CN110229214A - A kind of excretion body Sustained-release polypeptide hydrogel and its preparation method and application - Google Patents
A kind of excretion body Sustained-release polypeptide hydrogel and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of polypeptide hydrogels, it is polypeptide hydrogel made of being mixed with self-assembling peptides (SAP) aqueous solution and excretion body (Exosomes, Exos).The invention also discloses the preparation method of foregoing polypeptides hydrogel and purposes.Polypeptide hydrogel of the present invention can effectively slow down the release of Exos, extend its half-life period, and the Exos discharged has activity similar with the Exos newly separated, and HK2 cellular damage caused by anoxic can be effectively reduced, promote the recovery of blood vessel endothelium injury；Polypeptide hydrogel of the present invention is by reducing kidney cell apoptosis, inflammatory factor and injury of kidney marker protein-neutrophil leucocyte gelatinase correlation apolipoprotein (NGAL) expression quantity, extracellular matrix of kidney (ECM) synthesis secretion, acute renal injury can be effectively reduced, slow down the effect of renal fibrosis generation, potential applicability in clinical practice is good.
Description
Technical field
The present invention relates to technical field of biological material, and in particular to a kind of excretion body Sustained-release polypeptide water for promoting compromised kidney reparation
Gel.
Background technique
Acute kidney injury (acute kidney injury, AKI) is a kind of common clinical acute disease, with renal function it is rapid under
Clinical characters are reduced to, acute renal failure is eventually led to, or even cause other organ failures.The priming factors of AKI have very much, such as lack
Blood/Reperfu- sion, Operation comparison agent application, take drugs improper, rhabdomyolysis, infection etc..In recent years, the morbidity of AKI
Rate constantly increases, and inpatient AKI disease incidence reaches 1%-5%, and increases rapidly.Currently, still lacking in treatment clinical course
Effective AKI prevents and treats drug, after AKI occurs, so that renal function has been obtained certain improvement though patient carries out conventional therapy,
But the therapeutic purpose difficult to realize restored completely.And the incomplete reparation of compromised kidney will lead to chronic kidney inflammatory reaction, fibre
A series of pathological changes such as dimensionization increase its risk for lapsing to and occurring end-stage renal disease (ESRD) to chronic kidney disease (CKD).By
It can not have always been high any more in AKI disease incidence, induce CKD in growth trend year by year, caused to social economy and publilc health huge
Big influence.
The excretion body (Exosomes, Exos) in the source mescenchymal stem cell (MSCs) is the lipid bilayer of diameter 30-100nm
Film activity molecule can promote acute kidney injury tissue repair by transfer specific protein, m RNA, micro RNA.Although
Exos has shown that huge treatment AKI potential, but its be injected into vivo after half-life period in blood circulation system it is very short, and
And be easy to be removed by internal macrophage, therefore, it is necessary to design slow-released carrier to deliver Exos to promote its treatment and treat
Effect.
Self-assembling peptides are the nano materials prepared by amino acid by synthesis in solid state by artificially designing, in ion salt etc.
Induction is lower to be quickly converted to form the polypeptide hydrogel with three-D space structure from solution.Polypeptide hydrogel only forms when degrading
Amino acid will not generate adverse effect to body；After importing body, immune response and tissue inflammation will not be caused, be a kind of
The good pharmaceutical carrier slow-release material of application prospect.
MMP2 is a member in matrix metalloproteinase family MMPs, and can degrade extracellular matrix, has document report
MMP2 activity dramatically increases in nephridial tissue after AKI.Therefore, a kind of MMP2 responsive type polypeptide hydrogel is prepared, it can be special by MMP2
Property degradation, to regulate and control the rate of release of packaging medicine in hydrogel；It is released using hydrogel package Exos building intellectual drug
Place system is particularly important the curative effect for extending and promoted Exos treatment injury of kidney.
Summary of the invention
The present invention provides a kind of excretion body Sustained-release polypeptide hydrogel, and its preparation method and application.
The self-assembling peptides that the present invention designs are MMP2 to be prepared with it by the amino acid sequence of MMP2 selective degradation
Sensitive hydrogel, and system is discharged with hydrogel package Exos building intellectual drug, to extend and promote Exos treatment
The curative effect of injury of kidney.
The present invention provides a kind of self-assembling peptides, its amino acid sequence is KLDLPVGLIGKLDL (SEQ ID NO:1).
The present invention also provides a kind of polypeptide hydrogels, it is formed by the aqueous solution of self-assembling peptides above-mentioned.
Wherein, excretion body is also contained in the aqueous solution；Preferably, the excretion body is source for mesenchymal stem cells
Excretion body.
Wherein, it is made of the raw material of following weight proportion: excretion body 60-100 parts by weight, self-assembling peptides 100-300
Parts by weight add water to 20-40 parts by volume；
Parts by weight described in wherein: parts by volume is g: μ l of μ.
