CN105601723B - A kind of calcining Bone targeting combination polypeptide and its synthetic method and application - Google Patents
A kind of calcining Bone targeting combination polypeptide and its synthetic method and application Download PDFInfo
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- CN105601723B CN105601723B CN201510317322.3A CN201510317322A CN105601723B CN 105601723 B CN105601723 B CN 105601723B CN 201510317322 A CN201510317322 A CN 201510317322A CN 105601723 B CN105601723 B CN 105601723B
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Abstract
The invention discloses a kind of calcining Bone targeting combination polypeptide for belonging to field of biomedicine technology and its synthetic method and applications.It is with amino acid residue sequence shown in sequence table SEQ ID NO:1.Calcining Bone targeting combination polypeptide of the invention can effectively control BMP-2 active peptides in bone tissue part, avoid promoting the generation of osteocyte, the treatment especially suitable for osteoporosis with the attached combination of other histocyte parents, targeting.
Description
Technical field
The invention belongs to field of biomedicine technology, and in particular to a kind of calcining Bone targeting combination polypeptide and its synthetic method
With application.
Background technique
Currently, select the bioactive sequence wherein to play a role to be synthesized in vitro by the research to BMP mechanism of action,
And devise with targeting combine minerals bracket active region, obtain can with calcining ox bone bracket stable combine, delay
The BMP-2 functional polypeptide released, synthesis cost are lower than the 1% of the BMP-2 market price.In the active comparative experiments with BMP-2 albumen
In, BMP-2 functional polypeptide is superior to the facilitation of the key indexes such as the proliferation of osteoblast and alkaline phosphatase activities
BMP-2 albumen.Experimental evaluation in animal body is currently carried out, main result will be obtained in 1-2 months.The functional polypeptide is in bone
Also there is potential application prospect in the treatment of matter osteoporosis.
Major issue existing for current BMP-2 is cannot to stablize with timbering material compound, and release is lost fast in vivo, therefore
It needs to increase dosage in production, increases the side effects risk such as ectopic osteogenesis and inflammation, and at high cost.U.S.'s BMP-2 product
The eukaryotic expression system somewhat expensive of use, activity is preferably.Cost is relatively low for domestic prokaryotic expression system, poor activity.
The base group modification BMP in conjunction with collagen is obtained the binding ability with bone, however collagen is more in vivo by someone before
Ingredient common to kind tissue, such as the collagen component and bone tissue of tendon are completely the same, also rich in muscular fascia
In addition this collagen contains collagen component in the tissue such as blood vessel and skin even cartilage, the various fibers of meniscus.Therefore its peace
Full property is still a pending problem.
How effectively BMP to be controlled in bone tissue part, avoid with the attached combination of other histocytes parent, become a weight
The project wanted.
Summary of the invention
The purpose of the present invention is to provide a kind of calcining Bone targeting combination polypeptide and its synthetic method and applications.
A kind of calcining Bone targeting combination polypeptide, is the polypeptide of one of following amino acid residue sequences:
1) sequence table SEQ ID NO:1 (KIPKASSVPTELSAISTLYLEPRREVAEL), shown in amino acid residue sequence
Column；
2) by amino acid residue shown in sequence table SEQ ID NO:1 (KIPKASSVPTELSAISTLYLEPRREVAEL)
Sequence passes through the polypeptide replaced, missed or added and have calcining Bone targeting binding function of 1 to 3 amino acid residue.
Further, the present invention also provides the genes for encoding above-mentioned calcining Bone targeting combination polypeptide, for using gene
The method of engineering produces aforementioned polypeptides, and takes the common purification process in genetic engineering to be purified.
