CN108623670A - A kind of fell skin tissue endogenous polypeptide PDHPS1 and its application - Google Patents

A kind of fell skin tissue endogenous polypeptide PDHPS1 and its application Download PDF

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CN108623670A
CN108623670A CN201810517689.3A CN201810517689A CN108623670A CN 108623670 A CN108623670 A CN 108623670A CN 201810517689 A CN201810517689 A CN 201810517689A CN 108623670 A CN108623670 A CN 108623670A
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pdhps1
skin tissue
endogenous polypeptide
scar
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CN108623670B (en
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李俊
李景云
季晨博
曾新
崔县伟
付子毅
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Nanjing Maternity and Child Healthcare Hospital
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/00—Medicinal preparations containing peptides
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

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Abstract

The invention discloses a kind of fell skin tissue endogenous polypeptide PDHPS1 and its applications.A kind of fell skin tissue endogenous polypeptide PDHPS1, amino acid sequence is as shown in SEQ ID NO.1.Polypeptide PDHPS1 can inhibit the proliferation of scar dermal fibroblast, the mrna expression amount for inhibiting glue protogene COL1A1, COL1A2 in people's scar dermal fibroblast, significantly reduces the expressing quantity of myofibroblast outstanding feature object α SMA in people's scar dermal fibroblast.The fell skin tissue endogenous polypeptide PDHPS1 can be used for preparing the drug or reagent for inhibiting scar dermal fibroblast proliferation, reducing collagen content, reducing myofibroblast amount, to inhibit scar proliferation to provide new target spot.

