CN110229215A - A kind of medicament slow release polypeptide hydrogel and its preparation method and application - Google Patents
A kind of medicament slow 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 aqueous solution, heparin sodium water solution, hepatocyte growth factor (HGF) aqueous solution and TNF- ɑ antibody (anti-TNF- ɑ).The invention also discloses the preparation method of foregoing polypeptides hydrogel and purposes.Polypeptide hydrogel of the present invention has good medicament slow release effect and kidney biocompatibility, by adding heparin sodium, achieve the purpose that sequential successive release anti-TNF- ɑ and HGF, polypeptide hydrogel of the present invention can effectively inhibit acute kidney injury (AKI) metanephric tubule Apoptosis, reduce NGAL expression quantity, mitigate renal inflammation reaction, renal tubular cell is promoted to dedifferente and be proliferated, with extension anti-TNF- ɑ and HGF half-life period, protein drug is given full play to the different times the effect of, reduce injury of kidney degree, promote the effect of compromised kidney reparation, 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 medicament slow release polypeptide water-setting for treating Ischemic kieney injury
Glue.
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.Therefore the therapy for researching and developing a kind of effective rush kidney injury reparation is of great significance
Proinflammatory factor has played key effect, tumor necrosis factor-ɑ (tumor necrosis after AKI in injury of kidney
Factors- ɑ, TNF- ɑ) it is a kind of macrophage product generated by stimulations such as ischemia-reperfusion, lipopolysaccharides, endotoxins, it can also
To be synthesized by messangial cell, direct cytotoxicity, vessel retraction are played by Apoptosis mode Human Umbilical Vein Endothelial Cells
Renal function is damaged with the effects of reduction renal blood flow and aggregation neutrophil leucocyte and monocyte, therefore by inhibiting renal tissue
Middle TNF- ɑ expression and activity can be effectively improved the kidney injury of ischemia-reperfusion induction.
Hepatocyte growth factor (hepatocyte growth factor, HGF) is a kind of pleiotropic growth factor, mainly
It is generated by interstitial cell, epithelial cell, endothelial cell and interstitial cell itself is acted on by autocrine and paracrine mode,
Have the function of mitogenesis, promote cellular morphology formation and adjust cellular activity, so that the organ and tissue to damage carry out
It repairs.Have research it was demonstrated that given in acute kidney injury exogenous HGF can protect renal cells, rebuild kidney it is small
Pipe structure and maintenance renal function integrality.
Antibody and growth factor belong to protein medicaments, are widely used in biomedicine field.Due to albumen
Internal Various Tissues cell is possible to play a role, systematicness, which is excessively used, will lead to potential risk；And albumen is in body
Interior half-life period is shorter, easily loses bioactivity.Therefore, it is necessary to design slow-released carrier deliver antibody and growth factor to
It promotes it and treats curative effect.
Self-assembling peptides are designed by the mankind, the nano material prepared by amino acid by synthesis in solid state, in ion salt etc.
Induction is lower to be quickly converted to form the polypeptide hydrogel with three-D space structure from solution state.When polypeptide hydrogel is degraded only
Amino acid is formed, adverse effect will not be generated to body；After importing body, immune response and tissue inflammation will not be caused, be
A kind of good pharmaceutical carrier slow-release material of application prospect.
Although anti-TNF- ɑ and HGF has repair to injury of kidney, action time is not identical；anti-
TNF- ɑ mainly plays preferable anti-inflammatory effect in kidney injury early stage, and HGF repairs phase performance biology effect in kidney later
It answers.Therefore, a kind of polypeptide hydrogel is prepared, sequential successive release anti-TNF- ɑ and HGF reach while lowering renal inflammation damage
Wound, the dual purpose for promoting kidney reparation are particularly important treatment injury of kidney.
Summary of the invention
The present invention provides a kind of medicament slow release polypeptide hydrogel, and its preparation method and application.
