mL is added into fused polypeptide, 8 days angiogenesis are more after operation
The mouse of peptide treatment or so leg blood flow velocity ratio is 0.25, and mouse of fused polypeptide treatment or so leg velocity ratio is 0.35, control
The mouse of group treatment or so leg velocity ratio is 0.10, mouse of angiogenesis polypeptide treatment in 32 days or so leg blood flow velocity after operation
It is 0.43 than being the mouse of 0.33 fused polypeptide treatment or so leg velocity ratio, mouse of control group treatment or so leg velocity ratio is
0.11.Prove that the keyhole limpet hemocyanin promotes fused polypeptide and plays effect.
During injection, 0.001mg sweet potato starch is added into fused polypeptide, the mouse of angiogenesis polypeptide treatment in 8 days after operation
Left and right leg blood flow velocity ratio is 0.24, and mouse of fused polypeptide treatment or so leg velocity ratio is 0.34, the mouse of control group treatment
Left and right leg velocity ratio is 0.10, and mouse of angiogenesis polypeptide treatment in 32 days or so leg blood flow velocity ratio is 0.32 fusion after operation
Mouse of polypeptide therapeutic or so leg velocity ratio is 0.41, and mouse of control group treatment or so leg velocity ratio is 0.11.Prove this kind
Sweet potato starch promotes fused polypeptide and plays effect.
During injection, 0.001mg cocounut oil is added into fused polypeptide, mouse of angiogenesis polypeptide treatment in 8 days or so after operation
Leg blood flow velocity ratio is 0.24, and mouse of fused polypeptide treatment or so leg velocity ratio is 0.34, mouse of control group treatment or so
Leg velocity ratio is 0.10, and mouse of angiogenesis polypeptide treatment in 32 days or so leg blood flow velocity ratio is 0.32 fused polypeptide after operation
Mouse for the treatment of or so leg velocity ratio is 0.41, and mouse of control group treatment or so leg velocity ratio is 0.11.Prove that the cocounut oil promotees
Effect is played into fused polypeptide.
During injection, 0.0001mg crosslinked polyvinylpyrrolidone, 8 days angiogenesis polypeptides after operation are added into fused polypeptide
Mouse for the treatment of or so leg blood flow velocity ratio is 0.24, and mouse of fused polypeptide treatment or so leg velocity ratio is 0.34, control group
Mouse for the treatment of or so leg velocity ratio is 0.10, mouse of angiogenesis polypeptide treatment in 32 days or so leg blood flow velocity ratio after operation
Mouse or so the leg velocity ratio for being the treatment of 0.32 fused polypeptide is 0.41, and mouse of control group treatment or so leg velocity ratio is
0.11.Prove that crosslinked polyvinylpyrrolidone oil promotes fused polypeptide and plays effect.
The gene order of the fused polypeptide of the present invention and vascularization polypeptide is transferred to animal by the method for genetic engineering
Expression vector is transferred in Mice Body, obtains the cartilaginous tissue of mouse, and crack protein matter is detected by Western Blot and expressed
Fused polypeptide and BMP-2 active peptides amount, the fused polypeptide of the present embodiment, the degradation speed of cell is slow in vivo, than natural
Vascularization polypeptide stability improve 10 times or more, active duration is long.
Claims (4)
1. a kind of have the fused polypeptide for promoting bone uptake and vascularization, which is characterized in that promotes bone uptake effect by having
Polypeptide and with promote vascularization effect peptide fusion form, amino acid sequence such as SEQ ID NO：Shown in 3；It is described
Amino acid sequence such as SEQ ID NO with the polypeptide for promoting bone uptake effect：Shown in 1；It is described that there is promotion vascularization to make
Polypeptide such as SEQ ID NO：Shown in 2.
2. application of the fused polypeptide described in claim 1 in the drug for promoting bone uptake and vascularization is prepared.
3. a kind of pharmaceutical composition for including fused polypeptide described in claim 1.
4. pharmaceutical composition according to claim 3, which is characterized in that described pharmaceutical composition in addition to containing fused polypeptide,
Further include pharmaceutically acceptable carrier, excipient or adjuvant.
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| CN104861076B true CN104861076B (en) | 2018-06-19 |
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| CN111320682B (en) * | 2020-02-27 | 2022-07-08 | 广州领晟医疗科技有限公司 | Application of polypeptide in preparation of medicine for promoting cartilage repair and/or treating osteoarthritis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1752103A (en) * | 2005-10-27 | 2006-03-29 | 华中科技大学同济医学院附属协和医院 | Delicious peptide 2 bioactive peptides and preparation method and application |
| CN103665143A (en) * | 2013-12-16 | 2014-03-26 | 北京博恩康生物科技有限公司 | Induced osteogenesis polypeptide, and preparation method and purposes thereof |
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| US8420774B2 (en) * | 2009-12-01 | 2013-04-16 | Wisconsin Alumni Research Foundation | Decorating hydroxyapatite biomaterials with modular biologically active molecules |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1752103A (en) * | 2005-10-27 | 2006-03-29 | 华中科技大学同济医学院附属协和医院 | Delicious peptide 2 bioactive peptides and preparation method and application |
| CN103665143A (en) * | 2013-12-16 | 2014-03-26 | 北京博恩康生物科技有限公司 | Induced osteogenesis polypeptide, and preparation method and purposes thereof |
Non-Patent Citations (2)
| Title |
|---|
| Repair of 20-mm long rabbit radial bone defects using BMP-derived peptide combined with an α-tricalcium phosphate scaffold;Atsuhiro Saito等;《Journal of Biomedical Materials Research Part A》;20060320;第700-706页 * |
| 促血管生成功能化自组装多肽的筛选及细胞学评价;乔琳;《中国优秀硕士学位论文全文数据库医药卫生科技辑》;20130715(第07期);第1章至第5章 * |
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