Description

A kind of fell skin tissue endogenous polypeptide PDHPS1 and its application
Technical field
The present invention relates to wound healings and scar field, relate in particular to a kind of fell skin tissue endogenous polypeptide PDHPS1 and its application.
Background technology
Hyperplastic scar is common pathologic scar, is that excessive tissue is repaiied in the agglutination after dermal layer of the skin is impaired The extracellular matrixs over-deposits such as multiple, diseased region collagen as a result, its incidence is up to 70%.Clinically, Hypertrophic scar Trace often causes patient part itch, pain, and possible appearance is damaged and function limitation, body and soul to patient are brought greatly Injury, seriously affects the quality of life of patient.Currently, though operation excision, local hormone injection, laser intervention etc. are Hypertrophic scars The important means of trace treatment, however there is limitation or bad complication, still lack effectively preventing method so far.Cause This, it is very necessary actively to find scar prevention means that are novel, effective, having no toxic side effect.The formation of known hyperplastic scar Mechanism and the process of tissue reparation after skin injury are closely related, are related to inflammatory reaction, angiogenesis, re-epithelialization, granulation group It knits to be formed and be remolded etc..Wherein, scar dermal fibroblast hyper-proliferative, be activated and be divided into myofibroblast, heavy The extracellular matrixs such as the excessive collagen (I, type III) of product, are the major reasons of scar proliferation.
Invention content
Goal of the invention：The object of the present invention is to provide a kind of fell skin tissue endogenous polypeptide PDHPS1.
It is a further object of the present invention to provide the applications of the polypeptide.
It is yet another object of the invention to provide a kind of cell culture fluids containing the polypeptide.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of fell skin tissue endogenous polypeptide PDHPS1 of the present invention, amino acid sequence such as SEQ ID NO.1 institutes Show.
The gene of the coding fell skin tissue endogenous polypeptide PDHPS1.
The gene of the coding fell skin tissue endogenous polypeptide PDHPS1 preferably has core shown in SEQ ID NO.2 Nucleotide sequence.
The fell skin tissue endogenous polypeptide PDHPS1 inhibits the medicine of scar dermal fibroblast proliferation preparing Application in object or reagent.
The fell skin tissue endogenous polypeptide PDHPS1 is preparing reduction scar dermal fibroblast collagen content Drug or reagent in application.
The fell skin tissue endogenous polypeptide PDHPS1 reduces the drug or reagent of myofibroblast amount preparing In application.
Composition containing fell skin tissue endogenous polypeptide PDHPS1 described in claim 1.
In the composition, the fell skin tissue endogenous polypeptide PDHPS1 is present in composition with therapeutically effective amount In.
The polypeptide or the peptide composition can be prepared into pharmaceutically acceptable dosage form, including but not limited to spray, The dosage forms such as emulsion, emulsifiable paste, patch, gelling agent.
A kind of cell culture fluid contains the fell skin tissue endogenous polypeptide PDHPS1.
Recombinant expression carrier containing the gene.
The host microbial cell of the recombinant expression carrier transfection.
The acquisition of the polypeptide can be obtained according to amino acid sequence by solid phase synthesis process；Or pass through host microorganism Or cloning in vivo and the DNA fragmentation for expressing the nucleotide sequence for carrying one of coding said polypeptide, pass through existing recombinant DNA Technology prepares.Used expression vector and host cell are that recombinant technique is known to the public.Expression vector is for example PET carriers, pGEX carriers；Host cell such as Escherichia coli (E.coli), actinomyces (Actinomycetes), bacillus (Bacillus), streptomycete (Streptomyces), polypeptide of the present invention can use conventional enzymatic cleavage methods by it from host It is detached in cell, can also be detached and be purified by conventional liquid chromatography, above-mentioned isolation and purification method is all this Technology well known to field technology personnel.
Advantageous effect：
Fell skin tissue endogenous polypeptide PDHPS1 (IATTTASAATAAAIGATPRAK) is the sources NUMA1 peptide, by 21 A amino acid composition, there is no its correlation function to report at present.Bioinformatic analysis and experimental result are shown：1. the sources PDHPS1 2090-2110 amino acids in NUMA1 PROTEIN Cs end；2. ProtParam on-line analyses are found, the unstability index of PDHPS1 (Instability index) is 1.89, belongs to stable type polypeptide, can be stabilized；Fat coefficient (Aliphatic index) It is 80, average hydrophilic and hydrophobic (Grand average of hydropathicity) is 0.5, shows as hydrophobic lipophilic, is easy Entered by diffusion or encytosis intracellular；3. PDHPS1 is added in cell culture fluid, has and inhibit scar corium at fiber finer The function of born of the same parents' proliferation；Quantitative PCR experiment confirm PDHPS1 can reduce glue protogene COL1A1 in scar dermal fibroblast, The mrna expression amount of COL1A2；Protein immunoblotting (Western blot) experiment confirms that PDHPS1 can reduce flesh into fiber finer The expressing quantity of born of the same parents outstanding feature object α-SMA.Therefore, PDHPS1 can inhibit scar dermal fibroblast to increase preparing Grow, reduce collagen content, reduce myofibroblast amount drug or reagent in apply.
Description of the drawings
Fig. 1 is the figure that PDHPS1 inhibits scar dermal fibroblast proliferation in embodiment 2；