The self-assembling peptides that the present invention designs, the positive charge carried by arginine (R) and the heparin sodium for having negative electrical charge
(Hep) molecule be combined with each other, then by the affinity interaction between heparin and growth factor, reaches significant and extend hepatocyte growth factor
The effect of HGF release.
The present invention provides a kind of self-assembling peptides, its amino acid sequence is KLDLKLDLKLDLR (SEQ ID NO:1).
The present invention provides a kind of polypeptide hydrogels, it is formed by the aqueous solution of self-assembling peptides above-mentioned.
Wherein, heparin sodium, hepatocyte growth factor and TNF- ɑ antibody are also contained in the aqueous solution.
Wherein, it is made of the raw material of following weight proportion:
TNF- ɑ antibody-solutions 1-5 parts by volume, hepatocyte growth factor 0.3-1 parts by weight, heparin sodium 0.5-3 parts by weight, from
Assembled peptide 50-300 parts by weight, add water to 20-40 parts by volume；
Wherein, the concentration of the TNF- ɑ antibody-solutions is 0.5-10mg/ml；
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:
2 parts by volume of TNF- ɑ antibody-solutions, 0.5 parts by weight of hepatocyte growth factor, 1 parts by weight of heparin sodium, self-assembling peptides
100 parts by weight add water to 25 parts by volume；
Wherein, the concentration of the TNF- ɑ antibody-solutions is 1mg/ml.
The present invention also provides a kind of methods for preparing polypeptide hydrogel above-mentioned, it is comprised the following steps:
(1) 50-300 parts by weight self-assembling peptides dry powder is dissolved in 8-15 parts by volume water, is configured to self-assembling peptides aqueous solution；
0.5-3 parts by weight heparin sodium dry powder is dissolved in 0.5-3 parts by volume water, heparin sodium water solution is configured to；By 0.3-1 parts by weight liver
Porcine HGF dry powder is dissolved in 3-10 parts by volume water, prepares hepatocyte growth factor aqueous solution；Take the TNF- of 1-5 parts by volume
ɑ antibody-solutions；
(2) 4 kinds of solution that step (1) obtains are taken, 20-40 parts by volume is added water to, is mixed to get polypeptide hydrogel；
Wherein, the concentration of the TNF- ɑ antibody-solutions is 0.5-10mg/ml.
Wherein, it is comprised the following steps:
(1) 100 parts by weight self-assembling peptides dry powder are dissolved in 10 parts by volume sterile distilled waters, it is water-soluble is configured to self-assembling peptides
Liquid；1 parts by weight heparin sodium dry powder is dissolved in 1 parts by volume sterile distilled water, heparin sodium water solution is configured to；By 0.5 parts by weight
Hepatocyte growth factor dry powder is dissolved in 5 parts by volume sterile distilled waters, prepares dry hepatocyte growth factor aqueous solution；Take 2 volumes
The TNF- ɑ antibody-solutions of part；
(2) 4 kinds of solution that step (1) obtains are taken, adds the sterile distilled water of 7 parts by volume, is mixed, in total 25 parts by volume, i.e.,
Obtain polypeptide hydrogel；
Wherein, the concentration of the TNF- ɑ antibody-solutions is 1mg/ml.
The present invention finally provides a kind of use of polypeptide hydrogel in the polypeptide hydrogel of preparation treatment acute kidney injury
On the way.
Wherein, the polypeptide hydrogel can inhibit rat tubular cell apoptosis caused by acute kidney injury, reduce NGAL table
Up to amount, mitigates renal inflammation reaction, promote compromised kidney reparation.
Wherein, the polypeptide hydrogel can improve Ki67 cell positive rate, promote Pax2 expression, promote renal tubular cell
It dedifferentes and is proliferated.
