CN108434441A - Application of novel fibroblast growth factor -21 analogs of son in treating the chromic fibrous pneumonopathy of diffusivity - Google Patents
Application of novel fibroblast growth factor -21 analogs of son in treating the chromic fibrous pneumonopathy of diffusivity Download PDFInfo
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Abstract
The invention discloses a kind of application of 1 reforming technology of fibroblast growth factor 2 and sub 21 analogs of improved novel fibroblast growth factor in the chromic fibrous pneumonopathy treatment for the treatment of diffusivity and/or in preventing.It is 1 analog of fibroblast growth factor 2 that the present invention, which also protects a kind of drug treated and/or prevent the chromic fibrous pneumonopathy of diffusivity, active constituent,.Present invention discover that 1 analog of fibroblast growth factor 2 can effectively inhibit the generation of the chromic fibrous pneumonopathy of diffusivity, it can be used as drug therapy and/or prevent the chromic fibrous pneumonopathy of diffusivity, there is substantial worth for the treatment and prevention of the chromic fibrous pneumonopathy of diffusivity.
Description
Technical field
The present invention relates to fibroblast growth factor-21 reforming technologies, and pass through improved novel fibroblast cell
A kind of new application of 1 analog of growth factor-2, i.e. novel fibroblast growth factor -21 analogs of son are between treating diffusivity
Application in matter pneumonopathy.
Background technology
The chromic fibrous pneumonopathy of diffusivity is the common final result that numerous pulmonary diseases develop to late period, major pathologic features
It is chronic pulmonary interstitial inflammation, fibroblastic activation and proliferation, extracellular matrix（Extracellular matrixc, ECM）
Over-deposit, the institutional framework for eventually leading to lung is destroyed, function is impaired, respiratory failure, or even dead.
Studies have shown that the pathogenic factors of the chromic fibrous pneumonopathy of diffusivity is numerous, there are about 200 kinds at present, including itself exempts from
Epidemic disease disease, drug, environment, infection, heredity etc..Also the chromic fibrous pneumonopathy of part diffusivity because inducement it is unknown, be referred to as
Idiopathic pulmonary fibrosis（IPF）, severity is suitable with lung cancer, and 5 years survival rates only have 30%~50%.According to conservative estimation, I
Chromic fibrous 3,200,000 people of pneumonopathy patient of the existing diffusivity of state, average 10 patients of increase daily, dead 2, and incidence
Trend is steeply risen in presentation, has become one of the important diseases for seriously endangering health.
Be presently used for treatment the chromic fibrous pneumonopathy of diffusivity medicament categories mainly include anti-inflammatory drug, antioxidant,
Chinese medicine, anti-fibrosis medicine and genetic engineering antibody etc..The drug of specific anti-fibrosis only has pirfenidone and Ni Dani
Cloth, clinical efficacy are preferable.It is approved by the FDA in the United States in October, 2014, the lung function that can effectively slow down mild to moderate IPF patient declines
It moves back, delays progression of disease.But can both lead to apparent side effect, as pirfenidone leads to gastrointestinal tract and skin allergy
Reaction, Nintedanib cause diarrhea etc..So between clinically still lacking safe and effective specific treatment diffusivity at present
The drug of matter pneumonopathy.In addition, the gradual of pathogenic mechanism research with the chromic fibrous pneumonopathy of diffusivity gos deep into, research
Person develops the novel drugs and new way for the chromic fibrous pneumonopathy treatment of diffusivity again, but most of research is still in clinic
Preceding or clinical investigation phase.Therefore, more safely effectively drug becomes complete to the chromic fibrous pneumonopathy of research treatment diffusivity
One of the focus of competition of world's researcher.
The main molecules mechanism of the chromic fibrous pneumonopathy of diffusivity include inflammatory reaction, lung fibroblast to flesh at fiber
Transformation, Epithelial-mesenchymal transition（Epithelial-mesenchymal transition, EMT）, base
Matter metalloproteinases（Matrix metalloproteinase, MMPs）/ tissue inhibitors of metalloproteinases（Tissue
Inhibitor of metalloproteinase, TIMP）Unbalance, oxidative stress and the activation of relevant signal transduction pathway etc..