Fig. 2 is that PDHPS1 inhibits glue protogene COL1A1, COL1A2 in scar dermal fibroblast in embodiment 3 The figure of mrna expression amount；
Fig. 3 is that PDHPS1 inhibits myofibroblast outstanding feature object α-in scar dermal fibroblast in embodiment 4 The expressing quantity of SMA and the figure of gray analysis result.
Specific implementation mode
With reference to specific embodiment, invention is further explained.
RNA Reverse Transcriptase kits and RNA detection kits etc. are purchased from Thermo Fisher companies of the U.S., PCR kit TaKaRa LA Polymerase Hot-Start Version PCR are purchased from Takara companies of Japan, DMEM high sugar cultures Base is purchased from Sciencell companies of the U.S., holoprotein extraction agent box, PAGE gel electrophoresis kit, CCK-8 cell Proliferations Kit, Western Blot detection kits, ECL detection kits, Braford protein contents detection kit, pre-dyed egg White molecular weight and Ponceaux dyeing liquor are purchased from Nanjing of China Kai Ji biotechnologies Development Co., Ltd, developing solution, fixing solution Purchased from Chinese Wuxi City Ling Qi service articles factory.
Embodiment 1
The commission Shanghai bio tech ltd Ke Tai passes through polypeptide shown in the following SEQ ID NO.1 of Solid phase synthesis PDHPS1：
Ile Ala Thr Thr Thr Ala Ser Ala Ala Thr Ala Ala Ala Ile Gly Ala Thr Pro Arg Ala Lys
This is the polypeptide of 21 amino acid composition, and the principal biological of foregoing polypeptides sequence is obtained by online tool Parameter is as follows：
Isoelectric point (PI) is 11.0, molecular mass (Mw) 1915.18Da.
10mg polypeptides are taken, being diluted to 50mM-40 DEG C with aseptic double-distilled water stores for future use.
The influence that embodiment 2CCK-8 methods detection polypeptide PDHPS1 is proliferated people's scar dermal fibroblast
In 37 DEG C, 5%CO2In incubator, people's scar dermal fibroblast is cultivated, is connect by the density of 2000 cells/wells Kind is added 100 μ l culture mediums/every hole, is added afterwards is separately added into polypeptide PDHPS1 processing (final concentrations for 24 hours in 96 well culture plates Respectively 0.1,1,10,100 μM), control group is given solvent aseptic double-distilled water and is handled, every group of 5 multiple holes, is trained in 37 DEG C of incubators It supports.
96 orifice plates are taken out after 24 hours, per 10 μ l CCK-8 of the adherent addition in hole, are avoided bubble formation, are put back to 37 degree of incubators Continue to be incubated 2 hours.Microplate reader measures absorbance, is counted using wavelength 450nm, describes growth curve.
The results are shown in Figure 1, it is seen that polypeptide PDHPS1 has the function of inhibiting scar dermal fibroblast proliferation.
3 real-time quantitative PCR of embodiment (RT-PCR) detects polypeptide PDHPS1 to collagen in people's scar dermal fibroblast The influence of the mRNA expression of gene C OL1A1, COL1A2
In 37 DEG C, 5%CO2In incubator, people's scar dermal fibroblast is cultivated, by 105/ ml density is inoculated in 6 orifice plates In, when cell fusion degree is up to 60% or so, people's scar corium is added into fiber finer in polypeptide PDHPS1 (100 μM of final concentration) Born of the same parents use RNA extraction agent boxes (TRIzol methods) to extract cell total rna afterwards for 24 hours.
The people's scar dermal fibroblast for collecting polypeptide processing group and control group respectively, uses RNA extraction agent boxes (TRIzol methods) extracted total RNA, Nanodrop2.0 detect RNA concentration, and quantitative 150ng total serum IgE reverse transcriptions are cDNA, further Use the expression of corresponding primer detection people COL1A1, COL1A2.Realtime PCR reaction systems are：10 μ l of 2XSybr Mix, CDNA1 μ l, 1 μ l of primer, 8 μ l of distilled water, totally 20 μ l, run, condition is in ABI 7500：After 95 DEG C of 10min of pre-degeneration, 95 DEG C 15s, 60 DEG C of 1min repeat 40 cycles.The relative expression quantity of people COL1A1, COL1A2 are calculated with △ △ CT methods.As a result as schemed Shown in 2.
COL1A1 forward primers:5’-CCTAGTCTGTCCTGCGTCCTC-3’
Reverse primer:5’-GAGTCTTTTGCTTCCTCCCAC3-’
COL1A2 forward primers:5’-ACTACCTCAAAACACTTTCCCAT-3’
Reverse primer:5’-ACACCACACGATACAACTCAATAC-3’
Embodiment 4Western blot methods detect polypeptide PDHPS1 to flesh in people's scar dermal fibroblast into fiber finer The influence of the expressing quantity of born of the same parents outstanding feature object α-SMA
The people's scar dermal fibroblast for collecting polypeptide processing group and control group respectively, proceeds as follows：
The extraction of holoprotein
It is washed 2 times in cold PBS, each addition pre- Cold lysis buffer of 100ul ice, ice bath 30min after mixing, every 5min Be vortexed concussion 10s, fully cracks.Centrifugation：4 DEG C, 12000 × g, 10min.Supernatant is dispensed, is stored in -80 DEG C, is avoided repeatedly Freeze thawing.
Protein quantification
1. the configuration of protein standard liquid：Bovine serum albumin(BSA) (BSA) 50mg adds distilled water to 50ml, final concentration of 1 μ g/1 μl
2. the measurement of standard curve：
BSA(1mg/mL)(μL) 0 2 4 6 8 10
Distilled water (μ L) 10 8 6 4 2 0
Dye liquor (μ L) 990 990 990 990 990 990
A(595nm) 0 0.089 0.198 0.283 0.379 0.451
3. drawing standard curve：
A=0.0459C+0.004
R2=0.997
4. taking testing protein sample appropriate, Coomassie brilliant blue G250 dye liquor 990ml are added, mixing measures 595nm OD values, Then gained concentration is sought on standard curve.
SDS-PAGE electrophoresis