Polypeptide hydrogel of the present invention has slow releasing function to anti-TNF- ɑ and HGF, but rate of release is different.Hydrogel pair
The sustained release of anti-TNF- ɑ is to slow down molecule diffusion by the nanofibrous structures that it is crosslinked to realize, therefore release anti-TNF- ɑ
It puts comparatively fast, allows to play preferable anti-inflammatory effect in kidney injury early stage.Pass through the heparin combined in hydrogel and growth
The affinity interaction of the factor makes it repair phase performance biology in kidney later to keep the bioactivity and slow release of HGF
Effect.The present invention reaches sequential successive release anti-TNF- ɑ and HGF by self-assembling peptides/heparin sodium hydrogel of injectable
Purpose, thus have simultaneously lower renal inflammation damage, promotion kidney reparation double action.
Polypeptide hydrogel of the present invention has good medicament slow release effect and kidney biocompatibility, by adding heparin
Sodium achievees the purpose that sequential successive release anti-TNF- ɑ and HGF, polypeptide hydrogel of the present invention can effectively inhibit AKI to induce
Rat tubular cell apoptosis reduces NGAL expression quantity, mitigates renal inflammation reaction, renal tubular cell is promoted to dedifferente and be proliferated, have
Have extend anti-TNF- ɑ and HGF half-life period, give full play to protein drug the different times the effect of, reduce the damage journey of kidney
The effect of degree, potential applicability in clinical practice are 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
HGF and anti-TNF- ɑ Cumulative release profile in Fig. 1 hydrogel；
Fig. 2 Cy7 marks living body fluorescent image after the injection of HGF/Gel kidney peplos；
Pathological dyes after the injection of Fig. 3 hydrogel kidney peplos；
Fig. 4 hydrogel delays controlled release HGF and anti-TNF- ɑ slows down injury of kidney；
Fig. 5 hydrogel delays controlled release HGF and reacts with anti-TNF- ɑ reduction renal inflammation；
Fig. 6 hydrogel delays controlled release HGF and anti-TNF- ɑ and promotes the reparation of AKI metanephric tubule.
Specific embodiment
The preparation of the medicament slow release polypeptide hydrogel of the present invention of embodiment 1
Needed for treating according to the present invention, using hydrogel sustained release HGF and anti-TNF- ɑ, (controlled according to preliminary result
Therapeutic effect), experimental animal (C57 mouse) and site of administration (mouse kidney peplos) determine that slow-releasing system is as follows:
1) self-assembled short peptide KLD1R (sequence: n-KLDLKLDLKLDLR-c) is by upper hypo Thailand biotechnology synthesizing and purifying,
100ug dry powder is dissolved in 10 μ l sterile waters, is configured to the liquid storage of 10mg/ml, and 10 μ l is taken to be added in the EP pipe of 1.5ml；
2) 1ug heparin sodium (being purchased from Sigma company) dry powder is dissolved in 1 μ l sterile water, is configured to the liquid storage of 1mg/ml,
1 μ l is taken to be added in EP pipe；
3) 0.5ugHGF (being purchased from the Divine Land Yi Qiao biotech firm) dry powder is dissolved in 5 μ l sterile waters, is configured to 0.1mg/
The liquid storage of ml takes 5 μ l to be added in EP pipe；
4) 2 μ l anti-mouse TNF- ɑ antibody (purchased from Biolegend company, 1mg/ml) is taken to be added in EP pipe.
5) again in EP pipe, 7 μ l sterile waters are added, amount to 25 μ l, insulin syringe piping and druming mixes to get medicine of the present invention
Object Sustained-release polypeptide hydrogel.