In recent years, research finds that EMT is one of the important mechanisms that the chromic fibrous pneumonopathy of diffusivity occurs.In the chromic fibrous lungs of diffusivity
During disease occurs, alveolar epithelial cells loses the structure and function of its epithelium, is transformed into mesenchymal cell-flesh into fiber
Cell, and then secrete a large amount of ECM and be gathered in lung leads to lungs scar and stiff, to form the chromic fibrous lungs of diffusivity
Disease.When EMT occurs for alveolar epithelial cells, endothelial cell marker such as E- calcium mucin thereon（E-cadherin）, keratin
（keratin）Deng disappearance, and then there is α-smooth muscle actin（α-smooth muscle actin, α-SMA）, waveform egg
In vain（Vimentin）Equal mesenchymal cell markers.Numerous studies confirm outside Current Domestic, generation and hair of the TGF-β 1 in EMT
Important function is played in exhibition.Numerous studies find that the ROS that oxidative stress generates can promote the expression and activation of TGF-β 1.
Therefore, the oxidation resistance for improving body can inhibit the expression and activation of TGF-β 1, and then inhibit EMT, and diffusivity is effectively relieved
Chromic fibrous pneumonopathy.
Fibroblast growth factor-21 is because having the function of that adjusting glycolipid metabolism etc. becomes the focus of research.But it is acted on
Effect deficient in stability, exact effect need further to verify.The structure and activity of drug gene itself are to determine its work
One of an important factor for effect and stability.Therefore, to people source fibroblast growth factor-21 carry out molecular modification and
Optimal Expression, it has also become the hot spot of the field Recent study.
Invention content
The object of the present invention is to provide fibroblasts after fibroblast growth factor-21 reforming technology, and transformation to give birth to
Application of the long factor-21 analog in treating the chromic fibrous pneumonopathy of diffusivity.
The present invention also protects the drug of a kind for the treatment of and/or the prevention chromic fibrous pneumonopathy of diffusivity, and active constituent is
Fibroblast growth factor-21 analog.The drug further includes other pharmaceutically acceptable carriers or auxiliary material.The medicine
In object, other than the active constituent, the excipient pharmaceutically allowed, filler, sorbefacient, surface-active can be added
Agent, absorption carrier, synergist and additive etc..The administration form of the drug can be injection（Such as pulvis, aqua, finish）.Institute
Preparation is stated the known common preparation method of those skilled in the art all can be used and obtain.The administration route of the drug can be skin
Lower injection, intravenous injection or intramuscular injection.
Any description above fibroblast growth factor-21 analog is as follows（a）Or（b）：
（a）The protein that amino acid sequence forms shown in sequence in sequence table 1；
（b）By the amino acid sequence of sequence 1 by one or several amino acid residues substitution and/or lack and or add and
Have the function for the treatment of and/or preventing the protein derived from sequence 1 of the chromic fibrous pneumonopathy of diffusivity.
The fibroblast growth factor-21 analog is presented as the therapeutic effect of the chromic fibrous pneumonopathy of diffusivity
（c1）Extremely（c7）At least one of：
（c1）The chromic fibrous pneumonopathy of diffusivity is inhibited to occur；
（c2）Inhibit the chromic fibrous pneumonopathy degree of diffusivity；
（c3）Reduce the level of collagenous fibres and/or muscle fibre in lungs；
（c4）Reduce the level of hydroxyproline in lungs；
（c5）Reduce I-type collagen and/or the level of α-smooth muscle actin in lungs；
（c6）It is horizontal to reduce TGF-β 1 in lungs；
（c7）It is horizontal to improve Nrf2 in lungs, improves antioxidant ability of organism, inhibits TGF-β 1 horizontal.