Clean Bio-Rad1.5~glass plate, by specification is taken to be installed on gum-making rack；According to the molecule of destination protein It measures size and selects suitable gel strength, the separation gel (i.e. lower layer's glue) of SDS-PAGE is prepared according still further to following table；Carefully With pipette tips by separation gel implantation glass sheet separation, stopping at 2/3rds glass plate height about is being accounted for, is being added thereafter above Enter several milliliters of distilled waters, it is therefore an objective to prevent inhibiting effect of the air to gel polymerisation.After the completion of separation gel polymerization, outwell above glue The distilled water of covering, the liquid for the remaining that exhausted as possible with filter paper, not encounter separation gel carefully.
1 resolving polyacrylamide gel formula of table
The concentration glue of SDS-PAGE is prepared according to table 1 and injects the upper end of separation gel, it is careful to be inserted into and sheet thickness phase The sample comb of adaptation avoids generating bubble：
Table 2 configures the component table of different volumes SDS-PAGE concentration glue
After spacer gel polymerization, sample comb is carefully taken out, the electrophoretic buffer of lxTris-Gly is then added.
It draws appropriate amount of sample supernatant to be added in sample well, the albumen Marker of pre-dyed is added in the hole by sample, does not add In the hole for adding Sample supernatants, lxSDS sample-loading buffers are added and keep glue surface balance.
Power supply is opened, voltage starts setting up as 60V, and after protein sample enters separation gel, 90V can be improved in voltage.Ginseng According to the position of pre-dyed Marker, when purpose band enters gel optimal separation area (about the 2/3 of gel), stop electrophoresis.
In advance by the 4 DEG C of precoolings of transferring film liquid.Transfer box is opened on pallet, oneself is spread through with transferring film close to the inner face of cathode side What buffer solution soaked has hole dimension pad, puts three layers of Whatman3MM filter paper for being soaked with transferring film buffer solution thereon, pays attention to emptying bubble.
Glass plate carefully is pried open, glue is placed in the pallet containing transferring film liquid, the separation gel containing purpose band is cut, It is placed on filter paper with the immersion of transferring film liquid.10) the NC films soaked through methanol and transferring film liquid are spread on gel, between glue and film not Can have a bubble, film, filter paper and gel size, it is roughly the same.Put the Whatman filters of two layers of dipped transferring film liquid again on NC films Paper pays attention to emptying bubble.Second block of foam-rubber cushion is put, makes entirely to transfer interlayer and sequentially forms " fiber mat-filter paper-gel- NC films-filter paper-fiber mat " level closes transfer folder, is put into transfer groove, transferring film liquid is filled in slot.
Open power supply, current stabilization 200mA, 120min.After transferring film, takes out NC films and perform label, film is washed with TBST 10minX3 times.
Immunoblotting
1) it closes：NC films are put into plate, the confining liquid containing 5% skimmed milk power is added, shaking table vibrates 1.5-2h；
2) after closing, film 10min × 3 time are washed with TBST；
3) film is put into the plate containing primary antibody (using westem primary antibodies diluted), 4 DEG C of shaking table oscillation incubations are stayed overnight；
4) it takes out within second day, primary antibody is abandoned in shaken at room temperature 30min, suction, and TBST washes 10min × 3 time；
5) secondary antibody, room temperature shaker oscillating reactions 1-2h are diluted with 5% skimmed milk power confining liquid；
6) secondary antibody after reaction, recycles secondary antibody.Then film 5-10min × 3 time are washed with TBsT；
7) two kinds of liquid of A, B in EcL chemical luminescence reagent kits are pressed into l:L is mixed in equal volume, and it is spare to be configured to working solution；
8) NC films are taken out from TBST, gets rid of extra liquid, the film containing protein is face-up, it is placed on fresh-keeping Film is added dropwise appropriate working solution, is covered with preservative film, places in development folder, closes development folder；
9) use Britain syngene chemiluminescences gel imaging system (being purchased from Shanghai Ren Qi instrument and meters Co., Ltd) at Picture carries out gray analysis using Image J softwares to result.
Test result：As shown in figure 3, finding that the scar corium of polypeptide processing group is at fibre by Western blot experiments Tieing up the expressing quantity of myofibroblast outstanding feature object α-SMA in cell significantly reduces, and has dropped 61%, shows polypeptide PDHPS1 can significantly inhibit the expressing quantity of α-SMA in scar dermal fibroblast.
5 cell culture formula of liquid of embodiment
A kind of cell culture fluid, is formulated and is：DMEM high glucose mediums (Sciencell companies of the U.S.), 10% fetal calf serum (Gibco companies), 100 μM of polypeptide PDHPS1.
Embodiment 6
A kind of solution containing polypeptide PDHPS1, using distilled water as solvent, a concentration of 50 μM of polypeptide PDHPS1.
The above is only a preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.
Sequence table
<110>Nanjing Women and Children Healthcare Hospital
<120>A kind of fell skin tissue endogenous polypeptide PDHPS1 and its application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> PRT
<213>The mankind (Homo sapiens)
<400> 1
Ile Ala Thr Thr Thr Ala Ser Ala Ala Thr Ala Ala Ala Ile Gly Ala
1 5 10 15
Thr Pro Arg Ala Lys
20
<210> 2
<211> 63
<212> DNA
<213>The mankind (Homo sapiens)
<400> 2
attgccacca ccacagccag cgccgccact gctgccgcca ttggtgccac ccctcgagcc 60
aag 63