Wherein, it is made of the raw material of following weight proportion: 8 parts by weight of excretion body, 13 parts by weight of self-assembling peptides add water
To 25 parts by volume.
The present invention also provides a kind of methods for preparing polypeptide hydrogel above-mentioned, it is comprised the following steps:
(1) self-assembling peptides of 100-300 parts by weight are dissolved in 10-20 parts by volume water and are configured to self-assembling peptides aqueous solution；It will
The excretion body of 60-100 parts by weight is dissolved in 8-15 parts by volume water and is configured to excretion body aqueous solution；
(2) self-assembling peptides aqueous solution, the excretion body aqueous solution for taking step (1) to be prepared, add water to 20-40 parts by volume,
It is mixed to get polypeptide hydrogel.
Wherein, it is comprised the following steps:
(1) 130 parts by weight self-assembling peptides are dissolved in 13 parts by volume sterile distilled waters, are configured to self-assembling peptides aqueous solution；
80 parts by weight excretion bodies are dissolved in 10 parts by volume sterile distilled waters, excretion body aqueous solution is configured to；
(2) self-assembling peptides aqueous solution, the excretion body aqueous solution for taking step (1) to obtain, add 2 parts by volume sterile distilled waters, mix
, in total 25 parts by volume to get polypeptide hydrogel.
The present invention finally provides purposes of the foregoing polypeptides hydrogel in the polypeptide hydrogel of preparation treatment acute kidney injury.
Wherein, the polypeptide hydrogel can reduce HK2 cellular damage caused by anoxic, promote the extensive of blood vessel endothelium injury
It is multiple.
Wherein, the polypeptide hydrogel can be reduced kidney cell apoptosis, inflammatory factor and NGAL expression quantity, kidney ECM
Synthesize secretory volume.
Polypeptide hydrogel of the present invention can effectively slow down the release of Exos, extend its half-life period, and the Exos discharged has
Activity similar with the Exos newly separated, can be effectively reduced HK2 cellular damage caused by anoxic, promote the extensive of blood vessel endothelium injury
It is multiple；Polypeptide hydrogel of the present invention is by reducing kidney cell apoptosis, inflammatory factor and NGAL expression quantity, kidney ECM is synthesized and secreted,
The degree of injury of kidney can be effectively reduced, reach the purpose for the treatment of acute kidney injury, potential applicability in clinical practice is good.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
Detailed description of the invention
Package situation of Fig. 1 self-assembling peptides nanofiber to Exos；
Fig. 2 self-assembling peptides hydrogel discharges the inside and outside of Exos；
The bioactivity of Exos is discharged in Fig. 3 self-assembling peptides hydrogel；
Migrating at pipe, scratch and transwell for Fig. 4 SAP-Exos detects；
Fig. 5 self-assembling peptides hydrogel, which is sustained Exos, reduces ischemia-reperfusion injury of kidney；
Fig. 6 self-assembling peptides hydrogel, which is sustained Exos, reduces the expression of inflammatory factor；
Fig. 7 self-assembling peptides hydrogel, which is sustained Exos, reduces renal tissue collagen secretion.
Specific embodiment
The preparation of the Exosomes in the source MSCs of the present invention of embodiment 1
The preparation method is as follows:
1, MSCs originally culture: the disconnected neck of mouse is put to death, and 70% alcohol, which impregnates 10 minutes, to be sterilized, and bilateral tibial is separated, with nothing
Blood serum medium rinses marrow, collects flushing liquor, and centrifugation is resuspended, and obtains MSCs using adherent method.
2, culture medium is collected: using addition 10% without excretion body fetal calf serum (Exosome-depleted FBS, the U.S.
SBI company) DMEM culture medium (Gbico company, the U.S.) cultivate MSCs, periodically collect culture supernatant.
3, centrifugation is extracted: Exos is extracted using multiple centrifugal method, 2000g × 10 minute are centrifuged removal cell fragment,
10000g × 30 minute centrifugation removal microcapsule bubble (MV), 100000g × 60 minute centrifugation obtain Exos.
4, purify: the Exos that previous step obtains is cleaned once with PBS, and 100000g × 60 minute centrifugation obtains pure again
Exos, be placed in -80 degree refrigerators it is spare.
The preparation of the polypeptide hydrogel of the present invention of embodiment 2
Needed for treating according to the present invention, it is sustained Exos using the hydrogel, according to preliminary result (therapeutic effect) and in fact
It tests animal (C57 mouse) and site of administration (mouse kidney peplos) determines that slow-releasing system is as follows:
| Ingredient | Concentration (mg/ml) | Volume (μ l) |
| Exosomes(Exos) | 8 | 10 |
| Self-assembled short peptide KMP2 | 10 | 13 |
| Sterile water | 103 | 2 |
| It amounts to | 25 |
(1) self-assembled short peptide KMP2 (KLDLPVGLIGKLDL) is by upper hypo Thailand biotechnology synthesizing and purifying, dry powder dissolution
In sterile water, it is configured to the liquid storage of 10mg/ml, 13 μ l is taken to be added in the EP pipe of 1.5ml；
(2) it takes the Exos that 10 μ l concentration are the source for mesenchymal stem cells of 8mg/ml to be added in EP pipe, mixes spare.