The method system included the following steps specifically can be used in any description above fibroblast growth factor-21 analog
It is standby：
（1）Express the fusion protein of the fibroblast growth factor-21 analog and molecular chaperones；
（2）The fusion protein is collected, cuts off that obtain the fibroblast growth factor-21 after the molecular chaperones similar
Object.
Concretely molecule ubiquitin sample modifies albumen to the molecular chaperones（That is molecular chaperones SUMO）.
The realization side of " fusion protein for expressing the fibroblast growth factor-21 analog and molecular chaperones "
Method is as follows：The encoding gene of the fibroblast growth factor-21 analog is inserted into prokaryotic expression carrier pSUMO, is obtained
Recombinant plasmid；The recombinant plasmid is imported into Escherichia coli Rosetta (DE3), obtains recombinant bacterium；It ferments and the recombinant bacterium and adopts
It is induced with IPTG, obtains the fusion protein.
The realization method of described " cutting off the molecular chaperones " is as follows：The fusion protein is taken, with SUMO protease I
（The molar ratio of SUMO protease I and fusion protein concretely 1:50）And DTT（The initial concentration of the DTT can be 2mmol/
L）Cutting（Cutting condition concretely stay overnight by 4 DEG C of cuttings）.
The encoding gene of the fibroblast growth factor-21 analog concretely following 1）Or 2）Or 3）DNA
Molecule：
1）DNA molecular shown in sequence 2 in sequence table；
2）Under strict conditions with 1）The DNA sequence dna of restriction hybridizes and encodes the DNA molecular of albumen with the same function；
3）With 1）The DNA sequence dna of restriction has the DNA molecular of 90% or more homology and coding albumen with the same function.
The stringent condition is in 0.1 × SSPE（Or 0.1 × SSC）, 0.1% SDS solution in, it is miscellaneous under the conditions of 65 DEG C
It hands over and washes film.
Present invention discover that improved fibroblast growth factor-21 analog can effectively inhibit diffusivity chromic fibrous
The generation of pneumonopathy, and function and effect are significantly better than people source fibroblast growth factor-21, can be used as drug therapy and/
Or prevent the chromic fibrous pneumonopathy of diffusivity, there is substantial worth for the treatment and prevention of the chromic fibrous pneumonopathy of diffusivity.
Description of the drawings
Fig. 1 is the polyacrylate hydrogel electrophoretogram of fibroblast growth factor-21 analog.
Fig. 2 is HE coloration results.
Fig. 3 is Masson coloration results and superficial density.
Fig. 4 is hydroxyproline content testing result.
Fig. 5 is TGF-β 1, I-type collagen, α-smooth muscle actin and E- calcium Mucin gene with respect to β-actin bases
Because the expression of transcriptional level changes.
Fig. 6 is TGF-β 1, I-type collagen, α-smooth muscle actin and E- calcium Mucin gene with respect to β-actin bases
Because the expression of protein level changes.
Fig. 7 is that expression of the Nrf2 genes with respect to the transcriptional level of β-actin genes changes.
Fig. 8 is that expression of the Nrf2 genes with respect to the protein level of β-actin genes changes.
Specific implementation mode
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
Fibroblast growth factor-21 analog, abbreviation FGF-21 analogs, as shown in the sequence 1 of sequence table.FGF-
The encoding gene of 21 analogs is as shown in the sequence 2 of sequence table.
Prokaryotic expression carrier pSUMO（It is referred to as in the literature " pHisSUMO "）：Bibliography：Jiang Yuanyuan, Yin Chengkai, Lee
Jin Nan, Ren Guiping, Zhang Wei, Li Deshan.Utilize the research of SUMO emerging system high efficient expression soluble recombinant proteins.Northeast agricultural
College journal, 2008,39 (10): 57-62；Li Lu, Yin Chengkai, Li Deshan.The expression of high efficient expression soluble recombinant protein carries
Body --- pHisSUMO.Biotechnology, 2009,19 (3): 11-14.
Escherichia coli Rosetta (DE3)：Beijing Quanshijin Biotechnology Co., Ltd, catalog number (Cat.No.) CD801.