The synthetic method of above-mentioned calcining Bone targeting combination polypeptide carries out in accordance with the following steps:
(1) using Rink Amide resin or Fmoc-Gly-WANG resin as starting material, with fmoc-protected amino acid
For monomer, peptide reaction being carried out using condensing agent, connects amino acid one by one, the last one peptide chain uses the mercaptopropionic acid of protection,
After obtaining protection resin, synchronizes and carry out de- side chain protecting group and cut peptide；
(2) addition cuts peptide reagent and carries out cutting peptide；
(3) it using ether as solvent, precipitates and collects the polypeptide crude product for promoting ostosis；
(4) the polypeptide crude product of ostosis is soluble in water, pH to 7.5-10.0 is adjusted with ammonium hydroxide, collects and promotes ostosis
Polypeptide；
(5) polypeptide by the promotion ostosis that step (4) are collected is isolated and purified by HPLC, obtains target product.
The condensing agent is HBTU/HOBt, TBTU/HOBt, BOP/HOBt, TBTU/HOAt, HBTU/HOA or BOP/
HOAt。
The peptide reagent of cutting is TFA/EDT/HAc/TIS/EDT.
Application of the above-mentioned calcining Bone targeting combination polypeptide in the drug that preparation promotes bone synthesis.
Beneficial effects of the present invention: calcining Bone targeting combination polypeptide of the invention can effectively control BMP-2 albumen
In bone tissue part, avoid promoting the generation of osteocyte with the attached combination of other histocyte parents, targeting, dredging especially suitable for sclerotin
The treatment of loose disease.
Specific embodiment
The present invention will be further described combined with specific embodiments below.
BMP-2 protein sequence as used in the following examples are as follows: SEQ ID NO:3 QAKHKQRKRLKSSCKRHPLYVD
FSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKV
VLKNYQDMVVEGCGCR.Its gene order are as follows: SEQ ID NO:4 (CGCGGATCCCAGGCGAAACATAAACAACGTAAAC
GCCTGAAAAGTTCCTGCAAACGTCATCCGCTGTATGTTGATTTTAGCGACGTCGGCTGGAACGATTGGATTGTTGC
TCCGCCGGGCTATCATGCGTTCTACTGCCACGGTGAATGTCCGTTTCCGCTGGCCGACCATCTGAACTCTACCAAT
CACGCAATTGTTCAGACGCTGGTTAACAGTGTCAATTCCAAAATCCCGAAAGCGTGCTGTGTCCCGACCGAACTGT
CAGCGATCTCGATGCTGTACCTGGATGAAAATGAAAAAGTGGTCCTGAAAAACTATCAAGACATGGTGGTGGAAGG
CTGTGGCTGCCGCTGACTCGAGCGG)。
Embodiment 1
1. the synthesis of the present embodiment induced osteogenesis polypeptide
SEQ ID NO:1 (KIPKASSVPTELSAISTLYLEPRREVAEL).
The synthetic method of the polypeptide of above-mentioned promotion ostosis carries out in accordance with the following steps:
(1) using Rink Amide resin or Fmoc-Gly-WANG resin as starting material, with fmoc-protected amino acid
For monomer, peptide reaction is carried out using condensing agent (TBTU/HOAt), connects amino acid one by one, the last one peptide chain uses protection
Mercaptopropionic acid, after obtaining protection resin, synchronize and carry out de- side chain protecting group and cut peptide；
(2) addition cuts peptide reagent (TFA/EDT/HAc/TIS/EDT) and carries out cutting peptide；
(3) it using ether as solvent, precipitates and collects the polypeptide crude product for promoting ostosis；
(4) the polypeptide crude product of ostosis is soluble in water, pH to 7.5-10.0 is adjusted with ammonium hydroxide, collects and promotes ostosis
Polypeptide；
(5) polypeptide by the promotion ostosis that step (4) are collected is isolated and purified by HPLC, obtains target product.
2. test cell line
(1) related equipment is tested
Instrument designation Model manufacturer
Clean bench NU-126-006 Nuaire
Research grade inverted microscope IX41 OLYMPUS
Refrigerated centrifuge 5810R EPPENDORF
Carbon dioxide incubator NU4750E Nuaire
(2) associated materials are tested
Mono- plant of SD MSC (RASMX-01001), source: Cyagen
Cell state: cell state is good, and form is uniform.
