CN110368485A - Antler polypeptide is preparing the purposes in the drug for treating osteosarcoma - Google Patents
Antler polypeptide is preparing the purposes in the drug for treating osteosarcoma Download PDFInfo
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- CN110368485A CN110368485A CN201910614399.5A CN201910614399A CN110368485A CN 110368485 A CN110368485 A CN 110368485A CN 201910614399 A CN201910614399 A CN 201910614399A CN 110368485 A CN110368485 A CN 110368485A
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- cell
- osteosarcoma
- antler polypeptide
- antler
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention discloses antler polypeptides to prepare the purposes in the drug for treating osteosarcoma, wherein the amino acid sequence of the antler polypeptide is as shown in SEQ ID NO:1.Antler polypeptide can significantly inhibit the proliferation of human osteosarcoma cell MG-63 and U2OS, and induce human osteosarcoma cell's apoptosis, and concentration be 80 μ g/ml when effect it is the most significant.Antler polypeptide can be used as the potential drug of clinical treatment of osteosarcoma, slow down the growth of tumour, promote to cure osteosarcoma.
Description
Technical field
The invention belongs to biomedicine technical field, it is related to a kind of antler polypeptide and is preparing the drug for treating osteosarcoma
In purposes.
Background technique
Pilose antler is the traditional rare traditional Chinese medicine in China, has thousands of years medicinal histories so far, while being also mammal
Unique reproducible organ, scholar as the perfect regenerating model of the tissues such as nerve, blood vessel, bone and carries out in the world
In-depth study.Recent study personnel carry out in the extraction and identification discovery pilose antler of effective component pilose antler using modernism
Amino acid, growth factor, steroidal compounds and lipid rich in etc. improve the immunity of the human body treatment disease, nourish body
Body has good effect (Y.S.Huo, H.Huo, J.Zhang.The contribution of deer velvet antler
research to the modern biological medicine[J].Chin J Integr Med.2014；20(10):
723-8.)。
(Zhang Zhengyao, Duan Lengxin, Zhou Qiuli wait the chemical structure and bioactivity [J] of sika deer velvet antler polypeptide to Zhang Zhengyao etc.
Chemical Journal of Chinese Universities .2012；33 (9): 2000-4) a kind of antler polypeptide is disclosed, which is to be handed over using ion
The Measurements for Biochemistry such as chromatography, gel permeation chromatography and reversed-phase high performance liquid chromatography chromatography are changed, it is isolated from sika deer velvet antler
A kind of novel polypeptide, SDS-PAGE electrophoresis showed is a band, and HPLC map is simple spike, and MALDI-TOF MS provides the polypeptide
Accurate molecular weight be 3263.4, isoelectric point pI=8.15. primary structure is studies have shown that the polypeptide is residual by 32 amino acid
The straight-chain polypeptide of base composition, is free of cysteine, is rich in valine, lysine, leucine and glycine.
Osteosarcoma is the most common primary malignant bone tumor in Children and teenager, is mainly in distal femur and shin bone is close
End, and male's disease incidence is higher than women, this disease is still rarer, only accounts for the tight less than 1%, but after the onset of tumor patient
Ghost image rings ability to act or even the amputation of patient, and treatment method mostly uses chemotherapy, radiotherapy and the surgical operation side of combining at present
Method, but chemotherapy resistance is still the obstruction of clinical treatment of osteosarcoma.Treatment for osteosarcoma, it is most important that induction tumour cell withers
It dies, inhibit Growth of Osteosarcoma.The identification of apoptotic signal is proposed with the treatment that the research and development for promoting apoptotic agent are osteosarcoma
New strategy (effect [J] the Medical review of Liang Renzheng, Ji Guangrong autophagy and apoptosis in osteosarcoma, 2019,25 (05):
904-908.)。
Summary of the invention
To overcome problems of the prior art, the present invention provides antler polypeptide in preparation for treating osteosarcoma
Purposes in drug.
The technical solution of the present invention is as follows:
Antler polypeptide is preparing the purposes in the drug for treating osteosarcoma, wherein the amino acid of the antler polypeptide
Sequence is as shown in SEQ ID NO:1.Specifically, the amino acid sequence of antler polypeptide is VLSATDKTNVLAAWGKVGGNAPAFG
AEALERM；That is, Zhang Zhengyao, Duan Lengxin, Zhou Qiuli, wait chemical structure and bioactivity [J] high of sika deer velvet antler polypeptide etc. to learn
School chemistry journal .2012；33 (9): antler polypeptide disclosed in 2000-4.