Male C57BL/6 mouse：Shanghai Slac Experimental Animal Co., Ltd., animal quality quality certification number SCXK
(Shanghai) 2017-0005.
PBS buffer solution（pH 7.2）：Solvent is water, and solute and its concentration are as follows：NaCl 137 mmol/L, KCl 2.7
Mmol/L, Na2HPO410 mmol/L, KH2PO4 2 mmol/L。
Hematoxylin eosin staining method (hematoxylin-eosin staining), abbreviation HE decoration methods：Hematoxylin dye liquor
For alkalinity, endonuclear chromatin is mainly made hyacinthine with intracytoplasmic ribosomes；Yihong is acid dyes, is mainly made thin
Ingredient red coloration in cytoplasm and extracellular matrix.
Masson is dyed（It is mainly used for the differential staining of collagenous fibres and muscle fibre）：Collagenous fibres are in blue (by aniline
It is Lan Suoran) or green（It is contaminated by brilliant green）, muscle fibre takes on a red color and (contaminated by acid fuchsin and Ponceaux).
The preparation of embodiment 1, FGF-21 analogs
One, the structure of recombinant plasmid
1, in order to improve people source FGF-21 genes（As shown in 3 gene of sequence）The stability for giving expression to albumen does its gene
Following transformation：An alanine is added in the ends 5'（Ala）, extend the half-life period of recombinant protein, and by histidine（His）It is substituted for
Tyrosine（Tyr）, the ends 3' second-to-last alanine（Ala）It is substituted for threonine（Thr）, and it is inclined to the codon of gene
After good property optimizes, send to Services Co., Ltd of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd and synthesize full genome.
2, the genophore after synthesis is transferred in DH5 α competence and is expanded, then extract Plasmid DNA, use is restricted
Restriction endonuclease BsaI and BamHI double digestion recycles digestion products.
3, with restriction enzyme BsaI and BamHI double digestion prokaryotic expression carrier pSUMO, the carrier of about 5700bp is recycled
Skeleton.
4, the digestion products of step 2 are connected with the carrier framework of step 3, it is similar obtains recombinant plasmid pSUMO-FGF-21
Object.In recombinant plasmid pSUMO-FGF-21 analogs, the molecular chaperones on the encoding gene and carrier framework of FGF-21 analogs
The coded sequence of the coded sequence of SUMO and the His labels on carrier framework（Positioned at the upstream of the coded sequence of SUMO, by 6
A histidine residues composition）Fusion forms fusion, expressed fusion protein（Fusion protein is followed successively by His marks from N-terminal to C-terminal
Label, molecular chaperones SUMO and FGF-21 analog）.
The expection molecular weight of FGF-21 analogs is 21 kDa（Reckoning molecular weight is 21 kDa, SDS-PAGE electrophoresis showeds
Molecular weight is 25 kDa or so）, the expection molecular weight of molecular chaperones SUMO is 12 kDa（Reckoning molecular weight is 12 kDa, SDS-
PAGE electrophoresis showed molecular weight is 18 kDa or so）.
Two, the preparation and purification of FGF-21 analogs
1, recombinant plasmid pSUMO-FGF-21 analogs are imported into Escherichia coli Rossetta (DE3), obtains recombinant bacterium.
2, the single bacterium colony for the recombinant bacterium that step 1 obtains is seeded in 5 mL LB culture mediums, 37 DEG C, 120rpm oscillation trainings
10h is supported, bacterium solution is then taken, with 1:100 volume ratio is inoculated in the LB culture mediums of 500mL penicillin containing 50mg/mL, 37 DEG C,
120rpm shaken cultivation 2h, at this time OD600nmIt is 0.5 or so.
3, IPTG is added in the bacterium solution obtained to step 2 and makes its a concentration of 0.25mmol/L to be induced（25℃、
60rpm shaken cultivations 10h）, then 4 DEG C, 4000rpm centrifugation 30min collect thalline.