Claims (10)

1. a kind of fell skin tissue endogenous polypeptide PDHPS1, it is characterised in that amino acid sequence is as shown in SEQ ID NO.1.
2. the gene of coding fell skin tissue endogenous polypeptide PDHPS1 described in claim 1.
3. fell skin tissue endogenous polypeptide PDHPS1 described in claim 1 is preparing inhibition scar dermal fibroblast increasing Application in the drug or reagent grown.
4. fell skin tissue endogenous polypeptide PDHPS1 described in claim 1 is preparing reduction scar dermal fibroblast glue The drug of former content or the application in reagent.
5. fell skin tissue endogenous polypeptide PDHPS1 described in claim 1 reduces the drug of myofibroblast amount preparing Or the application in reagent.
6. the composition containing fell skin tissue endogenous polypeptide PDHPS1 described in claim 1.
7. composition according to claim 6, it is characterised in that fell skin tissue endogenous polypeptide described in claim 1 PDHPS1 is present in therapeutically effective amount in composition.
8. a kind of cell culture fluid, it is characterised in that contain fell skin tissue endogenous polypeptide PDHPS1 described in claim 1.
9. the recombinant expression carrier containing gene described in claim 2.
10. the host microbial cell that expression vector described in claim 9 transfects.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113817042A (en) * 2021-08-13 2021-12-21 南京市妇幼保健院 Human adipose-derived stem cell secreted polypeptide ADSCP2 and application thereof
CN114790234A (en) * 2022-04-08 2022-07-26 南京市妇幼保健院 Adipose-derived stem cell secreted endogenous polypeptide ADSCP5 and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002064736A2 (en) * 2001-01-04 2002-08-22 Myriad Genetics, Inc. Protein-protein interactions
CN1852974A (en) * 2003-06-09 2006-10-25 密歇根大学董事会 Compositions and methods for treating and diagnosing cancer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002064736A2 (en) * 2001-01-04 2002-08-22 Myriad Genetics, Inc. Protein-protein interactions
CN1852974A (en) * 2003-06-09 2006-10-25 密歇根大学董事会 Compositions and methods for treating and diagnosing cancer

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113817042A (en) * 2021-08-13 2021-12-21 南京市妇幼保健院 Human adipose-derived stem cell secreted polypeptide ADSCP2 and application thereof
CN113817042B (en) * 2021-08-13 2022-04-01 南京市妇幼保健院 Human adipose-derived stem cell secreted polypeptide ADSCP2 and application thereof
CN114790234A (en) * 2022-04-08 2022-07-26 南京市妇幼保健院 Adipose-derived stem cell secreted endogenous polypeptide ADSCP5 and application thereof
CN114790234B (en) * 2022-04-08 2022-11-18 南京市妇幼保健院 Adipose-derived stem cell secreted endogenous polypeptide ADSCP5 and application thereof

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