CN106366174B - A kind of antler polypeptide is reducing the application in mescenchymal stem cell adhesive force - Google Patents
A kind of antler polypeptide is reducing the application in mescenchymal stem cell adhesive force Download PDFInfo
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Abstract
The invention belongs to biomedicine technical field, disclosing a kind of antler polypeptide is reducing the application in mescenchymal stem cell adhesive force, and the amino acid sequence of antler polypeptide is VLSATDKTNVLAAWGKVGGNAPAFGAEALERM in the present invention.The antler polypeptide has the function of reducing fat mesenchymal stem cell (ADSCs) adhesion, especially when concentration is 50ug/ml, effect is the most significant, can play a significant role in tumour, thrombus, rheumatoid arthritis treatment and reduction immunity of organism rejection.
Description
Technical field
The invention belongs to biomedicine technical fields, and being related to antler polypeptide (VAPs) is reducing mescenchymal stem cell
(ADSCs) application in adhesive force.
Background technique
Pilose antler --- return liver and kidney channel, kidney-replenishing, benefiting essence-blood, strengthening the bones and muscles.Belong to one of traditional Chinese medicine.Chinese people's republicanism
State's pharmacopeia (version one in 2005) is by pilose antler function and cures mainly narration are as follows: invigorating kidney yang, benefiting essence-blood, strengthening the bones and muscles adjust punching to appoint, torr sore
Poison.For impotence involuntary emission, cold palace is infertile, and win is thin, and refreshing tired, chilly, dizziness and tinnitus is deaf, and waist and knee crymodynia, muscles and bones is cold soft, metrorrhagia and metrostaxis band
Under, negative subcutaneous ulcer is not held back.
Pilose antler is the animal for being under the jurisdiction of Chordata Mammalia Cervidae, it be sika deer or red deer stag it is unossified simultaneously
And the young horn of dense villus.Pilose antler history is loaded in Shennong's Herbal, it is indicated that :-pilose antler specially enters the gate of vitality, superintends and directs, it is simultaneous enter liver.Gan Xianqi
Temperature reports the matter of pure sun, the gas containing generation ", product in category.Li Shizhen (1518-1593 A.D.) is said :-tortoise, Lu Jieling and have the longevity.Deer nose Chang Fanxiang tail, can lead to
Governor vessel, therefore its angle is taken, to mend life, mend essence, tonifying Qi, all to establish the yang function.It is the profound micro- of physics, the what one is particularly good at of refreshing work ".Moreover, modern
The effects of pharmacological research also shows that pilose antler has anti-inflammatory effect, can inhibit and remove free radical, delays senescence, promotes wound healing,
The antler polypeptide extracted in pilose antler can promote the proliferation and differentiation of chondrocytes in vitro cell, osteoblast, inhibit cartilage cell's tune
It dies, meanwhile, literature research also prompts, and returns liver and kidney channel drug to use in the prescription for the treatment of " rheumatism " for representative with pilose antler etc.
Frequency accounts for 39.3%.
Contain more complicated chemical component in pilose antler, the type of amino acid there are 19 kinds or more, including human body itself
The essential amino acid that cannot be synthesized, 10 kinds of phospholipid compositions, 9 kinds of fatty acid (the strongest oleic acid of bioactivity, linoleic acid, linolenic acid
Content is higher), glycolipid, sugar, steroid, hormonelike substance, prostaglandin, cerebrin, ribonucleic acid, nuclifort, three phosphorus
Adenosine monophosphate, chondroitin sulfate, polyamines, peptides, lipoprotein, vitamin, enzyme and various microelements etc..
Antler polypeptide used in the present invention is to utilize ion-exchange chromatography, gel permeation chromatography and reversed-phase high performance liquid chromatography layer
The Measurements for Biochemistry such as analysis, 1 isolated novel polypeptide from sika deer velvet antler, SDS-PAGE electrophoresis showed are a band,
HPLC map is simple spike, and the accurate molecular weight that MALDI-TOF MS provides the polypeptide is 3263.4, isoelectric point pI=8.15.