4, the thalline for taking step 3 to obtain carries out ultrasonication（AMP 35%, 8-12min, work 1s stop 1s）, 4 DEG C,
12000rpm is centrifuged, and collects supernatant and precipitation respectively.
Supernatant and precipitation are subjected to 12% SDS-PAGE electrophoretic analysis respectively.The result shows that the purpose of solubility expression
Albumen accounts for 70% or more of thalline catalogue albumen.
5, the supernatant for taking step 4 to obtain carries out HisTrapTM FF crude colum affinity chromatographys.
Column model is：Column length 0.7cm, pillar height 2.5cm.
Applied sample amount is 10ml.
Elution process：First with the foreign protein eluent of 5 times of column volumes（Solvent is water, each solute containing following concentration：
40mmol/L imidazoles, 500mmol/L NaCl and 50mmol/L Na3PO4；pH7.4）Elution is to remove foreign protein, flow velocity 1ml/
min；Then with the destination protein eluent of 3 times of column volumes（Solvent is water, each solute containing following concentration：500mmol/L
Imidazoles, 500mmol/L NaCl and 50mmol/L Na3PO4；pH7.4）Elution, flow velocity 1ml/min, 280nm wavelength monitoring are received
Collect target peak（I.e. peak value is higher than the peak of 80mAU）, as fusion protein solution.
6, desalination is carried out using the fusion protein solution that 26/10 Desalting of HiPrepTM obtain step 5.
7, the solution for taking step 6 to obtain, with SUMO protease I（The molar ratio of SUMO protease I and fusion protein is 1:
50）4 DEG C of cuttings of DTT with final concentration of 2mmol/L are overnight.
8, the solution for taking step 7 to obtain carries out HisTrapTM FF crude colum affinity chromatographys.
Column model is：Column length 0.7cm, pillar height 2.5cm.
Applied sample amount is 15ml, and 280nm wavelength monitorings collect target peak（I.e. peak value is higher than the peak of 30mAU）, as FGF-21
Analog solution.
The polyacrylate hydrogel electrophoretogram of FGF-21 analog solution is shown in Fig. 1（Swimming lane 1 is molecular weight marker, and swimming lane 6 is FGF-
21 analog solution）.Recycling purpose band simultaneously carries out N-terminal sequencing, the results showed that, 15 amino acid residue such as sequence tables before N-terminal
Sequence 1 is from shown in the 1st to 15 amino acids residue of N-terminal.
The therapeutic effect of embodiment 2, FGF-21 analogs to the chromic fibrous pneumonopathy of diffusivity.
Experimental animal is six week old detergent male C57BL/6 mouse.
The FGF-21 analog solution prepared using embodiment 1 is tested.
SPF grades of C57BL/6 mouse of 92 14 week old males, weight about 25-30 g are 22 ± 2 DEG C in temperature, relatively wet
Degree is 55 ± 2 %, gives 12 h illumination daily, under conditions of giving the standard diet of rodent, after 5 d of adaptable fed,
It is randomly divided into 5 groups：Normal control（Normal control groups, N）Group 12；Model comparison（BLM）Group：20；People source
FGF-21 is treated（hFGF-21）Group：20；FGF-21 analogue treatments（FH）Group：20；Pirfenidone treatment（PFD）Group：20
Only.BLM uses the normal saline dilution of 0.9 %, and mouse is aseptically performed the operation, BLM groups, hFGF-21 groups, FH groups
With 2 mg/ml BLM of PFD groups difference intratracheal instillation.The isometric physiological saline of N group mouse intratracheal instillations.Mouse is being instiled
After 7 d of BLM, BLM groups, hFGF-21 groups, FH groups and PFD groups take the successful mouse of modeling 12 respectively, carry out subsequent experimental,
HFGF-21 groups and FH groups mouse start that FGF-21 analog albumen is subcutaneously injected respectively, and dosage is respectively 5 mg/kg, note daily
It penetrates 1 time, continuous injection 3 weeks.According to the literature, PFD groups mouse uses 500 mg/kg of gastric infusion after 7 d of instillation BLM
Same volume PBS is subcutaneously injected in PFD, N group mouse and BLM groups mouse daily.Terminate within 4th week, puts to death each group mouse, take mouse lung
Dirty tissue.