(3) 2 μ l sterile waters are added, piping and druming mixes spare.
The preparation of the polypeptide hydrogel of the present invention of embodiment 2
(1) self-assembled short peptide KMP2 (KLDLPVGLIGKLDL) is by upper hypo Thailand biotechnology synthesizing and purifying, certainly by 100ug
Assembled peptide is dissolved in 10 μ l water and is configured to self-assembling peptides aqueous solution；60ug excretion body is dissolved in 8 μ l water and is configured to excretion body aqueous solution；
It is added in EP pipe；
(2) self-assembling peptides aqueous solution, the excretion body aqueous solution obtained step (1), adds 2 μ l water, and 20 μ l, is mixed in total,
Up to polypeptide hydrogel.
The preparation of the polypeptide hydrogel of the present invention of embodiment 3
(1) self-assembled short peptide KMP2 (KLDLPVGLIGKLDL) is by upper hypo Thailand biotechnology synthesizing and purifying, certainly by 300ug
Assembled peptide is dissolved in 20 μ l water and is configured to self-assembling peptides aqueous solution；100ug excretion body is dissolved in 15 μ l water, and to be configured to excretion body water-soluble
Liquid；It is added in EP pipe；
(2) self-assembling peptides aqueous solution, the excretion body aqueous solution obtained step (1), adds 5 μ l water, and 40 μ l, is mixed in total,
Up to polypeptide hydrogel.
Beneficial effects of the present invention are proved below by way of specific pharmacodynamic experiment:
Package situation of the 1 self-assembling peptides nanofiber of test example to Exos
1 experimental material
High speed freezing desk centrifuge (Optima XPN-100 μ ltracentrifuge, Beckmann, the U.S.)
2000 Spectrophotometer of NanoDrop (Thermo Scientific, the U.S.)
Laser light scattering nano particle size instrument (Zetasizer Nano S, Malvern)
Transmission electron microscope (H-600, Hitachi)
Protein concentration BCA kit (CWBIO, China)
Sterile disposable filter (0.22 μm of aperture, Millipore)
Mono- antiantibody of Western blot: TSG101 (Proteintech, USA), HSP70 (ABclonal, China)
2 experimental methods
(1) mesenchymal stem cell (MSCs) routine culture, after cell fusion degree reaches 70%, replacement removal Exos
Culture medium continuously cultivate 10-12h after collect supernatant；
(2) 300g is centrifuged 15min, after 2000g is centrifuged 15min, sterile disposable filter (0.22 μm of aperture) filtering；
(3) 100,000g are centrifuged 1h, are resuspended after tri-distilled water washing；
(4) BCA kits protein concentration, -80 DEG C of preservations are continued to employ after packing；
(5) Exos specific marker object TSG101 and HSP70 are identified using western blot；
(6) partial size after laser light scattering nano particle size instrument analysis Exos, self-assembling peptides, self-assembling peptides package Exos；
(7) form after transmission electron microscope detection Exos, self-assembling peptides and self-assembling peptides package Exos.
3 experimental results
Western blot the result shows that, Exos being capable of specific expressed marker TSG101 and HSP70 (Figure 1A).Transmission
The Exos of Electronic Speculum (TEM) observation discovery (Figure 1B), MSC source is micro-capsule minibody structure, and rounded or oval, diameter is in 30-
100nm or so, and self-assembling peptides (SAP) form staggered nanofibrous structures；Exos is mixed with SAP as it can be seen that SAP Nanowire
Dimension can wrap Exos in netted；Laser particle analyzer testing result shows (Fig. 1 C), and the diameter of Exos is mainly distributed on 30-
Between 100nm, SAP nanofiber is in 10-20nm or so, and SAP granularity then dramatically increases after Exos is added.
From the above it is found that SAP nanofiber can wrap Exos well.
Inside and outside release experiment of the medicament slow release hydrogel of the present invention of test example 2 to Exos
1 experimental material
Self-assembled short peptide KMP2
The Exos of DIO label
The Exos of Cy7 label
1.5ml EP manages (Becton, Dickinson and Company)
10 μ l, 200 μ l, 1ml liquid-transfering gun pipette tips (Eppendorf)
PBS solution (0.1M, pH=7.2)
NaHCO3Solution (1M)
It is sustained buffer (PBS for containing 0.3% (w/v) BSA)
C57 BL/6J mouse (8 week old)
Yellow Jackets (Merck)
Zoopery related equipment (1ml syringe, shave, Iodophor, eye scissors, ophthalmic tweezers, cotton swab, gauze, operation
Towel, sewing needle, suture etc.)