Primary structure is free of cysteine studies have shown that the straight-chain polypeptide that the polypeptide is made of 32 amino acid residues, is rich in figured silk fabrics ammonia
Acid, lysine, leucine and glycine.(the such as Zhang Zhengyao " chemical structure and bioactivity of sika deer velvet antler polypeptide " high
School chemistry journal 33.9 (2012): 2000-2004.).
Fat mesenchymal stem cell is by extracting adipose tissue and by being separately cultured a kind of fiber-like obtained
Cell is a kind of adult stem cell, and from mesoderm as bone marrow stroma stem cell, surface antigen is thin with marrow mesenchymal stem
Cell phase seemingly, and in vitro condition of culture, growth conditions and expression marker it is also essentially identical with bone marrow stroma stem cell
(ZukPA,ZhuM,AshjianP,etal.Humanadiposetissue is asourceofmultipotentstem
Cells [J] .MolBiolCell, 2002,13:4279 1 is 4295.).It is wide with source, richness is high, damage is small, is easy to cultivate
The advantages that.Fat mesenchymal stem cell has various effects, it has proved that has in terms for the treatment of a variety of diseases huge
Potentiality can induce as neuron cell, treat type-1 diabetes mellitus, promote skin wound healing, reduce acute and chronic renal damage
Wound etc..Marina Burgos-Silva1's et al. studies have shown that adipose tissue-derived mesenchymal stem cell transplantation can reduce serum urea,
The renal function of kidney failure mouse is set to be significantly improved (Burgos-Silva M, Semedo-Kuriki P, Donizetti-
Oliveira C,Costa PB,Cenedeze MA,Hiyane MI,et al.(2015)Adipose Tissue-Derived
Stem Cells Reduce Acute and Chronic Kidney Damage in Mice.PLoS ONE 10(11):
E0142183.doi:10.1371/journal.pone.0142183), after treatment in 4 weeks, kidney fibrosis can be mitigated, mitigated slow
Property inflammation.Fat mesenchymal stem cell (ADSCs) plays positive protective effect in the treatment of Nephrotoxicity, can pass through
Cell cycle regulation inhibits inflammation, mitigates kidney damage, improves renal function, inhibits organ fibrosis and improve immune tune for a long time
Section effect.By carrying out fat mesenchymal stem cell treatment in the rodent of injury of kidney, it was demonstrated that can be used to reduce group
Tissue inflammation and injury of kidney (Morigi M, Introna M, Imberti B, Corna D, Abbate M, Rota C, et al.
(2008)Human bone marrow mesenchymal stem cells accelerate recovery of acute
renal injury and prolong survival in mice.Stem Cells 26:2075–2082.doi:
10.1634/stemcells.2007-0795PMID:18499895；Furuichi K,Shintani H,Sakai Y,Ochiya
T,Matsushima K,Kaneko S,et al.(2012)Effects of adiposederived mesenchymal
cells on ischemia-reperfusion injury in kidney.Clin Exp Nephrol 16:679–
689.doi:10.1007/s10157-012-0614-6PMID:22398959).A large amount of experiment is it has been proved that different types of
Stem cell all has apparent effect (Fang TC, Pang CY, Chiu SC, Ding DC, Tsai in terms for the treatment of injury of kidney
RK(2012)Renoprotective effect of human umbilical cordderivedmesenchymal stem
Cells in immunodeficient mice suffering from acute kidney injury.PLoS One 7:
E46504.doi:10.1371/journal.pone.0046504 PMID:23029541；Fang TC,Alison MR,Cook
HT,Jeffery R,Wright NA,Poulsom R.(2005)Proliferation of bone
marrowderivedcells contributes to regeneration after folic acid-induced acute
tubular injury.J Am Soc Nephrol16:1723–1732.PMID:15814835).Moreover, most of experiment
Statistics indicate that the main mechanism of stem-cell therapy is inflammation process (the Stagg J (2007) in control damage generating process
Immune regulation by mesenchymal stem cells:two sides to the coin.Tissue
Antigens 69:1–9.PMID:17212702)。
Cell adhesion refers to the sticking phenomenon to interact between cell and cell and between cell and extracellular matrix,
Be between cell transmit information a kind of form (Parsons JT, Horwitz AR, Schwartz MA.Cell adhesion:
integratingcytoskeletal dynamics and cellular tension.Nat Rev Mol Cell Biol
2010；11:633-43.).Cell adhesion plays an important role in body normal physiological activity.The transfer and recurrence of tumour
It is the key factor of tumor lethal.In the transfer of tumour cell, there are many adhesion molecule participate in the falling off, stick of cancer cell,
Transition process (Zigler M, Dobroff AS, Bar-Eli M.Cell adhesion:implication in tumor
progression.Minerva Med 2010；101:149-62.).Tumour cell is needed by sticking and infiltration penetrates substrate
Film and sticking between lymphocyte could complete transfer process.Therefore, Anti-adhesive treatment is possible to inhibit turning for tumour
Move (Ofek I, Hasty DL, Sharon N.Anti-adhesion therapy of bacterial diseases:
prospects and problems.FEMS Immunol Med Microbiol 2003；38:181-91.).Thrombosis master
If due to platelet activating factor and sticking chemokine mediated platelet activation, sticks and assemble.And sticking to become
Change under factor effect, leucocyte has also assisted in the formation of inflammation and thrombus, therefore in the equal observable of inflammation or thrombosis position
To presence (Nieswandt B, Pleines I, the Bender M.Platelet adhesion and of leucocyte and blood platelet
activation mechanisms in arterial thrombosis and ischaemic stroke.J Thromb
Haemost2011；9Suppl 1:92-104.).Many, which sticks chemotactic factor (CF), anti-inflammatory and anti thrombotic action.In trnasplantion immunity
In rejection, the phenomenon that increase there are the vascular remodeling of donor organ and expression of adhesion molecule, this is because blood follows
The result of antibody in ring to identification and the reaction of graft antigen.The activation of endothelial cell and the increase of expression of adhesion molecule can
Promote leucocyte to the organ chemotactic of transplanting and invade profit, is one of an important factor for leading to immunological rejection.Therefore, resist and stick
The interaction that can block granulocyte and endothelial cell is treated, trnasplantion immunity rejection (Simmons is thus prevented and treat
DL.Anti-adhesion therapies.Curr Opin Pharmacol 2005；5:398-404).Autoimmune disease
Pathogenesis and cell adhesion it is closely related.In patient with rheumatoid arthritis, stick chemokine mediated T cell infiltration
And the immunization inflammatory reaction that infiltration of the inflammatory cell in synovial tissue of joint part is caused is the important mechanisms fallen ill.This
Outside, in the morbidity of the autoimmunity diseases such as lupus erythematosus and inflammatory enteritis, stick chemokine expression and cell adhesion increase all
It is important pathogenic factor (Norman MU, Kubes P.Therapeutic intervention in inflammatory
diseases:a time and place for anti-adhesion therapy.Microcirculation 2005；12:
91-8.).It can be seen that reducing cell adhesion in tumour, thrombus, rheumatoid arthritis treatment and reducing immunity of organism row
It plays an important role in different reaction, the drug for reducing cell adhesion has great importance for the treatment of disease.
Summary of the invention
To overcome problems of the prior art, the present invention provides a kind of antler polypeptides to reduce mescenchymal stem cell
Application in adhesive force.
The technical solution of the present invention is as follows:
A kind of amino acid sequence of antler polypeptide, sequence
VLSATDKTNVLAAWGKVGGNAPAFGAEALERM。
Above-mentioned antler polypeptide is reducing the application in mescenchymal stem cell adhesive force, includes the following steps:
The first step, cell culture；Culture cell is mescenchymal stem cell, using low-sugar type culture medium, in 37 DEG C, 5%CO2
It is cultivated in incubator.
Second step, cell dissociation；The mescenchymal stem cell in logarithmic growth phase is taken, conventional digestion is carried out, obtains slender
Born of the same parents' suspension.