Cell Sterility testing result: negative
(3) related reagent is tested
Human embryonic stem cell medium mTeSR1 (Canadian Stemcell company), 1 × PBS (PBS-10001-100),
Human mesenchymal stem cell Osteoinductive differentiation culture medium C P1202 (Wei Tong biotech firm, Shenzhen), 0.25%
Trypsin-0.0,4%EDTA (TEDTA-10001-100), BMP-2 (Beijing Bioisystech Co., Ltd, Yi Qiao divine boat)
Induced osteogenesis polypeptide alkaline phosphatase assay kit of the invention (Science and Technology Ltd. is built up in Nanjing).
(4) test procedure
I, cell prepares: when SD MSC cell fusion is up to 80%, cell being passed on, is inoculated in through 0.1% gelatin
In 24 orifice plates being coated with, it to be used for osteogenic induction.
A. cell dissociation, inoculation:
A. it discards supernatant, is cleaned cell 2 times with 1 × PBS, 1mlPBS and 1ml 0.25%Trypsin-0.04% is added
EDTA vitellophag；
B. it digesting 1-2 minutes, visible cell gap increases under microscope, and cell rounding pats the wall of culture vessel with hand,
The SDMSC complete medium that 2mL is added immediately terminates 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 bottle ware bottom
Wall moves into cell in centrifuge tube, then 1 × PBS is added into culture bottle and washes 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, is mixed well, 3 is averagely inoculated in and was coated in 12 orifice plates of gelatin,
It shakes up, is placed 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.The sterile water of 1ml is added and dissolves 1mg osteogenin in centrifuge tube,
Filtering, is sub-packed in EP pipe, is stored in -80 DEG C of refrigerator-freezers for using.
III, induced osteogenesis:
A. just induction: 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: the culture of mescenchymal stem cell complete culture solution is used；2 hole positive controls: 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 culture；Between induced osteogenesis polypeptide of the invention of 2 holes containing 10ug/ml
Mesenchymal stem cells complete culture solution culture；Of the invention induced osteogenesis polypeptide mescenchymal stem cell 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, guarantees that 50ul cell re-suspension liquid contains
Have and is greater than 5X105Cell.Cell is resuspended with 0.05ml buffer, sequentially adds corresponding solution: 0.05mL is added in measurement pipe
Cell suspension, 0.05mL buffer, 0.05mL matrix liquid；0.05mL 0.1mg/ml phenol Standard Applying Solution is added in standard pipe,
0.05mL buffer, 0.05mL matrix liquid；0.05mL distilled water, 0.05mL buffer, 0.05mL matrix liquid are added in blank tube.
37 DEG C of water-baths 15 minutes are mixed well, 1.5mL developing solution is added, are mixed, 520nm, 0.5cm 1cm optical path colorimetric, blank tube
Zeroing, surveys each pipe absorbance.
V. experimental result
A. two weeks alkaline phosphatase assay:
| Measurement | Alkaline phosphatase (Jin Shi/100ml) |
| Negative control | 2.57 |
| Positive control | 6.79 |
| 10ug/ml(BMP-2) | 7.52 |
| 30ug/ml(BMP-2) | 13.78 |
| The induced osteogenesis polypeptide of 10ug/ml the present embodiment | 10.16 |
| The induced osteogenesis polypeptide of 30ug/ml the present embodiment | 18.86 |
B. three weeks alkaline phosphatase assay:
Substance containing pure natural promotion bone mass cells growth in above-mentioned Herba Cirsii extract, is extracted with the following method:
Take 20 meshes dry field thistle leaf powder be added petroleum ether degreasing after collected residue, after residue is dried by liquid material mass ratio be 5~
Be added ethanol solution that volume fraction be 40~90% at 35: 1, ultrasonic wave extraction 3 times under the conditions of 125~250W, 55~80 DEG C,
10~60min every time is filtered, merging filtrate, vacuum-concentrcted, recycles ethyl alcohol, and decoloration obtains field thistle with deionized water dissolving and mentions
Liquid is taken, is evaporated, white Herba Cirsii extract is obtained.