In intratracheal injection bleomycin（BLM）It induces mouse lung fibrosis model after a week, FGF-21 is injected intraperitoneally
Analog carries out treatment three weeks, then puts to death mouse, takes lungs, carries out HE dyeing.Lungs are sliced HE coloration results and show, model
Group（M）Mouse lung damage is serious, and the alveolar space of mouse part lungs disappears, and has a large amount of inflammatory cell infiltration in alveolar space
（Shown in arrow a）, fibroblast（Shown in arrow b）, lungs lose its original structure.Compared with model group, PFD intervention groups are small
Only have a small amount of inflammatory cell and fibroblast in mouse lungs, observable sees the more neat alveolar structure of complete arrangement.
FGF-21 analogue treatment group results are similar to PFD treatment results, slightly alleviate after the treatment of hFGF-21 groups, but FGF-21 is similar
Object therapeutic effect is significantly better than hFGF-21, as shown in Figure 2.
After experiment, the lungs of each group mouse is taken to carry out Masson dyeing.The results show that model group（M）Mouse lung
The content of collagenous fibres（Blue region）It is significantly increased compared to Normal group；Collagenous fibres in PFD intervention group mouse lungs
Content（Blue region）Compared to model group（M）It is remarkably decreased, FGF-21 analog intervention group results and PFD intervention group results
It is similar, slightly alleviate after the treatment of hFGF-21 groups, but FGF-21 analogue treatment significant effects are better than hFGF-21, as shown in Figure 3.
Using the hydroxyproline content in hydroxyproline kit detection lungs, the results show that model group（M）Lungs hydroxyl dried meat
Propylhomoserin level is compared to Normal group（N）Significantly increase, and PFD intervention group mouse lung hydroxyprolin levels are compared to model
Group（M）It being remarkably decreased, is on close level with Normal group, FGF-21 analogue treatments group is close with PFD intervention group results,
Slightly alleviate after the treatment of hFGF-21 groups, but FGF-21 analogue treatment significant effects are better than hFGF-21, as shown in Figure 4.
It extracts each group mouse lung portion of tissue and extracts RNA, reverse transcription is fine using Real-time PCR detections at cDNA
Dimensionization related gene TGF-β 1, I-type collagen, α-smooth muscle actin and E- calcium mucins transcriptional expression are horizontal, use
β-actin genes are reference gene.Primer pair for detecting 1 gene of TGF-β is：5'-ATGCTAAAGAGGTCACCCGC-
3', 5'-TGCTTCCCGAATGTCTGACG-3'.The primer pair of I-type collagen gene is：5'-
CGCCATCAAGGTCTACTGC-3', 5'-GAATCCATCGGTCATGCTCT-3'.The primer of α-smooth muscle actin gene
To for：5'-CCGAGATCTCACCGAC-3', 5'-TCCAGAGCTACATGACACAG-3'.E- calcium Mucin gene primer pairs are
5'-GCAGTTCTGCCAGAGAAACC-3', 5'-TGGATCCAAGATGGTGATGA-3 '.The results show that model group（M）Lungs
Intgf-β1、col IAndα-smaMRNA level in-site is expressed compared to Normal group（N）Significantly increase,e-cadherin mRNA
Horizontal expression is compared to Normal group（N）It significantly reduces, and in PFD intervention group mouse lungstgf-β1、col IAndα-sma
MRNA expressions are compared to model group（M）It significantly reduces（P<0.05）,e-cadherinMRNA level in-site expression significantly increases,
It is close with normally organizing.The mRNA expressions of said gene and PFD intervention group phases in FGF-21 analogue treatment group mouse lungs
It is similar, slightly alleviate after the treatment of hFGF-21 groups, but FGF-21 analogue treatment significant effects are better than hFGF-21, as shown in Figure 5.