In a specific embodiment, the drug is by inhibiting the proliferation of osteosarcoma cell and/or inducing bone
Sarcoma cell apoptosis and treat osteosarcoma.
In a specific embodiment, TGF-β 1, p-smad2/3 in the antler polypeptide up-regulation osteosarcoma cell
With the expression of Bax, the expression of BCL-2 in osteosarcoma cell is lowered.
In a specific embodiment, the expression for being expressed as protein level.
In a specific embodiment, the osteosarcoma is the osteosarcoma in mammal.
In a specific embodiment, the mammal is people.
In a specific embodiment, osteosarcoma cell is U2OS and/or MG-63 cell line.
The beneficial effects of the present invention are: the invention discloses antler polypeptides in preparing the drug for treating osteosarcoma
Purposes, which can significantly inhibit the proliferation of osteosarcoma cell, and induce apoptosis in osteosarcoma cells, especially people's bone
Sarcoma cell U2OS, when concentration is 80 μ g/ml, effect is the most significant.Antler polypeptide can be used as the potential medicine of clinical treatment of osteosarcoma
Object slows down the growth of tumour, promotes to cure osteosarcoma.
Detailed description of the invention
Influence Fig. 1 shows antler polypeptide to MG-63 cell Proliferation；
Fig. 2 indicates influence that antler polypeptide is proliferated MG-63 for 24 hours；
Fig. 3 indicates influence 48h of the antler polypeptide to MG-63 cell Proliferation；
Fig. 4 indicates influence 72h of the antler polypeptide to MG-63 cell Proliferation；
Fig. 5 indicates influence of the antler polypeptide to U2OS cell Proliferation；
Fig. 6 indicates influence of the antler polypeptide to U2OS cell Proliferation for 24 hours；
Fig. 7 indicates influence 48h of the antler polypeptide to U2OS cell Proliferation；
Influence 72h Fig. 8 shows antler polypeptide to U2OS cell Proliferation；
Fig. 9 indicates antler polypeptide regulation osteosarcoma U 2OS apoptosis-related protein and TGF-β signal path correlation egg
It is white.
Specific embodiment
Below in conjunction with attached drawing, the present invention is further illustrated by embodiment, but not as limitation of the present invention.It mentions below
Specific material and its source used in embodiment of the present invention are supplied.However, it should be understood that these are only example
Property, it is not intended to the limitation present invention, it is same or similar with the type of following reagent and instrument, model, quality, property or function
Material may be incorporated for implement the present invention.Experimental method used in following embodiments is routine unless otherwise specified
Method.The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1: detection antler polypeptide inhibits the proliferation of osteosarcoma cell MG-63 and U2OS
(1) cell recovery: being adjusted to 37 DEG C for water bath temperature in iuntercellular, by freeze-stored cell (MG-63 cell and U2OS
Cell is purchased from Chinese Academy of Sciences's Shanghai cell bank) after being taken out in liquid nitrogen container, it is immediately placed in 37 DEG C of water-baths, gently shakes jelly
Pipe is deposited, it is made all to melt in 1 minute and (be no more than 3 minutes).Cells frozen storing liquid is transferred in centrifuge tube after melting, and is carried out
The centrifugation 5min of 1000rpm, discards supernatant liquid after centrifugation, is resuspended with containing 10%FBS (v/v) cell culture fluid, cell is mixed
After move into 25cm2Tissue Culture Flask is put into 5%CO2, 37 DEG C of constant incubators are cultivated.
(2) cell culture: osteosarcoma cell MG-63 uses MEM culture solution, and U2OS uses 1640 culture medium, is added 10%
Fetal calf serum, 2% dual anti-(Pen .- Strep) are put into constant incubator and are cultivated, and after cell culture fluid discoloration, discard original
The culture solution come, and cleaned with PBS solution and new culture solution is added twice, it is grown in cell and occupies 80% face of Tissue Culture Flask
Passage or plating procedure are carried out when product.