The result of the induced osteogenesis polypeptide of the present embodiment and positive control sample is carried out pair it can be seen from the above results
Than it can be found that the induced osteogenesis polypeptide of the present embodiment is fully able to induced cell growth.And it is induced osteogenesis of the invention is more
The result of the sample of peptide and BMP-2 compares, and induces bone thin it can be found that the induced osteogenesis polypeptide of the present embodiment can be realized
The effect of intracellular growth, and effect is substantially better than natural BMP-2, and auxiliary material Herba Cirsii extract, better effect is added.
BMP-2 albumen can be controlled effectively in bone tissue part, be kept away by the calcining Bone targeting combination polypeptide of the present embodiment
Exempt to promote the generation of osteocyte, the treatment especially suitable for osteoporosis with the attached combination of other histocyte parents, targeting.
Embodiment 2
1. the synthesis of the present embodiment induced osteogenesis polypeptide
SEQ ID NO:2 (KIPKASSVPAELSAISTLYLEPRRAVAEL).
The synthetic method of the polypeptide of above-mentioned promotion ostosis carries out in accordance with the following steps:
(1) using Rink Amide resin or Fmoc-Gly-WANG resin as starting material, with fmoc-protected amino acid
For monomer, peptide reaction is carried out using condensing agent (TBTU/HOBt), connects amino acid one by one, the last one peptide chain uses protection
Mercaptopropionic acid, after obtaining protection resin, synchronize and carry out de- side chain protecting group and cut peptide；
(2) addition cuts peptide reagent (TFA/EDT/HAc/TIS/EDT) and carries out cutting peptide；
(3) it using ether as solvent, precipitates and collects the polypeptide crude product for promoting ostosis；
(4) the polypeptide crude product of ostosis is soluble in water, pH to 7.5-10.0 is adjusted with ammonium hydroxide, collects and promotes ostosis
Polypeptide；
(5) polypeptide by the promotion ostosis that step (4) are collected is isolated and purified by HPLC, obtains target product.
2. test cell line
(1) related equipment is tested
Instrument designation Model manufacturer
Clean bench NU-126-006 Nuaire
Research grade inverted microscope IX41 OLYMPUS
Refrigerated centrifuge 5810R EPPENDORF
Carbon dioxide incubator NU4750E Nuaire
(2) associated materials are tested
Mono- plant of SD MSC (RASMX-01001), source: Cyagen
Cell state: cell state is good, and form is uniform.
Cell Sterility testing result: negative
(3) related reagent is tested
Human embryonic stem cell medium mTeSR1 (Canadian Stemcell company), 1 × PBS (PBS-10001-100),
Human mesenchymal stem cell Osteoinductive differentiation culture medium C P1202 (Wei Tong biotech firm, Shenzhen), 0.25%
Trypsin-0.0,4%EDTA (TEDTA-10001-100), BMP-2 (Beijing Bioisystech Co., Ltd, Yi Qiao divine boat)
Induced osteogenesis polypeptide alkaline phosphatase assay kit of the invention (Science and Technology Ltd. is built up in Nanjing).
(4) test procedure
I, cell prepares: when SD MSC cell fusion is up to 80%, cell being passed on, is inoculated in through 0.1% gelatin
In 24 orifice plates being coated with, it to be used for osteogenic induction.