The preparation of the medicament slow release polypeptide hydrogel of the present invention of embodiment 2
1) self-assembled short peptide KLD1R (sequence: n-KLDLKLDLKLDLR-c) is by upper hypo Thailand biotechnology synthesizing and purifying,
50ug dry powder is dissolved in 8 μ l sterile waters, is configured to self-assembling peptides aqueous solution, is added in the EP pipe of 1.5ml；
2) 0.5ug heparin sodium (being purchased from Sigma company) dry powder is dissolved in 0.5 μ l sterile water, is configured to heparin sodium water
Solution is added in EP pipe；
3) 0.3ug HGF (being purchased from the Divine Land Yi Qiao biotech firm) dry powder is dissolved in 3 μ l sterile waters, prepares hepatoblast
Growth factor aqueous solution is added in EP pipe；
4) 1 μ l anti-mouse TNF- ɑ antibody (purchased from Biolegend company) is taken, concentration 0.5mg/ml is added in EP pipe；
5) again in EP pipe, 7.5 μ l sterile waters are added, amount to 20 μ l, insulin syringe piping and druming mixes to get the present invention
Medicament slow release polypeptide hydrogel.
The preparation of the medicament slow release polypeptide hydrogel of the present invention of embodiment 3
1) self-assembled short peptide KLD1R (sequence: n-KLDLKLDLKLDLR-c) is by upper hypo Thailand biotechnology synthesizing and purifying,
300ug dry powder is dissolved in 15 μ l sterile waters, is configured to self-assembling peptides aqueous solution, is added in the EP pipe of 1.5ml；
2) 3ug heparin sodium (being purchased from Sigma company) dry powder is dissolved in 3 μ l sterile waters, is configured to heparin sodium water solution,
It is added in EP pipe；
3) 1ug HGF (being purchased from the Divine Land Yi Qiao biotech firm) dry powder is dissolved in 10 μ l sterile waters, prepares hepatoblast
Growth factor aqueous solution is added in EP pipe；
4) 5 μ l anti-mouse TNF- ɑ antibody (purchased from Biolegend company) is taken, concentration 10mg/ml is added in EP pipe；
5) again in EP pipe, 7 μ l sterile waters are added, amount to 40 μ l, insulin syringe piping and druming mixes to get medicine of the present invention
Object Sustained-release polypeptide hydrogel.
Beneficial effects of the present invention are proved below by way of specific pharmacodynamic experiment:
The sustained release of the medicament slow release polypeptide hydrogel of the present invention of test example 1 is tested
1 experimental material
Self-assembled short peptide KLD1R (sequence: n-KLDLKLDLKLDLR-c)
FITC marks anti-mouse TNF- ɑ antibody (Biolegend)
It recombinates HGF (Divine Land Yi Qiao biotech firm)
Heparin sodium (Sigma)
HGF detects ELISA kit (Da Kewei company)
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)
Cy7 fluorescent dye (Solarbio)
Balb/c 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 external sequential release anti-TNF- ɑ and HGF
50 μ l drug hydrogels are prepared by embodiment 1, are placed in 1.5ml EP pipe tube bottom, are carefully added into 200 μ l sustained release
Buffer is placed in 37 DEG C of insulating boxs and is protected from light incubation, carefully draws supernatant in set time point and detects its HGF and anti-TNF- ɑ
Content, and the fresh sustained release buffer of 200 μ l is added again.Wherein HGF content is operated by ELISA kit specification,
Anti-TNF- ɑ content reads fluorescent value using fluorescence microplate reader and is calculated.According to HGF in supernatant and anti-TNF- ɑ content
It calculates release rate and draws release profiles.
HGF is sustained in 2.2 self assembly polypeptide hydrogel bodies
According to experimental cost and experiment meaning, this part only shows that self-assembling peptides are sustained HGF in animal body.
Animal packet: tri- groups of Free-HGF, Gel-HGF, Gel
1) HGF and hydrogel are marked according to Cy7 fluorescent dye specification respectively, and free fluorescence is removed by ultrafiltration and is visited
Needle.
2) Balb/c 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.
5) mouse notch is sutured, is adopted in carrying out first time image on mouse living imaging system (In-Vivo Xtreme)
Collection, and it is recorded as experimental result when 0.
6) it after Image Acquisition is complete, sends mouse back to animal house and normally raises.Again respectively at for 24 hours, abdominal cavity is infused after 72h, 120h
It penetrates and carries out living imaging after giving yellow Jackets anesthetized mice.