Each group mouse lung is taken, total protein is extracted, it is flat to detect TGF-β 1, I-type collagen, α-by Western blot
The protein expression of sliding flesh actin, E- calcium mucin and reference gene β-actin（For detecting TGF-β 1, type i collagen
Albumen, α-smooth muscle actin, E- calcium mucin and β-actin genes primary antibody be purchased from Abcam companies.Western blot
Photo and the TGF-β 1, I-type collagen, α-smooth muscle actin, the E- calcium mucins that are obtained by gray analysis it is opposite
The protein level of β-actin, is as a result shown in Fig. 6：Model group（M）Lungs TGF-β 1, Col I, α-SMA protein expressions compared to
Normal group（N）It significantly increases, E-cadherin expressions are compared to Normal group（N）It significantly reduces.And PFD intervenes
TGF-β 1, Col I, α-SMA protein expressions are compared to model group in group mouse lung（M）It significantly reduces, E-cadherin
Expression significantly increases.The result of FGF-21 analog intervention groups is close with PFD intervention group results, after the treatment of hFGF-21 groups
Slightly alleviate, but FGF-21 analogue treatment significant effects are better than hFGF-21.The result shows that FGF-21 analogs can be notable
Inhibit the generation of the chromic fibrous pneumonopathy of diffusivity, and improved therapeutic effect dramatically increases.
Mouse lung fibrosis model is induced in intratracheal injection BLM after a week, and intraperitoneal injection FGF-21 analogs carry out
Treatment three weeks.Nrf-2 expressions in lungs RNA, Real-time PCR detection each group mouse lungs are extracted, as a result（Such as Fig. 7）
It has been shown that, FGF-21 analogue treatments group and PFD intervention groups（PFD）Nrf-2 mRNA expressions are compared to model comparison in lungs
Group（M）And Normal group（N）It significantly increases, is slightly increased after the treatment of hFGF-21 groups, but FGF-21 analog function and effect
It is significantly better than hFGF-21.
Mouse lung fibrosis model is induced in intratracheal injection BLM after a week, and intraperitoneal injection FGF-21 analogs carry out
Treatment three weeks.Lungs albumen is extracted, Western blotting detect Nrf-2 protein expressions in each group mouse lung, knot
Fruit shows, FGF-21 analogs intervention group and PFD intervention groups（PFD）Nrf-2 expressions are compared to model control group in lungs
（M）And Normal group（N）It significantly increases, is slightly increased after the treatment of hFGF-21 groups, but FGF-21 analog function and effect are aobvious
It writes and is better than hFGF-21.As shown in Figure 8.
Sequence table
<110>Harbin Bo'ao Biopharmaceutical Technology Development Co., Ltd.
<120>Application of novel fibroblast growth factor -21 analogs of son in treating the chromic fibrous pneumonopathy of diffusivity
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
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<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Ala Thr Pro Ile Pro Ala Ser Ser Pro Leu Leu Gly Pro Gly Gly Gly