A. cell dissociation, inoculation:
A. it discards supernatant, is cleaned cell 2 times with 1 × PBS, 1mlPBS and 1ml 0.25%Trypsin-0.04% is added
EDTA vitellophag；
B. it digesting 1-2 minutes, visible cell gap increases under microscope, and cell rounding pats the wall of culture vessel with hand,
The SDMSC complete medium that 2mL is added immediately terminates 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 bottle ware bottom
Wall moves into cell in centrifuge tube, then 1 × PBS is added into culture bottle and washes 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, is mixed well, 3 is averagely inoculated in and was coated in 12 orifice plates of gelatin,
It shakes up, is placed 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.The sterile water of 1ml is added and dissolves 1mg osteogenin in centrifuge tube,
Filtering, is sub-packed in EP pipe, is stored in -80 DEG C of refrigerator-freezers for using.
III, induced osteogenesis:
A. just induction: 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: the culture of mescenchymal stem cell complete culture solution is used；2 hole positive controls: 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 culture；Between induced osteogenesis polypeptide of the invention of 2 holes containing 10ug/ml
Mesenchymal stem cells complete culture solution culture；Of the invention induced osteogenesis polypeptide mescenchymal stem cell 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, guarantees that 50ul cell re-suspension liquid contains
Have and is greater than 5X105Cell.Cell is resuspended with 0.05ml buffer, sequentially adds corresponding solution: 0.05mL is added in measurement pipe
Cell suspension, 0.05mL buffer, 0.05mL matrix liquid；0.05mL 0.1mg/ml phenol Standard Applying Solution is added in standard pipe,
0.05mL buffer, 0.05mL matrix liquid；0.05mL distilled water, 0.05mL buffer, 0.05mL matrix liquid are added in blank tube.
37 DEG C of water-baths 15 minutes are mixed well, 1.5mL developing solution is added, are mixed, 520nm, 0.5cm 1cm optical path colorimetric, blank tube
Zeroing, surveys each pipe absorbance.
V. experimental result
A. two weeks alkaline phosphatase assay:
| Measurement | Alkaline phosphatase (Jin Shi/100ml) |
| Negative control | 2.57 |
| Positive control | 6.79 |
| 10ug/ml(BMP-2) | 7.52 |
| 30ug/ml(BMP-2) | 13.78 |
| The induced osteogenesis polypeptide of 10ug/ml the present embodiment | 16.16 |
| The induced osteogenesis polypeptide of 30ug/ml the present embodiment | 31.86 |
B. three weeks alkaline phosphatase assay:
| Measurement | Alkaline phosphatase (Jin Shi/100ml) |
| Negative control | 6.54 |
| Positive control | 12.86 |
| 10ug/ml(BMP-2) | 15.52 |
| 30ug/ml(BMP-2) | 17.81 |
| The induced osteogenesis polypeptide of 10ug/ml the present embodiment | 23.11 |
| The induced osteogenesis polypeptide of 30ug/ml the present embodiment | 37.81 |
The result of the induced osteogenesis polypeptide of the present embodiment and positive control sample is carried out pair it can be seen from the above results
Than it can be found that the induced osteogenesis polypeptide of the present embodiment is fully able to induced cell growth.And by the induced osteogenesis of the present embodiment
The result of the sample of polypeptide and BMP-2 compares, it can be found that the induced osteogenesis polypeptide of the present embodiment can be realized induction bone
The effect of cell growth, and inducing effect is more preferable than embodiment 1.
BMP-2 albumen can be controlled effectively in bone tissue part, be kept away by the calcining Bone targeting combination polypeptide of the present embodiment
Exempt to promote the generation of osteocyte, the treatment especially suitable for osteoporosis with the attached combination of other histocyte parents, targeting.
Embodiment 3
1. the synthesis of the present embodiment induced osteogenesis polypeptide
SEQ ID NO:5 (KIPKASSVPTELSAISTLYL DSSDSSDSSDSSDSSDSS).