2 experimental methods
2.1 self assembly polypeptide hydrogels are sustained Exos in vitro
(1) Dio label Exos (DIO-Exos) of 10 μ l, 1 μ g/ml is mixed gently with 50 μ l self-assembling peptides hydrogels,
It is placed in EP bottom of the tube, after bubble removing is removed in centrifugation, 37 DEG C are incubated for 10 minutes；
(2) 150 μ l PBS (0.22 μm of membrane filtration) is added on pipe top, is put into 37 DEG C of incubators, in 2h, 4h, 8h,
For 24 hours, 48h, 72h, 96h, 120h, 144h, 168h collect 100 μ l of supernatant, add PBS (100 μ l) after collecting every time；
(3) Supernatant samples collected are put into 4 DEG C and detect to unified.Fluorescent value is read using fluorescence microplate reader to be calculated.
Exos is sustained in 2.2 self assembly polypeptide hydrogel bodies
Animal packet: two groups of Free-Exo, Gel-Exo
(1) Exos is marked according to Cy7 fluorescent dye specification respectively, is removed using high speed centrifugation (100000g, 1h) free
Dyestuff, Cy7 label Exos (Cy7-Exos) purified.
(2) C57BL/6J mouse, yellow Jackets intraperitoneal injection, anesthesia, iodophor disinfection preserved skin.
(3) abdomen median line notch is done, integumentary musculature, exposure kidney are successively separated.
(4) according to grouping, every group 3, give the administration of mouse list kidney (left kidney) kidney peplos respectively, it is as follows that ingredient is administered in each group
Shown in table.
| Free-Exos | Gel-Exos | |
| Cy7-Exos(8mg/ml) | 10 | 10 |
| Self-assembled short peptide (SAP, 10mg/ml) | 0 | 10 |
| Sterile water | 15 | 5 |
| Total volume | 25 | 20 |
(5) mouse notch is sutured, after excessive anesthetic euthanasia mouse is injected respectively at for 24 hours, after 48h, 72h, collects kidney
Dirty, liver, spleen, small intestine is imaged at mouse living imaging system (In-Vivo Xtreme).
3 experimental results
In-vitro release curves show (Fig. 2A), and the Exos rate of release being wrapped in self-assemble gels is more relatively slow, until
Still there is within 5th day the release of Exos.As shown in EP pipe fluorescence imaging (Fig. 2 B), at 4-5 days still it is observed that remaining in water-setting
Exos is not discharged in glue, demonstrates above-mentioned sustained release result from another point of view.By free Cy7-Exos or it is wrapped in hydrogel
Cy7-Exos is injected in mouse kidney peplos respectively, and liver, spleen, double kidneys and small intestine is taken to carry out fluorescence imaging respectively in different time points.
The result shows that free Exos very fast metabolite clearance in Mice Body, living imaging is carried out after 24 hours can't detect Cy7 label
Exos, and be wrapped in the Exos in hydrogel and remain to detect signal (Fig. 2 C) after 3 days, show that hydrogel effectively slows down
The release of Exos in animal body.
From the above it is found that medicament slow release hydrogel of the present invention can effectively slow down Exos in vitro and animal is intracorporal releases
It puts, has the function of extending Exos half-life period.
The medicament slow release hydrogel of the present invention of test example 3 discharges the bioactivity verifying of Exos
1 experimental material
Matrigel glue (BD Biosciences, the U.S.)
Anti-ICAM-1 antibody (Proteintech, the U.S.)
Anti-IL-1 β antibody (Proteintech, the U.S.)
Anti-OCLN antibody (ABclonal, the U.S.)
Transwell (8 μm, BD FALCON, the U.S.)
PKH26 (Sigma, the U.S.)
Hochest33342 (the green skies, China)
2 experimental methods
(1) Exos cellular uptake is tested: by the Exos (1 μ g/ μ l) and people's renal cells of 20 μ l Pkh26 label
(HK2) training 12h is mixed, cell Hochest dyes 5min, and observation Exos by cellular uptake situation and takes pictures under fluorescence microscope.
(2) Exos (SAP-Exos) of hydrogel release is collected: by 10 μ l, 1 μ g/ μ l Exos and 50 μ l self-assembling peptides water
Gel mixes gently, and is placed in EP bottom of the tube, leaves away after bubble removing in wink, and 37 DEG C are incubated for 10 minutes.150 μ l are gently added on pipe top
DF12 culture medium is put into 37 DEG C of incubators, 72h hours release supernatants before collecting.
(3) reoxygenation of HK2 cell hypoxia/again is tested: be divided into Con (normal oxygen group), H/R (cell hypoxia for 24 hours again reoxygenation 2h),
Exos (cell H/R+Exos 10 μ l, 1 μ g/ μ l), SAP-Exos (cell H/R+ wraps up Exos hydrogel and discharges supernatant) group.