7) mouse is put to death after advanced action object entirety living imaging after 120h, and collects its liver, spleen, kidney, small intestine
It is imaged.
3 experimental results
3.1 self assembly polypeptide hydrogels external sequential release anti-TNF- ɑ and HGF
The hydrogel for being enclosed with heparin sodium, anti-TNF- ɑ and HGF is placed in sustained release buffer, is detected on each time point
Anti-TNF- ɑ and HGF in clear, draw release profiles.The result shows that hydrogel has good delay to anti-TNF- ɑ and HGF
Release effect.Anti-TNF- ɑ rate of release is significantly higher than HGF, when for 24 hours preparation reach enter after 50%, 48h it is flat
The platform phase, and presentation of the HGF in hydrogel continues slowly to discharge.(as shown in Figure 1)
HGF is sustained in 3.2 self assembly polypeptide hydrogel bodies
The HGF (Free-HGF) that Cy7 is marked is wrapped in HGF (Gel-HGF) and the Cy7 label of the Cy7 label of hydrogel
Hydrogel (Gel) be injected in mouse subrenal capsule respectively, and carry out living body fluorescent imaging in each test point.The result shows that trip
It is very fast in Mice Body intracellular metabolite from HGF, it can't detect the HGF of Cy7 label after 24 hours.The HGF being wrapped in hydrogel exists
It remains within 3-5 days detect, the Cy7 visible 0-5 days fluorescence of hydrogel marked is concentrated on into always renal tract (Fig. 2A).After 5 days
Materials, by mouse liver, spleen, small intestine, kidney progress fluorescence imaging, it is seen that the hydrogel of Cy7 label resides at kidney, hydrogel
The long period injection site can be remained in after injection.Although water Gel-HGF group is in abdominal cavity location detection to fluorescence signal, kidney
Fluorescence is not detected, shows that hydrogel effectively slows down the release of HGF in animal body.(as shown in Figure 2 B)
From the above it is found that medicament slow release polypeptide hydrogel of the present invention is in vivo and in vitro to anti-TNF- ɑ and HGF
With good slow releasing function.
The kidney biocompatibility of the medicament slow release polypeptide hydrogel of the present invention of test example 2
1 experimental material
C57 mouse (6-8 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.)
Histopathology detects related reagent (tissue is fixed, and is embedded, and is sliced, dyeing)
2 experimental methods
In order to study the self assembly polypeptide hydrogel biocompatibility, mouse kidney peplos give hydrogel injection, and pass through
Its influence of histopathology staining evaluation.Concrete operations are as follows:
1) hydrogel prepares (system of a kidney), the self assembly polypeptide liquid storage for the 10mg/ml for taking 10 μ l to prepare, and is added 10
μ l sterile water is mixed stand-by with insulin syringe piping and druming.
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) mouse is randomly divided into control group (3), Gel group (9), gives 20 μ l of mouse list kidney (left kidney) kidney peplos respectively
Physiological saline or hydrogel.
5) respectively at it is postoperative for 24 hours, 72h, 120h put to death three mouse of Gel group, normal group is put to death when 120h, collection mouse
Kidney.
6) mouse kidney extracts total serum IgE, carries out quantitative fluorescent PCR (qPCR) and detects inflammatory factor expression situation.
7) mouse kidney carries out HE and Masson dyeing, and microscopy evaluates kidney structure and lesion situation.
3 experimental results
Self-assembling peptides hydrogel injection mouse give kidney peplos after, respectively at it is postoperative for 24 hours, 48h, 72h draw materials and carry out
QPCR detection and HE dyeing, the results showed that hydrogel injection to kidney normal configuration and inflammatory factor such as HMGB1, IL-1 β and
ICAM-1 all has no significant effect, and Masson coloration result also indicates that hydrogel injection does not influence the expression of renal collagen fiber.(as schemed
Shown in 3)
From the above it is found that medicament slow release polypeptide hydrogel of the present invention has no significant effect kidney normal configuration, have
Good kidney biocompatibility.