(3) cell passes on: the good osteosarcoma cell of the upgrowth situation of logarithmic growth phase discards culture solution, is first added
2ml PBS cleans cell, after cleaning twice, 1ml trypsase is added, is put into 37 DEG C, 5%CO2It is digested in incubator,
Equivalent culture medium is added when cell shrinkage and terminates digestion for microscopically observation.Cell bottle is blown and beaten with disposable sterilized plastic tube
Wall blows and beats cell from cell culture bottle wall, moves into and is centrifuged 1000rpm 5min in centrifuge tube, after centrifugation, abandons
Supernatant is removed, 1ml culture solution is added, cell is resuspended, so that cell is uniformly dispersed with liquid-transfering gun piping and druming cell precipitation, cell is distinguished
It is put into addition 4ml culture solution in two culture bottles, is put into constant temperature cell incubator and cultivates.
(4) cell cryopreservation: the osteosarcoma cell of logarithmic growth phase discards original cell culture fluid, and PBS solution is added
Cleaning, digests the osteosarcoma cell of adherent growth with trypsase after cleaning, moves into centrifuge tube and is centrifuged
1000rpm 5min after centrifugation, is resuspended cell precipitation with 900 μ l culture solutions, is transferred in cryopreservation tube, 100 μ l are added
DMSO has simultaneously marked Cell Name, date and name in cryopreservation tube, is put into freezing storing box -80 DEG C after 24 hours, moves to liquid nitrogen
It is saved for a long time in tank.
(5) MTT experiment: mtt assay is a kind of a kind of method for detecting cell relative populations and cell relative viability, is used extensively
In cytotoxicity experiment and cell proliferation experiment, principle is that MTT reagent can be taken off by succinic acid is generated in living cells mitochondria
The reduction of hydrogen enzyme, and then formation is not soluble in water, is dissolved in the bluish violet Jie Jing formazan of organic solvent, utilizes DMSO dissolution first in experiment
Za, by microplate reader detect absorbance can indirectly react living cells quantity (MTT concentration be 5mg/ml, need to be kept in dark place
And use 0.22 μm of membrane filtration degerming).
It takes logarithmic phase tumour cell to be digested, equivalent culture solution is added and terminates digestion, moves into centrifuge tube and is centrifuged
1000rpm 5min is added cell culture fluid and cell precipitation is resuspended after centrifugation, the cell suspension that will be resuspended passes through cell
Tally counts, and adjusting number of cells is 10 × 104/ ml, take 100 μ l cell liquid be added 96 orifice plates, 6 holes of each concentration, four
Zhou Kongzhong is added 200 μ l and contains dual anti-sterile PBS.37 DEG C of constant incubators are put into, is added is dissolved with corresponding culture solution afterwards for 24 hours
And diluted antler polypeptide (synthesis of Shanghai De Chi Biotechnology Co., Ltd) make its final concentration of 0,0.625,1.25,2.5,5,
10,96 orifice plates are put into 37 DEG C of CO after dosing by 20,40,80 μ g/ml2Cultivated respectively in incubator for 24 hours, 48h, 72h.Accordingly
Time point takes out the every hole of 96 orifice plates and 200 μ l of MTT solution is added, and is put into CO2Cell incubator continues to be incubated for 4h, abandons after incubation
Fall supernatant in orifice plate, 150 μ l DMSO are added in every hole, and concussion 96 orifice plate 10 minutes dissolving crystallized, detect 490nm with microplate reader
Locate each hole OD value (light absorption value).
As a result referring to Fig. 1 to Fig. 8 and the following table 1 and table 2, antler polypeptide starts to significantly inhibit MG-63 cell in 1.25ug/ml
Proliferation, and inhibitory effect is more obvious, and U2OS need to be than the antler polypeptide of MG-63 higher concentration, i.e., as concentration increases
In 2.5ug/ml, antler polypeptide starts to significantly inhibit the proliferation of U2OS, while brighter also with concentration raising inhibitory effect
It is aobvious.