Third step, centrifugation；Single cell suspension is put into centrifuge tube and is centrifuged；Supernatant is sucked, culture medium is added and blows
Cell is beaten, cell is made to be uniformly dispersed.
4th step, cell count；Adjusting number of cells is 3.3 × 105A/ml.
5th step plants plate；By finely dispersed cell inoculation in orifice plate, at 37 DEG C, 5%CO2It is cultivated in incubator.
Antler polypeptide is added in 6th step；When mescenchymal stem cell is in logarithmic growth phase, culture medium is sucked out, is added dense
Degree is the antler polypeptide of 0~50ug/ml, at 37 DEG C, 5%CO212h is cultivated in incubator.
Step 7: carrying out trypsin digestion experiment；Partial medium is stayed, pancreatin is then added；A Zhang Zhao is clapped every 15s
Piece observes different time, the variation of cellular morphology.
The mescenchymal stem cell is fat mesenchymal stem cell.
The culture medium is DMEM culture medium.
The beneficial effects of the present invention are: the present invention develops a kind of antler polypeptide reduction with specific amino acid sequence
The application of fat mesenchymal stem cell (ADSCs) adhesion, especially when concentration is 50ug/ml, effect is the most significant, can
It plays a significant role in tumour, thrombus, rheumatoid arthritis treatment and reduction immunity of organism rejection.
Detailed description of the invention
After acting on ADSCs cell 12h Fig. 1 shows 50ug/ml antler polypeptide, the variation of cellular morphology when 30s.
After Fig. 2 indicates that 5ug/ml antler polypeptide acts on ADSCs cell 12h, the variation of cellular morphology when 45s.
After Fig. 3 indicates that 1ug/ml antler polypeptide acts on ADSCs cell 12h, the variation of cellular morphology when 60s.
Specific embodiment
In the following with reference to the drawings and specific embodiments, the present invention is further explained.
Embodiment 1
Trypsinized experiment detection antler polypeptide concentration is 50ug/ml, after acting on 12h, to the shadow of cell adhesion ability
It rings.
Step 1: cell culture.Cell culture uses low-sugar type DMEM culture medium, and serum content 10% uses 75cm2
Tissue Culture Flask, in 37 DEG C, 5%CO2It is cultivated in incubator.
Step 2: cell dissociation.Take the good fat mesenchymal stem cell of upgrowth situation in logarithmic growth phase
(ADSCs), conventional digestion.When digestion, 4ml PBS is first added, fat mesenchymal stem cell (ADSCs) is cleaned, cleaning two
It is secondary, remaining culture medium is washed away, prevents remaining medium and pancreatin from neutralization occurs.Then 4ml pancreatin is added, is put into 37
DEG C, 5%CO21min is digested in incubator.Equivalent culture medium is added when cell shrinkage and terminates digestion.With disposable sterilized plastic tube
Cell bottle wall is blown and beaten, cell is blown and beaten from cell culture bottle wall, single cell suspension is made.
Step 3: centrifugation.Single cell suspension is put into centrifuge tube and is centrifuged, 1000rpm/min is centrifuged 5 minutes.It inhales
Supernatant is removed, culture medium is added and blows and beats cell, cell is made to be uniformly dispersed.
Step 4: cell count.Counted using cell counting board, adjust number of cells be every 1.5ml cell liquid contain 5 ×
105A cell.
Step 5: kind plate.By cell inoculation in 6 orifice plates, every hole is inoculated with 1.5ml, and number of cells is every hole 5 × 105It is a,
Altogether plus 2 holes.It is placed in 37 DEG C, 5%CO2And 12h is cultivated in the incubator of saturated humidity.
Step 6: antler polypeptide is added.After 12h, original culture medium is sucked out, 1.5ml culture is separately added into two holes
Base, then the culture medium 100ul that antler polypeptide concentration is 80ug/100ul is added in a hole thereto, equivalent training is added in second hole
Base is supported, as blank control.And Cell Name is marked, and antler polypeptide concentration, bed board and administration timing of drug.Continue to cultivate 12h.