The medicament slow release polypeptide hydrogel of the present invention of test example 3 is sustained anti-TNF- ɑ and HGF treatment ischemic acute renal damage
Wound verifying
1 experimental material
Self-assembled short peptide KLD1R
Anti-mouse TNF- ɑ antibody (Biolegend)
It recombinates HGF (Divine Land Yi Qiao biotech firm)
Heparin sodium (Sigma)
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 medium and slice immunofluorescence dyeing (tissue is fixed, and is closed, dyeing)
TUNEL staining kit (Promega)
Immunofluorescence dyeing primary antibody: IL-1 β (ABclonal), ICAM-1 (Proteintech), FN (ABclonal), α-
SMA (Abcam), Pax2 (ABclonal), Ki67 (GeneTex), NGAL (ABclonal)
Immunofluorescence dyeing secondary antibody: FITC/TRITC marks goat antirabbit/mouse fluorescence secondary antibody RNA extraction and RT-PCR phase
Close reagent
2 experimental methods
In order to study the effect of self assembly polypeptide hydrogel sustained release anti-TNF- ɑ and HGF treatment ischemic acute injury of kidney
Fruit closes building ischemia-reperfusion acute kidney injury model by C57 Mouse Kidney artery clamp, and gives to intervene in kidney peplos, passes through
Histopathology dyeing, its therapeutic effect of the overall merits such as Real time-PCR and histogenic immunity fluorescent staining.Concrete operations are such as
Under:
1) according to the form below prepares each group 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, after folder closes
It can be seen that kidney is because of ischemic blackening.
5) artery clamp is removed after 30min, it is seen that renal blood flow restores, 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 and AKI group injecting normal saline.
7) suture operation notch, after wound disinfection, mouse sends animal house back to and continues normal feed.
8) 72h, 120h put to death mouse for 24 hours respectively at postoperative, collect mice serum and kidney.
9) automatic biochemical detector (Roche) detects mice serum creatinine (CREA), urea nitrogen (UREAL) concentration.
10) mouse kidney carries out HE, Masson dyeing, and microscopy evaluates kidney structure lesion situation and fibrosis water
It is flat.
11) kidney carries out frozen section, carries out TUNEL dyeing, evaluates rat tubular cell apoptosis situation.12) renal tissue
RNA is extracted, RT-PCR detects inflammation, and fibrosis GAP-associated protein GAP rna expression is horizontal.
13) renal tissue frozen section progress immunofluorescence dyeing, detection kidney ey injury markers NGAL, renal inflammation,
Fibrosis correlative protein expression is horizontal.
14) renal tissue takes paraffin section to carry out immunohistochemical staining, detection renal inflammation, renal tubule regeneration and fibrosis
Talk about protein expression.