The synthetic method of the polypeptide of above-mentioned promotion ostosis carries out in accordance with the following steps:
(1) using Rink Amide resin or Fmoc-Gly-WANG resin as starting material, with fmoc-protected amino acid
For monomer, peptide reaction is carried out using condensing agent (TBTU/HOAt), connects amino acid one by one, the last one peptide chain uses protection
Mercaptopropionic acid, after obtaining protection resin, synchronize and carry out de- side chain protecting group and cut peptide；
(2) addition cuts peptide reagent (TFA/EDT/HAc/TIS/EDT) and carries out cutting peptide；
(3) it using ether as solvent, precipitates and collects the polypeptide crude product for promoting ostosis；
(4) the polypeptide crude product of ostosis is soluble in water, pH to 7.5-10.0 is adjusted with ammonium hydroxide, collects and promotes ostosis
Polypeptide；
(5) polypeptide by the promotion ostosis that step (4) are collected is isolated and purified by HPLC, obtains target product.
2. test cell line
(1) related equipment is tested
Instrument designation Model manufacturer
Clean bench NU-126-006 Nuaire
Research grade inverted microscope IX41 OLYMPUS
Refrigerated centrifuge 5810R EPPENDORF
Carbon dioxide incubator NU4750E Nuaire
(2) associated materials are tested
Mono- plant of SD MSC (RASMX-01001), source: Cyagen
Cell state: cell state is good, and form is uniform.
Cell Sterility testing result: negative
(3) related reagent is tested
Human embryonic stem cell medium mTeSR1 (Canadian Stemcell company), 1 × PBS (PBS-10001-100),
Human mesenchymal stem cell Osteoinductive differentiation culture medium C P1202 (Wei Tong biotech firm, Shenzhen), 0.25%
Trypsin-0.0,4%EDTA (TEDTA-10001-100), BMP-2 (Beijing Bioisystech Co., Ltd, Yi Qiao divine boat)
Induced osteogenesis polypeptide alkaline phosphatase assay kit of the invention (Science and Technology Ltd. is built up in Nanjing).
(4) test procedure
I, cell prepares: when SD MSC cell fusion is up to 80%, cell being passed on, is inoculated in through 0.1% gelatin
In 24 orifice plates being coated with, it to be used for osteogenic induction.
A. cell dissociation, inoculation:
A. it discards supernatant, is cleaned cell 2 times with 1 × PBS, 1mlPBS and 1ml 0.25%Trypsin-0.04% is added
EDTA vitellophag；
B. it digesting 1-2 minutes, visible cell gap increases under microscope, and cell rounding pats the wall of culture vessel with hand,
The SDMSC complete medium that 2mL is added immediately terminates 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 bottle ware bottom
Wall moves into cell in centrifuge tube, then 1 × PBS is added into culture bottle and washes 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, is mixed well, 3 is averagely inoculated in and was coated in 12 orifice plates of gelatin,
It shakes up, is placed 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.The sterile water of 1ml is added and dissolves 1mg osteogenin in centrifuge tube,
Filtering, is sub-packed in EP pipe, is stored in -80 DEG C of refrigerator-freezers for using.
III, induced osteogenesis:
A. just induction: 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: the culture of mescenchymal stem cell complete culture solution is used；2 hole positive controls: 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 culture；Between induced osteogenesis polypeptide of the invention of 2 holes containing 10ug/ml
Mesenchymal stem cells complete culture solution culture；Of the invention induced osteogenesis polypeptide mescenchymal stem cell 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, guarantees that 50ul cell re-suspension liquid contains
Have and is greater than 5X105Cell.Cell is resuspended with 0.05ml buffer, sequentially adds corresponding solution: 0.05mL is added in measurement pipe
Cell suspension, 0.05mL buffer, 0.05mL matrix liquid；0.05mL 0.1mg/ml phenol Standard Applying Solution is added in standard pipe,
0.05mL buffer, 0.05mL matrix liquid；0.05mL distilled water, 0.05mL buffer, 0.05mL matrix liquid are added in blank tube.
37 DEG C of water-baths 15 minutes are mixed well, 1.5mL developing solution is added, are mixed, 520nm, 0.5cm 1cm optical path colorimetric, blank tube
Zeroing, surveys each pipe absorbance.