Step 7: trypsinized is tested.After 12h, pancreatin is added and carries out trypsinized experiment.In this experiment,
In order to extend digestion time, the variation of cellular morphology after pancreatin is added convenient for observation, there is no all take when drawing culture medium
Out, but 1000ul culture medium is taken out, stays 600ul culture medium, 400ul pancreatin is then added.It takes a picture, sees every 15s
Examine different time, the variation of cellular morphology.Experiment is in triplicate.
Experimental result as shown in Figure 1, when antler polypeptide concentration be 50ug/ml when, it is complete in 30s cell dissociation, 30s it
Cellular morphology no longer occurs significantly to change afterwards.
Embodiment 2
Trypsinized experiment detection antler polypeptide concentration is 5ug/ml, after acting on 12h, to the shadow of cell adhesion ability
It rings.
Step 1: cell culture.Cell culture uses low-sugar type DMEM culture medium, and serum content 10% uses 75cm2
Tissue Culture Flask, in 37 DEG C, 5%CO2It is cultivated in incubator.
Step 2: cell dissociation.Take the good fat mesenchymal stem cell of upgrowth situation in logarithmic growth phase
(ADSCs), conventional digestion.When digestion, 4ml PBS is first added, fat mesenchymal stem cell (ADSCs) is cleaned, cleaning two
It is secondary, remaining culture medium is washed away, prevents remaining medium and pancreatin from neutralization occurs.Then 4ml pancreatin is added, is put into 37
DEG C, 5%CO21min is digested in incubator.Equivalent culture medium is added when cell shrinkage and terminates digestion.With disposable sterilized plastic tube
Cell bottle wall is blown and beaten, cell is blown and beaten from cell culture bottle wall, single cell suspension is made.
Step 3: centrifugation.Single cell suspension is put into centrifuge tube and is centrifuged, 1000rpm/min is centrifuged 5 minutes.It inhales
Supernatant is removed, culture medium is added and blows and beats cell, cell is made to be uniformly dispersed.
Step 4: cell count.Counted using cell counting board, adjust number of cells be every 1.5ml cell liquid contain 5 ×
105A cell.
Step 5: kind plate.By cell inoculation in 6 orifice plates, every hole is inoculated with 1.5ml, and number of cells is every hole 5 × 105It is a,
Altogether plus 2 holes.It is placed in 37 DEG C, 5%CO2And 12h is cultivated in the incubator of saturated humidity.
Step 6: antler polypeptide is added.After 12h, original culture medium is sucked out, 1.5ml culture is separately added into two holes
Base, then the culture medium 100ul that antler polypeptide concentration is 8ug/100ul is added in a hole thereto, equivalent training is added in second hole
Base is supported, as blank control.And Cell Name is marked, and antler polypeptide concentration, bed board and administration timing of drug.Continue to cultivate 12h.
Step 7: trypsinized is tested.After 12h, pancreatin is added and carries out trypsinized experiment.In this experiment,
In order to extend digestion time, the variation of cellular morphology after pancreatin is added convenient for observation, there is no all take when drawing culture medium
Out, but 1000ul culture medium is taken out, stays 600ul culture medium, 400ul pancreatin is then added.It takes a picture, sees every 15s
Examine different time, the variation of cellular morphology.Experiment is in triplicate.
Experimental result as shown in Fig. 2, when antler polypeptide concentration be 5ug/ml when, it is complete in 45s cell dissociation, 45s it
Cellular morphology no longer occurs significantly to change afterwards.
Embodiment 3
Trypsinized experiment detection antler polypeptide concentration is 1ug/ml, after acting on 12h, to the shadow of cell adhesion ability
It rings.
Step 1: cell culture.Cell culture uses low-sugar type DMEM culture medium, and serum content 10% uses 75cm2
Tissue Culture Flask, in 37 DEG C, 5%CO2It is cultivated in incubator.