Table 1: various concentration antler polypeptide influences n=6 to MG-63 cell Proliferation
Table 2: various concentration antler polypeptide influences n=6 to U2OS cell Proliferation
Embodiment 2: protein level detects antler polypeptide and induces human osteosarcoma cell U2OS apoptosis
1, it chooses the good osteosarcoma cell of logarithmic phase growth conditions to be digested, osteosarcoma U 2OS is transferred to 6
It is cultivated in orifice plate, cell culture fluid is the same as embodiment 1.It is counted before carrying out bed board, keeps each hole cell number identical, every hole
Cell number is 2-5 × 105It is a.When osteosarcoma cell grows into 70%-80% in six orifice plates, osteosarcoma U 2OS is divided into three
Group discards original cell culture fluid, after being cleaned twice with PBS solution, is added new culture solution and antler polypeptide, every group according to
0 μ g/ml, 20 μ g/ml, 80 μ g/ml, tri- concentration carry out interfering effects of drug respectively, and various concentration antler polypeptide is added in every group of two holes
It is marked on six orifice plate lids afterwards, is put into constant temperature CO2Cell incubator cultivates 48h.
2, culture solution in six orifice plates is sucked out with pipettor, PBS cleaning is added once, 500 μ l trypsase are added in each hole
Digestion, after cell shrinkage is rounded plus culture solution of the equivalent containing 10% (v/v) fetal calf serum terminates digestion, anti-with 1ml pipettor
Attached cell is blown and beaten again, so that cell is suspended completely, is then transferred into centrifuge tube, and 1000rpm is centrifuged 5min, and centrifugation terminates
After discard supernatant liquid, 1ml PBS solution is added, cell precipitation is resuspended, be centrifuged again after resuspension, 2000rpm is centrifuged 5min, discards
PBS solution in centrifuge tube as far as possible removes remaining PBS, according to 100:1 ratio be added RIPA lysate (P0013B,
Beyotime) and protease inhibitors PMSF (ST506, Beyotime), 4 DEG C of refrigerators are put into crack 1 hour, after cracking,
Centrifuge tube is put into high speed freezing centrifuge 12000rpm centrifugation 15min, moves into supernatant in new centrifuge tube after centrifugation,
Precipitating is discarded, protein concentration is calculated according to BCA quantitative (P0012, Beyotime), remaining albumen is mixed into 5 × loading
Buffer is put in -20 DEG C of storages after boiling 10min.
3, SDS-Page electrophoresis
(1) installation protein electrophoresis device prepares concentration glue and separation gel according to table 3, by the U2OS egg of extraction after being gelled admittedly
White carry out electrophoresis, adjusts the voltage and current of electrophoresis, stops electrophoresis when bromophenol blue is migrated to separation gel bottom.
The separation gel of table 3:SDS-PAGE and the formula of concentration glue
(2) transferring film: preparing ten filter paper, two pieces of sponges and a pvdf membrane, (size of film is identical as separation gel size, makes
5min is impregnated in methyl alcohol with preceding), the above article impregnates about 10min with transferring film liquid using preceding.After electrophoresis, according to pre-staining
The gel comprising target protein is cut in the position of label, is put into the immersion of 1 × transferring film liquid.Sponge is successively put in white board,
Then three layers of filter paper, pvdf membrane, separation gel, three layers of filter paper, sponge cover black plate and slightly squeeze, by assembled transferring film plate
It is put into transferring film electrophoresis tank, glue surface is towards cathode, and film surface is to anode.Constant current 200mA, transferring film 100min.Wherein pvdf membrane with point
It to be fitted closely from glue and avoid the occurrence of bubble.
(3) it closes
Pvdf membrane is put into confining liquid after transferring film, 3h is closed on room temperature shaker.
(4) primary antibody is incubated for
The diluted monoclonal antibody of pvdf membrane confining liquid (rabbit-anti people's GAPDH antibody, the rabbit-anti people BCL- that closing is terminated
2 antibody, rabbit-anti people's BAX antibody, rabbit against human T GF- beta 1 antibodies, rabbit-anti people's p-smad2/3 antibody are purchased from Abcam company, Britain)
It is incubated for, is incubated overnight under the conditions of 4 DEG C, primary antibody is recycled after incubation.
(5) film is washed
The pvdf membrane that antibody incubation terminates is cleaned on shaking table with TBST solution, is cleaned 5 times, each 5min.
(6) secondary antibody is incubated for
The pvdf membrane that TBST is washed is used to secondary antibody (goat-anti rabbit secondary antibody, the life of Beijing Zhong Shan Golden Bridge diluted after washing film
Object Technology Co., Ltd.) incubation at room temperature 1h.