(4) Real time-PCR: group of cells extracts total serum IgE, detects inflammation apoptosis fibrosis GAP-associated protein GAP rna expression
It is horizontal；
(5) Western blot: group of cells extract total protein, gel electrophoresis, transferring film, be incubated for primary antibody secondary antibody after, chemistry
Luminescence reagent box detects protein expression level.
(6) human umbilical vein endothelial cell (HUVECs) scratch experiment: it is divided into Con (normal oxygen group), H/R (cell hypoxia
Reoxygenation 2h for 24 hours), Exos (cell H/R+Exos10 μ l, 1 μ g/ μ l), SAP-Exos (cell H/R+ wrap up Exos hydrogel release
Supernatant) group.Each group treated cell is collected, is inoculated in 6 orifice plates, after adherent, vertically marked and streaked with 200 μ l pipette tips
Cell, PBS wash away floating cells, in the random inverted microscope observation of 0h and 6h.
(7) HUVECs cell Transwell is tested: collecting each group treated cell, adjustment cell density is 5 × 105
A/ml takes 200 μ l cell suspension inoculations to the small interior Transwell, 3 multiple holes of every group of setting, and in 24 orifice plates (lower room)
Endothelium culture medium of the 600 μ l containing 2%FBS is added as chemotactic factor (CF).Cell, 0.1% crystal violet dye are taken out after cell culture 5h
Color liquid dyes 30min, washes away residual dye with PBS, observes under inverted phase contrast microscope.
(8) HUVECs cell is tested at pipe: Matrigel glue is placed in 4 DEG C of refrigerator overnights, is allowed to freeze thawing；By Matrigel glue
With the DL culture medium of serum-free, 96 orifice plates of pre-cooling are added with the ratio of 1:1, whole process carries out on ice；37 DEG C of incubations are solidifying
Gu plastic；Each group is taken treated that single cell suspension is made in HUVECs, with 2 × 104The cell inoculation in/hole is in being coated with Matrigel
96 orifice plates of glue；It observes and counts under inverted microscope after incubation 2h in 37 DEG C of incubators.
3 experimental results
The Exos of PKH26 label is added in HK2 cell after cultivating, it is seen that a large amount of red fluorescence label Exos are in endochylema
Aggregation prompts Exos that can be absorbed (Fig. 3 A) by HK2 cell.Real-time PCR from SAP hydrogel the results show that discharge
Exos (SAP-Exos) can effectively inhibit Bax gene, the FAS of hypoxia inducible, inflammation-related gene IL-1 β,
MCP-1 and fibrosis associated genes Fn, α-SMA expression, curative effect and the Exos newly separated are similar (Fig. 3 B).Western blot
As a result it also turns out, SAP-Exos can reduce reconciliation OCLN expression on ICAM-1, IL-1 β of hypoxia inducible and lower (Fig. 3 C), save
The intrinsic bioactivity of Exos, to reduce HK2 cellular damage caused by anoxic.
In addition, AKI also can induce renal blood vessels endothelial cell damage, therefore we carry out into respectively pipe, scratch and
Transwell migration experiment has detected protective effect (Fig. 4) of the SAP-Exos to HUVECs.The result shows that normal HUVECs can phase
It connects, is formed compared with multi-pipeline spline structure, and cell forms pipeline spline structure reduced capability after anoxic treatment, tubule is shorter and not
Continuously, cell is restored at pipe ability after SAP-Exos and Exos is handled.In scratch experiment compared with normal group, lack
The migration distance of oxygen group cell significantly shortens, and after SAP-Exos processing, the migration distance of cell is dramatically increased compared with anoxic group,
Transwell experiment has also obtained similar result.Exos energy used above to confirm, being discharged from self-assembling peptides hydrogel
Its bioactivity is kept, prompting hydrogel is a kind of good slow-released carrier.
From the above it is found that the Exos of medicament slow release hydrogel of the present invention release is with similar with the Exos newly separated
Activity, can not only be effectively reduced HK2 cellular damage caused by anoxic, also have the function of that blood vessel endothelium injury is promoted to restore.
The verifying one of the medicament slow release hydrogel of the present invention of test example 4 reduction ischemia-reperfusion injury of kidney
1 experimental material
Self-assembled short peptide KMP2 (sequence: n-KLDLPVGLIGKLDL-c)
1.5ml EP manages (Becton, Dickinson and Company)
10 μ l, 200 μ l, 1ml liquid-transfering gun pipette tips (Eppendorf)
Insulin syringe
C57 mouse (6-8 week old)
Yellow Jackets (Merck)
Zoopery related equipment (1ml syringe, shave, Iodophor, eye scissors, ophthalmic tweezers, artery clamp, timer, cotton
Label, gauze, operation towel, sewing needle, suture etc.)