Step 2: cell dissociation.Take the good fat mesenchymal stem cell of upgrowth situation in logarithmic growth phase
(ADSCs), conventional digestion.When digestion, 4ml PBS is first added, fat mesenchymal stem cell (ADSCs) is cleaned, cleaning two
It is secondary, remaining culture medium is washed away, prevents remaining medium and pancreatin from neutralization occurs.Then 4ml pancreatin is added, is put into 37
DEG C, 5%CO21min is digested in incubator.Equivalent culture medium is added when cell shrinkage and terminates digestion.With disposable sterilized plastic tube
Cell bottle wall is blown and beaten, cell is blown and beaten from cell culture bottle wall, single cell suspension is made.
Step 3: centrifugation.Single cell suspension is put into centrifuge tube and is centrifuged, 1000rpm/min is centrifuged 5 minutes.It inhales
Supernatant is removed, culture medium is added and blows and beats cell, cell is made to be uniformly dispersed.
Step 4: cell count.Counted using cell counting board, adjust number of cells be every 1.5ml cell liquid contain 5 ×
105A cell.
Step 5: kind plate.By cell inoculation in 6 orifice plates, every hole is inoculated with 1.5ml, and number of cells is every hole 5 × 105It is a,
Altogether plus 2 holes.It is placed in 37 DEG C, 5%CO2And 12h is cultivated in the incubator of saturated humidity.
Step 6: antler polypeptide is added.After 12h, original culture medium is sucked out, 1.5ml culture is separately added into two holes
Base, then the culture medium 100ul that antler polypeptide concentration is 1.6ug/100ul is added in a hole thereto, equivalent is added in second hole
Culture medium, as blank control.And Cell Name is marked, and antler polypeptide concentration, bed board and administration timing of drug.Continue to cultivate 12h.
Step 7: trypsinized is tested.After 12h, pancreatin is added and carries out trypsinized experiment.In this experiment,
In order to extend digestion time, the variation of cellular morphology after pancreatin is added convenient for observation, there is no all take when drawing culture medium
Out, but 1000ul culture medium is taken out, stays 600ul culture medium, 400ul pancreatin is then added.It takes a picture, sees every 15s
Examine different time, the variation of cellular morphology.Experiment is in triplicate.
Experimental result as shown in figure 3, when antler polypeptide concentration be 1ug/ml when, it is complete in 60s cell dissociation, 60s it
Cellular morphology no longer occurs significantly to change afterwards.
The amino acid sequence of antler polypeptide are as follows:
VLSATDKTNVLAAWGKVGGNAPAFGAEALERM
Claims (3)
1. a kind of antler polypeptide is reducing the application in mescenchymal stem cell adhesive force, which comprises the steps of:
The first step, cell culture；Culture cell is fat mesenchymal stem cell, using low-sugar type culture medium, in 37 DEG C, 5%CO2
It is cultivated in incubator；Second step, cell dissociation；The mescenchymal stem cell in logarithmic growth phase is taken, conventional digestion is carried out,
Obtain single cell suspension；
Third step, centrifugation；Single cell suspension is put into centrifuge tube and is centrifuged；Supernatant is sucked, it is thin that culture medium piping and druming is added
Born of the same parents make cell be uniformly dispersed；
4th step, cell count；Adjusting number of cells is 3.3 × 105A/ml；
5th step plants plate；By finely dispersed cell inoculation in orifice plate, at 37 DEG C, 5%CO2It is cultivated in incubator；
The antler polypeptide of amino acid sequence VLSATDKTNVLAAWGKVGGNAPAFGAEALERM is added in 6th step；Work as mesenchyma
When stem cell is in logarithmic growth phase, culture medium is sucked out, the antler polypeptide that concentration is 0~50ug/ml is added, at 37 DEG C, 5%
CO212h is cultivated in incubator；
Step 7: carrying out trypsin digestion experiment；Partial medium is stayed, pancreatin is then added；It is taken a picture every 15s,
Observe different time, the variation of cellular morphology.
2. a kind of antler polypeptide according to claim 1 is reducing the application in mescenchymal stem cell adhesive force, feature
It is, the antler polypeptide that concentration is 50ug/ml is added in the 6th step.
3. a kind of antler polypeptide according to claim 1 or 2 is reducing the application in mescenchymal stem cell adhesive force, special
Sign is that the culture medium is DMEM culture medium.
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