V. experimental result
A. two weeks alkaline phosphatase assay:
| Measurement | Alkaline phosphatase (Jin Shi/100ml) |
| Negative control | 2.57 |
| Positive control | 6.79 |
| 10ug/ml(BMP-2) | 7.52 |
| 30ug/ml(BMP-2) | 13.78 |
| The induced osteogenesis polypeptide of 10ug/ml the present embodiment | 10.22 |
| The induced osteogenesis polypeptide of 30ug/ml the present embodiment | 18.45 |
B. three weeks alkaline phosphatase assay:
| Measurement | Alkaline phosphatase (Jin Shi/100ml) |
| Negative control | 6.54 |
| Positive control | 12.86 |
| 10ug/ml(BMP-2) | 15.52 |
| 30ug/ml(BMP-2) | 17.81 |
| The induced osteogenesis polypeptide of 10ug/ml the present embodiment | 18.44 |
| The induced osteogenesis polypeptide of 30ug/ml the present embodiment | 25.87 |
The result of the induced osteogenesis polypeptide of the present embodiment and positive control sample is carried out pair it can be seen from the above results
Than it can be found that the induced osteogenesis polypeptide of the present embodiment is fully able to induced cell growth.And it is induced osteogenesis of the invention is more
The result of the sample of peptide and BMP-2 compares, and induces bone thin it can be found that the induced osteogenesis polypeptide of the present embodiment can be realized
The effect of intracellular growth, and effect is substantially better than natural BMP-2.
BMP-2 albumen can be controlled effectively in bone tissue part, be kept away by the calcining Bone targeting combination polypeptide of the present embodiment
Exempt from and the attached combination of other histocyte parents, the generation of targeting promotion osteocyte, especially in the skeletonization region binding force of low mineralising
Relatively high, the region of new bone formation, targeting combines effect obvious, the treatment especially suitable for osteoporosis.
Embodiment 4
1. the preparation of the present embodiment induced osteogenesis albumen
SEQ ID NO:6
The method of above-mentioned albumen traditionally genetic engineering, carrier construction, transformation gene engineering cell are cultivated, and cracking is pure
Change is prepared.
2. test cell line
(1) related equipment is tested
Instrument designation Model manufacturer
Clean bench NU-126-006 Nuaire
Research grade inverted microscope IX41 OLYMPUS
Refrigerated centrifuge 5810R EPPENDORF
Carbon dioxide incubator NU4750E Nuaire
(2) associated materials are tested
Mono- plant of SD MSC (RASMX-01001), source: Cyagen
Cell state: cell state is good, and form is uniform.
Cell Sterility testing result: negative
(3) related reagent is tested
Human embryonic stem cell medium mTeSR1 (Canadian Stemcell company), 1 × PBS (PBS-10001-100),
Human mesenchymal stem cell Osteoinductive differentiation culture medium C P1202 (Wei Tong biotech firm, Shenzhen), 0.25%
Trypsin-0.0,4%EDTA (TEDTA-10001-100), BMP-2 (Beijing Bioisystech Co., Ltd, Yi Qiao divine boat)
Induced osteogenesis polypeptide alkaline phosphatase assay kit of the invention (Science and Technology Ltd. is built up in Nanjing).
(4) test procedure
I, cell prepares: when SD MSC cell fusion is up to 80%, cell being passed on, is inoculated in through 0.1% gelatin
In 24 orifice plates being coated with, it to be used for osteogenic induction.
A. cell dissociation, inoculation:
A. it discards supernatant, is cleaned cell 2 times with 1 × PBS, 1mlPBS and 1ml 0.25%Trypsin-0.04% is added
EDTA vitellophag；
B. it digesting 1-2 minutes, visible cell gap increases under microscope, and cell rounding pats the wall of culture vessel with hand,
The SDMSC complete medium that 2mL is added immediately terminates 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 bottle ware bottom
Wall moves into cell in centrifuge tube, then 1 × PBS is added into culture bottle and washes 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, is mixed well, 3 is averagely inoculated in and was coated in 12 orifice plates of gelatin,
It shakes up, is placed in 37 DEG C of carbon dioxide incubator and cultivates.