Histopathology dyes related reagent (tissue is fixed, and is embedded, and is sliced, dyeing)
Reagent needed for tissue freezing section's embedding machine and slice immunofluorescence dyeing (tissue is fixed, and is closed, dyeing)
TUNEL staining kit (Promega)
Immunofluorescence dyeing primary antibody: NGAL (ABclonal)
Immunofluorescence dyeing secondary antibody: FITC/TRITC marks goat antirabbit/mouse fluorescence secondary antibody
RNA is extracted and RT-PCR related reagent
2 experimental methods
In order to evaluate the effect of self-assembling peptides hydrogel sustained release Exos treatment Ischemic kieney injury, closed by kidney arteriovenous folder
Mouse kidney ischemia-reperfusion renal injury model is constructed, is administered in kidney peplos to treat, passes through histopathology dyeing, Real
The overall merits curative effect such as time-PCR and histogenic immunity fluorescent staining.Concrete operations are as follows:
(1) according to the form below prepares each group intervention reagent respectively, is mixed with insulin syringe piping and druming stand-by.
(2) C57 mouse, yellow Jackets intraperitoneal injection, anesthesia, iodophor disinfection preserved skin.
(3) abdomen median line notch is done, integumentary musculature, exposure kidney are successively separated.
(4) according to grouping other than Con group, remaining group mouse gives the bis- kidney arteriovenous folders of 30min and closes 30min, and folder closes
Visible kidney is because of ischemic blackening afterwards.
(5) artery clamp is removed after 30min, it is seen that renal blood flow is replied, and kidney color is gradually recovered normally, shows that modeling is complete
At.
(6) intervention system of each group, progress kidney peplos injection, Con group also give physiological saline according to the table.
(7) suture operation notch, after wound disinfection, mouse sends animal house back to and continues normal feed.
(8) respectively at it is postoperative for 24 hours, 72h, 120h put to death mouse, collect mouse kidney.
(9) mouse 72h serum is collected, creatinine (CREA) is detected and urea nitrogen (BUN) is horizontal.
(10) mouse kidney carries out HE dyeing, and microscopy evaluates kidney structure and lesion situation.
(11) kidney carries out frozen section, carries out TUNEL dyeing, evaluates rat tubular cell apoptosis situation.
(12) renal tissue extracts RNA, and it is horizontal that RT-PCR detects apoptosis-related protein rna expression.
(13) renal tissue frozen section carries out immunofluorescence dyeing, detects kidney ey injury markers NGAL protein expression water
It is flat.
3 experimental results
Ischemia-reperfusion (I/R) causes kidney to be badly damaged, and mouse kidney function biochemical indicator BUN and CREA level increases (figure
5A).The visible I/R mouse tubular ectasia of HE dyeing, lumen inner cell fragment and cast, renal cells swelling and degeneration,
Cell is downright bad in the form of sheets, and TUNEL positive apoptotic cells increased significantly (Fig. 5).Compared with Exos group, the kidney of SAP-Exos group is damaged
Hurt degree reduces significantly, and the ratio for being embodied in BUN and CREA level and apoptotic cell is lower (Fig. 5 A, C).Real-
Time PCR detects Bax gene and FAS, has also obtained same result (Fig. 5 B).Neutrophil leucocyte gelatinase phase
The secretory protein that lipocalin protein (NGAL) is a kind of small-molecular-weight is closed, can be generated largely in impaired renal cells
NGAL, therefore by as one of renal damage mark.Immunofluorescence results show that I/R group renal tubule NGAL expression is significant and increase
Add, and SAP-Exos intervention group expression quantity is lower compared with I/R and Exos group, shows that kidney injury degree reduces (Fig. 5 C).
From the above it is found that medicament slow release hydrogel of the present invention can significantly reduce kidney function biochemical indicator, it is thin to reduce kidney
Born of the same parents' apoptosis and NGAL expression quantity, can be effectively reduced the degree of injury of kidney.
The medicament slow release hydrogel of the present invention of test example 5 reduces renal inflammation factor expression after AKI
1 experimental material
Self-assembled short peptide KMP2 (sequence: n-KLDLPVGLIGKLDL-c)
1.5ml EP manages (Becton, Dickinson and Company)
10 μ l, 200 μ l, 1ml liquid-transfering gun pipette tips (Eppendorf)
Insulin syringe
Physiological saline
C57 mouse (6-8 week old)
Yellow Jackets (Merck)
Zoopery related equipment (1ml syringe, shave, Iodophor, eye scissors, ophthalmic tweezers, artery clamp, timer, cotton
Label, gauze, operation towel, sewing needle, suture etc.)
Histopathology dyes related reagent (tissue is fixed, and is embedded, and is sliced, dyeing)
Reagent needed for tissue freezing section's embedding machine and slice immunofluorescence dyeing (tissue is fixed, and is closed, dyeing)
Immunofluorescence dyeing primary antibody: IL-1 β (ABclonal), ICAM-1 (Proteintech), TNF-α (CST),
Immunofluorescence dyeing secondary antibody: FITC/TRITC marks goat antirabbit/mouse fluorescence secondary antibody RNA extraction and RT-PCR phase
Close reagent
2 experimental methods
(1) in order to study the effect that self assembly polypeptide hydrogel sustained release Exos treats ischemic acute injury of kidney, pass through
Arteria renalis folder closes building ischemia-reperfusion acute kidney injury, and gives to intervene in kidney peplos, and is dyed by histopathology, RT-
Its therapeutic effect of the overall merits such as PCR and its histogenic immunity fluorescent staining.Concrete operations are as follows:
(2) according to the form below prepares each group intervention reagent, is mixed with insulin syringe piping and druming stand-by.