(7) the same step of film (5) is washed
(8) shine development
Luminescent solution A, B are mixed into (1:1) by ECL kit (P0018, Beyotime) specification, it will with tweezers
Pvdf membrane is placed into detection plate, and luminescent solution is added dropwise, detection is exposed in gel imager.
Experimental result is as shown in figure 9, antler polypeptide can raise TGF-β 1 and p-smad2/ in 20 μ g/ml, 80 μ g/ml
3, and then osteosarcoma cell anti-apoptotic proteins BCL-2 is lowered, and raise pro apoptotic protein Bax, it can thus be appreciated that antler polypeptide may
The apoptosis of osteosarcoma U 2OS is induced by regulation TGF-β signal path.
The description that foregoing exemplary embodiment is presented is merely illustrative of the technical solution of the present invention, and is not intended to become
Without missing, it is also not intended to limit the invention to described precise forms.Obviously, those skilled in the art's root
Many changes are made according to above-mentioned introduction and variation is all possible.The exemplary embodiment was chosen and described for the sake of explanations
Certain principles and practical application of the invention, so that others skilled in the art are easy to understand, realize and utilize
Various illustrative embodiments of the invention and its various selection forms and modification.Protection scope of the present invention is intended to by institute
Attached claims and its equivalents are limited.
Claims (7)
1. antler polypeptide is preparing the purposes in the drug for treating osteosarcoma, wherein the amino acid sequence of the antler polypeptide
Column are as shown in SEQ ID NO:1.
2. purposes according to claim 1, wherein the drug is by inhibiting the proliferation of osteosarcoma cell and/or luring
It leads apoptosis in osteosarcoma cells and treats osteosarcoma.
3. purposes according to claim 2, wherein TGF-β 1, p-smad2/3 in the drug up-regulation osteosarcoma cell
With the expression of Bax, the expression of BCL-2 in osteosarcoma cell is lowered.
4. purposes according to claim 3, wherein the expression for being expressed as protein level.
5. purposes described in any one of -4 according to claim 1, wherein the osteosarcoma is the bone and flesh in mammal
Tumor.
6. purposes according to claim 5, wherein the mammal is people.
7. purposes according to claim 6, wherein osteosarcoma cell is U2OS and/or MG-63 cell line.
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| CN201910614399.5A CN110368485A (en) | 2019-07-09 | 2019-07-09 | Antler polypeptide is preparing the purposes in the drug for treating osteosarcoma |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116808111A (en) * | 2023-08-21 | 2023-09-29 | 长春中医药大学 | Hydrogel film loaded with traditional Chinese medicine, preparation method thereof and application of hydrogel film in resisting osteosarcoma |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102925522A (en) * | 2012-10-31 | 2013-02-13 | 王�华 | Method for preparing pilose antler peptide dry powder by enzymolysis method |
| CN107354128A (en) * | 2017-07-14 | 2017-11-17 | 大连理工大学 | A kind of rush osteoblast conversion purposes of antler polypeptide |
-
2019
- 2019-07-09 CN CN201910614399.5A patent/CN110368485A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102925522A (en) * | 2012-10-31 | 2013-02-13 | 王�华 | Method for preparing pilose antler peptide dry powder by enzymolysis method |
| CN107354128A (en) * | 2017-07-14 | 2017-11-17 | 大连理工大学 | A kind of rush osteoblast conversion purposes of antler polypeptide |
Non-Patent Citations (3)
| Title |
|---|
| ZHANG, ZHENGYAO等: "Velvet Antler compounds targeting major cell signaling pathways in osteosarcoma - a new insight into mediating the process of invasion and metastasis in OS", 《OPEN CHEMISTRY》 * |
| 王冉冉: "重金属及农药残留对鹿茸有效成分抗乳腺癌活性影响的研究", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 王华等: "鹿茸寡肽的制备及其促成骨细胞的增殖作用", 《高等学校化学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116808111A (en) * | 2023-08-21 | 2023-09-29 | 长春中医药大学 | Hydrogel film loaded with traditional Chinese medicine, preparation method thereof and application of hydrogel film in resisting osteosarcoma |
| CN116808111B (en) * | 2023-08-21 | 2023-12-26 | 长春中医药大学 | Hydrogel film loaded with traditional Chinese medicine, preparation method thereof and application of hydrogel film in resisting osteosarcoma |
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Application publication date: 20191025 |