3 experimental results
The sequential release anti-TNF- ɑ and HGF of 3.1Gel slows down injury of kidney
Acute renal injury in mice (AKI) model is established by renal ischemia/reperfusion injury, subrenal capsule injects HGF/anti-
TNF- ɑ (Free group) or the HGF/anti-TNF (Gel group) being wrapped in hydrogel are intervened, and materials are examined after 72 hours
It surveys.Blood biochemistry is the results show that Gel group mouse renal function index serum creatinine and urea nitrogen are below AKI model group and Free group (figure
4A).HE the result shows that, AKI group mouse kidney renal tubule lumen distention, protein cast is formed, and part tubule brush border falls off, and
Gel group renal damage degree significantly reduces, and is lower than Free group.TUNEL dyeing display AKI group renal tubular cell is largely positive
Property, show a large amount of apoptosis of its renal tubular cell；Compared with Free group, Gel group TUNEL positive apoptotic cells quantity is significantly reduced,
Show that it is sustained the rat tubular cell apoptosis that anti-TNF- ɑ effectively inhibits inflammation-induced.NGAL (neutrophil leucocyte gelatinase phase
Close apolipoprotein -2) it is early stage and sensitive injury of kidney biomarker.Immunofluorescence dyeing the result shows that, in normal kidney tissue
Its expression quantity is extremely low, AKI group renal tubule great expression, and Gel group NGAL expression quantity significantly reduces compared with Free group, instruction kidney damage
Hurt degree reduction.(as shown in Figure 4)
3.2Gel sequential release anti-TNF- ɑ and HGF reduce renal inflammation reaction
72 hours mouse materials after operation, and inflammation in nephridial tissue is detected by Real time-PCR and promotees apoptogene
Expression.The result shows that promoting the significant raising of apoptogene Bax expression in AKI group nephridial tissue, it coincide with TUNEL coloration result.Together
When AKI group mouse kidney great expression inflammatory factor IL-1 β and IL-6, immunofluorescence dyeing also indicates that AKI induction renal tubule is high
Express IL-1 β.Showed by immune group result, in Gel group CD68 positive macrophage order of magnitude chemotactic factor (CF) MCP-1 expression compared with
Free group and AKI group are substantially reduced.In conclusion the renal inflammation reaction of AKI can more be effectively reduced compared to it for Gel group Free group
And apoptosis degree.(as shown in Figure 5)
3.3Gel sequential release anti-TNF- ɑ and HGF promote kidney reparation
Impaired kidney has certain self-repairing capability, depends on dedifferenting and increasing for the intrinsic cell of kidney
It grows.Immunofluorescence dyeing detects renal tissue Ki67 expression, the results showed that normal kidney tissue cell does not rise in value substantially, AKI group
A small amount of Ki67 positive is presented in tubule cells, shows that the renal tubular cell of remaining starts to be proliferated, the starting of kidney repair process.Gel is dry
Pre- group Ki67 cell positive rate is significantly higher than AKI and Free group.Pax2 is the marker that renal tubular cell dedifferentes, normal kidney
Cell is not expressed, and damage kidney is expressed, and the expression of Gel intervention group is significantly increased compared with AKI and Free group.ImmunohistochemistryResults Results
It shows, the PCNA positive proliferative cell and Pax2 positive cell number in Gel group mouse kidney tissue are above Free group.This part
It dedifferentes and is proliferated the experimental results showed that being sustained HGF by self-assembling peptides hydrogel and can effectively facilitate renal tubular cell.(such as Fig. 6
It is shown)
From the above it is found that medicament slow release hydrogel of the present invention can effectively inhibit the rat tubular cell apoptosis of AKI, reduce
NGAL expression quantity mitigates renal inflammation reaction, effectively facilitates renal tubular cell and dedifferente and be proliferated, promotes compromised kidney reparation
Effect.
To sum up, polypeptide hydrogel of the present invention has good medicament slow release effect and kidney biocompatibility, passes through addition
Heparin achievees the purpose that sequential successive release anti-TNF- ɑ and HGF, polypeptide hydrogel of the present invention can effectively inhibit the kidney of AKI
Tubule cells apoptosis reduces NGAL expression quantity, mitigates renal inflammation reaction, promotes renal tubular cell to dedifferente and be proliferated, have
Extend anti-TNF- ɑ and HGF half-life period, give full play to protein drug the different times the effect of, reduce injury of kidney degree, promote
Into the effect of compromised kidney reparative regeneration, potential applicability in clinical practice is good.
[0001] sequence table
<110>Huaxi Hospital Attached to Sichuan Univ
<120>a kind of medicament slow release polypeptide hydrogel and its preparation method and application
<130> GY026-18P1091
<141> 2018-03-05
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 13
<212> PRT
<213>artificial synthesized sequence (artificial synthesis)
<400> 1
Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu Asp LeuArg
1 5 10
Claims (10)
1. a kind of self-assembling peptides, it is characterised in that:
Its amino acid sequence is KLDLKLDLKLDLR (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 heparin sodium, hepatocyte growth factor and TNF- ɑ antibody in the aqueous solution.