(3) C57 mouse, yellow Jackets intraperitoneal injection, anesthesia, iodophor disinfection preserved skin.
(4) abdomen median line notch is done, integumentary musculature, exposure kidney are successively separated.
(5) according to grouping other than Con group, remaining group mouse gives the bis- kidney arteriovenous folders of 30min and closes 30min, and folder closes
Visible kidney is because of ischemic blackening afterwards.
(6) artery clamp is removed after 30min, it is seen that renal blood flow is replied, and kidney color is gradually recovered normally, shows that modeling is complete
At.
(7) intervention system of each group, progress kidney peplos injection, Con group also give physiological saline according to the table.
(8) suture operation notch, after wound disinfection, mouse sends animal house back to and continues normal feed.
(9) respectively at it is postoperative for 24 hours, 72h, 120h put to death mouse, collect mouse kidney.
(10) renal tissue extracts RNA, and it is horizontal that RT-PCR detects inflammation related proteins rna expression.
(11) renal tissue frozen section carries out immunofluorescence dyeing, detects the protein expression level of inflammation related proteins.
3 experimental results
Ischemic and subsequent Reperfu- sion can cause serious damage to tissue and organ, and wherein inflammatory reaction plays
Key player.Real-time PCR the results show that proinflammatory inflammation factor ICAM-1, MCP-1 and TNF-α in the high expression of I/R group, and
Compared with Exos group, the proinflammatory factor expression and inflammatory cell infiltration reduction in SAP-Exos group mouse kidney tissue are more, with
I/R and free Exos group, which are compared, has significant difference, this result also with immunohistochemistry CD68 positive macrophage and chemotactic
Factor M CP-1 is reduced and immunofluorescence dyeing IL-1 β expresses reduced result and coincide (Fig. 6).
From the above it is found that medicament slow release hydrogel of the present invention can significantly reduce the expression of the renal inflammation factor after AKI,
The inflammation damnification degree of kidney can be effectively reduced.
The medicament slow release hydrogel of the present invention of test example 6 reduces renal fibrosis factor expression after AKI
1 experimental material
Self-assembled short peptide KMP2 (sequence: n-KLDLPVGLIGKLDL-c)
1.5ml EP manages (Becton, Dickinson and Company)
10 μ l, 200 μ l, 1ml liquid-transfering gun pipette tips (Eppendorf)
Insulin syringe
Physiological saline
C57 mouse (6-8 week old)
Yellow Jackets (merck)
Zoopery related equipment (1ml syringe, shave, Iodophor, eye scissors, ophthalmic tweezers, artery clamp, timer, cotton
Label, gauze, operation towel, sewing needle, suture etc.)
Histopathology dyes related reagent (tissue is fixed, and is embedded, and is sliced, dyeing)
Reagent needed for tissue freezing section's embedding machine and slice immunofluorescence dyeing (tissue is fixed, and is closed, dyeing)
Immunofluorescence dyeing primary antibody: FN (ABclonal), α-SMA (Abcam)
Immunofluorescence dyeing secondary antibody: FITC/TRITC marks goat antirabbit/mouse fluorescence secondary antibody
RNA is extracted and RT-PCR related reagent
2 experimental methods
In order to study the effect of self assembly polypeptide hydrogel sustained release Exoss treatment ischemic acute injury of kidney, pass through kidney
Artery clamp closes building ischemia-reperfusion acute kidney injury, and gives to intervene in kidney peplos, and is dyed by histopathology, RT-PCR
And its overall merits such as histogenic immunity fluorescent staining its therapeutic effect.Concrete operations are as follows:
(1) according to the form below prepares each group intervention system respectively, is mixed with insulin syringe piping and druming stand-by.
(2) C57 mouse, yellow Jackets intraperitoneal injection, anesthesia, iodophor disinfection preserved skin.
(3) abdomen median line notch is done, integumentary musculature, exposure kidney are successively separated.
(4) according to grouping other than Con group, remaining group mouse gives the bis- kidney arteriovenous folders of 30min and closes 30min, and folder closes
Visible kidney is because of ischemic blackening afterwards.
(5) artery clamp is removed after 30min, it is seen that renal blood flow is replied, and kidney color is gradually recovered normally, shows that modeling is complete
At.
(6) intervention system of each group according to the table carries out kidney peplos injection, Con group injecting normal saline.
(7) suture operation notch, after wound disinfection, mouse sends animal house back to and continues normal feed.