The preparation of II, induced osteogenesis albumen: the weighing of induced osteogenesis albumen 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.The sterile water of 1ml is added and dissolves 1mg osteogenin in centrifuge tube,
Filtering, is sub-packed in EP pipe, is stored in -80 DEG C of refrigerator-freezers for using.
III, induced osteogenesis:
A. just induction: 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: the culture of mescenchymal stem cell complete culture solution is used；2 hole positive controls: 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 culture；Between induced osteogenesis polypeptide of the invention of 2 holes containing 10ug/ml
Mesenchymal stem cells complete culture solution culture；Of the invention induced osteogenesis polypeptide mescenchymal stem cell 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, guarantees that 50ul cell re-suspension liquid contains
Have and is greater than 5X105Cell.Cell is resuspended with 0.05ml buffer, sequentially adds corresponding solution: 0.05mL is added in measurement pipe
Cell suspension, 0.05mL buffer, 0.05mL matrix liquid；0.05mL 0.1mg/ml phenol Standard Applying Solution is added in standard pipe,
0.05mL buffer, 0.05mL matrix liquid；0.05mL distilled water, 0.05mL buffer, 0.05mL matrix liquid are added in blank tube.
37 DEG C of water-baths 15 minutes are mixed well, 1.5mL developing solution is added, are mixed, 520nm, 0.5cm 1cm optical path colorimetric, blank tube
Zeroing, surveys each pipe absorbance.
V. experimental result
A. two weeks alkaline phosphatase assay:
B. three weeks alkaline phosphatase assay:
| Measurement | Alkaline phosphatase (Jin Shi/100ml) |
| Negative control | 6.54 |
| Positive control | 12.86 |
| 10ug/ml(BMP-2) | 15.52 |
| 30ug/ml(BMP-2) | 17.81 |
| The induced osteogenesis albumen of 10ug/ml the present embodiment | 18.39 |
| The induced osteogenesis albumen of 30ug/ml the present embodiment | 25.99 |
The result of the induced osteogenesis albumen of the present embodiment and positive control sample is carried out pair it can be seen from the above results
Than it can be found that the induced osteogenesis albumen of the present embodiment is fully able to induced cell growth.And by induced osteogenesis egg of the invention
The result of white and BMP-2 sample compares, and induces bone thin it can be found that the induced osteogenesis albumen of the present embodiment can be realized
The effect of intracellular growth, and effect is substantially better than natural BMP-2.The calcining Bone targeting binding protein of the present embodiment, can be effective
By the control of BMP-2 albumen in bone tissue part, avoid and the attached combination of other histocytes parent, targeting promote the life of osteocyte
At especially relatively high in the skeletonization region binding force of low mineralising, the region of new bone formation, targeting combines effect obvious, especially
Treatment suitable for osteoporosis.
Claims (1)
1. a kind of composition for promoting ostosis, which is characterized in that including 30 μ g/ml BMP-2,30 μ g/ml forging bone targets
To in conjunction with polypeptide, 30 μ g/ml Herba Cirsii extracts；
Amino acid sequence amino acid residue sequence as shown in sequence table SEQ ID NO:1 of the calcining Bone targeting combination polypeptide
Column；
The extracting method of the Herba Cirsii extract are as follows: collected after taking the dry field thistle leaf powder of 20 meshes that petroleum ether degreasing is added residual
Slag by liquid material mass ratio is the ethanol solution that 5~35: 1 addition volume fraction is 40~90% after drying residue, 125~
250W, ultrasonic wave extraction 3 times under the conditions of 55~80 DEG C, 10~60min, filters, merging filtrate, vacuum-concentrcted every time, returns
Ethyl alcohol is received, decoloration obtains field thistle extracting solution with deionized water dissolving, is evaporated, obtains white Herba Cirsii extract.
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