4. polypeptide hydrogel according to claim 2 or 3, it is characterised in that: it is the raw material group matched by following weight
At:
TNF- ɑ antibody-solutions 1-5 parts by volume, hepatocyte growth factor 0.3-1 parts by weight, heparin sodium 0.5-3 parts by weight, self assembly
Peptide 50-300 parts by weight, add water to 20-40 parts by volume；
Wherein, the concentration of the TNF- ɑ antibody-solutions is 0.5-10mg/ml.
5. polypeptide hydrogel according to claim 4, it is characterised in that: it is made of the raw material that following weight matches:
2 parts by volume of TNF- ɑ antibody-solutions, 0.5 parts by weight of hepatocyte growth factor, 1 parts by weight of heparin sodium, 100 weight of self-assembling peptides
Part is measured, 25 parts by volume are added water to；
Wherein, the concentration of the TNF- ɑ antibody-solutions is 1mg/ml.
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) 50-300 parts by weight self-assembling peptides dry powder is dissolved in 8-15 parts by volume water, is configured to self-assembling peptides aqueous solution；It will
0.5-3 parts by weight heparin sodium dry powder is dissolved in 0.5-3 parts by volume water, is configured to heparin sodium water solution；0.3-1 parts by weight liver is thin
Intracellular growth factor dry powder is dissolved in 3-10 parts by volume water, prepares hepatocyte growth factor aqueous solution；Take the TNF- ɑ of 1-5 parts by volume
Antibody-solutions；
(2) 4 kinds of solution that step (1) obtains are taken, 20-40 parts by volume is added water to, is mixed to get polypeptide hydrogel；
Wherein, the concentration of the TNF- ɑ antibody-solutions is 0.5-10mg/ml.
7. preparation method according to claim 6, which is characterized in that it is comprised the following steps:
(1) 100 parts by weight self-assembling peptides dry powder are dissolved in 10 parts by volume sterile distilled waters, are configured to self-assembling peptides aqueous solution；
1 parts by weight heparin sodium dry powder is dissolved in 1 parts by volume sterile distilled water, heparin sodium water solution is configured to；0.5 parts by weight liver is thin
Intracellular growth factor dry powder is dissolved in 5 parts by volume sterile distilled waters, prepares dry hepatocyte growth factor aqueous solution；Take 2 parts by volume
TNF- ɑ antibody-solutions；
(2) 4 kinds of solution that step (1) obtains are taken, adds the sterile distilled water of 7 parts by volume, is mixed, 25 parts by volume are in total to get more
Peptide hydrogel；
Wherein, the concentration of the TNF- ɑ antibody-solutions is 1mg/ml.
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 inhibit acute kidney injury to cause
Rat tubular cell apoptosis, reduce NGAL expression quantity, mitigate renal inflammation reaction, promote compromised kidney reparation.
10. purposes according to claim 8, it is characterised in that: the polypeptide hydrogel can improve Ki67 cell positive
Rate promotes Pax2 expression, and renal tubular cell is promoted to dedifferente and be proliferated.
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| CN113321709A (en) * | 2021-05-18 | 2021-08-31 | 南方医科大学 | Self-assembled polypeptide, slow-release exosome polypeptide hydrogel and preparation method and application thereof |
| CN114931547A (en) * | 2022-05-16 | 2022-08-23 | 四川大学华西医院 | Polypeptide hydrogel for treating alveolar bone injury and preparation method and application thereof |
| CN114931547B (en) * | 2022-05-16 | 2023-04-28 | 四川大学华西医院 | Polypeptide hydrogel for treating alveolar bone injury and preparation method and application thereof |
| CN116983321A (en) * | 2023-09-04 | 2023-11-03 | 浙江大学医学院附属邵逸夫医院 | Polypeptide hydrogel precursor solution loaded with microRNA and preparation method and application thereof |
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