(8) Yu Shuhou 120h puts to death mouse, collects mouse kidney.
(9) mouse kidney carries out Masson dyeing, and microscopy evaluates kidney structure and fibrosis lesion situation.
(10) renal tissue extracts RNA, and RT-PCR detects inflammation, and fibrosis GAP-associated protein GAP rna expression is horizontal.
3 experimental results
It can induce renal fibrosis signal activation, fiber in mechanism and the incomplete repair process of kidney after acute kidney injury
The heavy rephasing of cell hyperproliferation and extracellular matrix (ECM) is closed.Renal tissue is sliced Masson dyeing discovery, after damage five days
Renal collagen synthesis secretion degree obviously increases, and renal tissue collagen secretion is substantially reduced after Exos is treated, and SAP-Exos
Group effect is more preferable (Fig. 7).In addition, being found through α-SMA and Fn immunofluorescence dyeing, the expression of the two five days tables after ischemic perfusion
Up to increase, and expression quantity declines after Exos is treated, SAP-Exos group better effect (Fig. 7).
From the above it is found that medicament slow release hydrogel of the present invention can significantly reduce kidney ECM synthesis secretion after AKI, energy
The degree of injury of kidney is effectively reduced.
To sum up, polypeptide hydrogel of the present invention can effectively slow down the release of Exos, extend Exos half-life period, and discharge
Exos has activity similar with the Exos newly separated, and HK2 cellular damage caused by anoxic can be effectively reduced, and promotes blood vessel endothelium
The recovery of damage；Polypeptide hydrogel of the present invention is by reducing kidney cell apoptosis, inflammatory factor and NGAL expression quantity, kidney ECM
Synthesis secretion, can be effectively reduced the degree of injury of kidney, slow down renal fibrosis, potential applicability in clinical practice is good.
Sequence table
<110>Huaxi Hospital Attached to Sichuan Univ
<120>a kind of self-assembling peptides
<130> GY026-18P1090
<141> 2018-03-05
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 14
<212> PRT
<213>artificial synthesized sequence (artificial synthesis)
<400> 1
Lys Leu Asp Leu Pro Val Gly Leu Ile Gly Lys Leu Asp Leu
1 5 10
Claims (10)
1. a kind of self-assembling peptides, it is characterised in that:
Its amino acid sequence is KLDLPVGLIGKLDL (SEQ ID NO:1).
2. a kind of polypeptide hydrogel, it is characterised in that:
It is formed by the aqueous solution of self-assembling peptides described in claim 1.
3. polypeptide hydrogel according to claim 2, it is characterised in that: also contain excretion body in the aqueous solution；It is excellent
Selection of land, the excretion body are the excretion body of source for mesenchymal stem cells.
4. polypeptide hydrogel according to claim 2 or 3, it is characterised in that: it is made of following raw materials:
Excretion body 60-100 parts by weight, self-assembling peptides 100-300 parts by weight, add water to 20-40 parts by volume.
5. polypeptide hydrogel according to claim 4, it is characterised in that: it is made of the raw material that following weight matches:
80 parts by weight of excretion body, 130 parts by weight of self-assembling peptides, add water to 25 parts by volume.
6. a kind of method for preparing polypeptide hydrogel described in claim 2-5 any one, it is characterised in that: it includes as follows
Step:
(1) self-assembling peptides of 100-300 parts by weight 10-20 parts by volume water is dissolved in be configured to from group
Fill peptide aqueous solution；The excretion body of 60-100 parts by weight is dissolved in 8-15 parts by volume water and is configured to excretion body aqueous solution；
(2) self-assembling peptides aqueous solution, the excretion body aqueous solution for taking step (1) to be prepared, add water to 20-40 parts by volume, are mixed,
Up to polypeptide hydrogel.
7. the method according to claim 6 for preparing polypeptide hydrogel, it is characterised in that: it is comprised the following steps:
(1) 130 parts by weight self-assembling peptides are dissolved in 13 parts by volume sterile distilled waters, are configured to self-assembling peptides aqueous solution；By 80
Parts by weight excretion body is dissolved in 10 parts by volume sterile distilled waters, is configured to excretion body aqueous solution；
(2) self-assembling peptides aqueous solution, the excretion body aqueous solution for taking step (1) to obtain, add 2 parts by volume sterile distilled waters, are mixed, always
Totally 25 parts by volume are to get polypeptide hydrogel.
8. purposes of any one polypeptide hydrogel of claim 2-5 in the polypeptide hydrogel of preparation treatment acute kidney injury.
9. purposes according to claim 8, it is characterised in that: the polypeptide hydrogel can reduce HK2 caused by anoxic
Cellular damage promotes the recovery of blood vessel endothelium injury.
10. purposes according to claim 8, it is characterised in that: the polypeptide hydrogel can be reduced kidney cell apoptosis,
Inflammatory factor and NGAL expression quantity, kidney ECM synthesize secretory volume.
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