1 5 10 15
Val Ala Gly Ala Thr Leu Thr Thr Ala Ala Ala Gly Gly Thr Gly Ala
20 25 30
His Leu Gly Ile Ala Gly Ala Gly Thr Val Gly Gly Ala Ala Ala Gly
35 40 45
Ser Pro Gly Ser Leu Leu Gly Leu Leu Ala Leu Leu Pro Gly Val Ile
50 55 60
Gly Ile Leu Gly Val Leu Thr Ser Ala Pro Leu Cys Gly Ala Pro Ala
65 70 75 80
Gly Ala Leu Thr Gly Ser Leu His Pro Ala Pro Gly Ala Cys Ser Pro
85 90 95
Ala Gly Leu Leu Leu Gly Ala Gly Thr Ala Val Thr Gly Ser Gly Ala
100 105 110
His Gly Leu Pro Leu Ala Leu Pro Gly Leu Ala Ser Pro Ala Gly Ala
115 120 125
Ala Thr Ser Thr Gly Pro Val Ala Pro Leu Pro Met Pro Gly Leu Leu
130 135 140
His Gly Pro Gly Ala Gly Ala Gly Pro Leu Pro Pro Gly Pro Pro Ala
145 150 155 160
Val Gly Ser Ser Ala Pro Leu Ser Met Val Gly Pro Ser Gly Gly Ala
165 170 175
Ser Pro Ser Thr Thr Ser
180
<210> 2
<211> 549
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<213>Artificial sequence (Artificial Sequence)
<400> 2
gcttacccga tcccggactc ttctccgctg ctgcagttcg gtggtcaggt tcgtcagcgt 60
tacctgtaca ccgacgacgc tcagcagacc gaagctcacc tggaaatccg tgaagacggt 120
accgttggtg gtgctgctga ccagtctccg gaatctctgc tgcagctgaa agctctgaaa 180
ccgggtgtta tccagatcct gggtgttaaa acctctcgtt tcctgtgcca gcgtccggac 240
ggtgctctgt acggttctct gcacttcgac ccggaagctt gctctttccg tgaactgctg 300
ctggaagacg gttacaacgt ttaccagtct gaagctcacg gtctgccgct gcacctgccg 360
ggtaacaaat ctccgcaccg tgacccggct ccgcgtggcc cagctaggtt cctgccgctg 420
ccaggtctgc cgccggctct gccggaaccg ccgggtatcc tggctccgca gccgccggac 480
gttggttctt ctgacccgct gtctatggtt ggtccgtctc agggtcgttc tccgtcttac 540
acctcttga 549
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<213>Genus Homo people (Homo sapiens)
<400> 3
His Pro Ile Pro Ala Ser Ser Pro Leu Leu Gly Pro Gly Gly Gly Val
1 5 10 15
Ala Gly Ala Thr Leu Thr Thr Ala Ala Ala Gly Gly Thr Gly Ala His
20 25 30
Leu Gly Ile Ala Gly Ala Gly Thr Val Gly Gly Ala Ala Ala Gly Ser
35 40 45
Pro Gly Ser Leu Leu Gly Leu Leu Ala Leu Leu Pro Gly Val Ile Gly
50 55 60
Ile Leu Gly Val Leu Thr Ser Ala Pro Leu Cys Gly Ala Pro Ala Gly
65 70 75 80
Ala Leu Thr Gly Ser Leu His Pro Ala Pro Gly Ala Cys Ser Pro Ala
85 90 95
Gly Leu Leu Leu Gly Ala Gly Thr Ala Val Thr Gly Ser Gly Ala His
100 105 110
Gly Leu Pro Leu Ala Leu Pro Gly Leu Ala Ser Pro Ala Gly Ala Ala
115 120 125
Thr Ser Thr Gly Pro Val Ala Pro Leu Pro Met Pro Gly Leu Leu His
130 135 140
Gly Pro Gly Ala Gly Ala Gly Pro Leu Pro Pro Gly Pro Pro Ala Val
145 150 155 160
Gly Ser Ser Ala Pro Leu Ser Met Val Gly Pro Ser Gly Gly Ala Ser
165 170 175
Pro Ser Thr Ala Ser
180
<210> 4
<211> 546
<212> DNA
<213>Genus Homo people (Homo sapiens)
<400> 4
caccccatcc ctgactccag tcctctcctg caattcgggg gccaagtccg gcagcggtac 60
ctctacacag atgatgccca gcagacagaa gcccacctgg agatcaggga ggatgggacg 120
gtggggggcg ctgctgacca gagccccgaa agtctcctgc agctgaaagc cttgaagccg 180
ggagttattc aaatcttggg agtcaagaca tccaggttcc tgtgccagcg gccagatggg 240
gccctgtatg gatcgctcca ctttgaccct gaggcctgca gcttccggga gctgcttctt 300
gaggacggtt acaatgtgta ccagtctgaa gcccatggcc tgcccctgcg tctgcctcag 360
aaggactccc caaaccagga tgcaacatcc tggggacctg tgcgcttcct gcccatgcca 420
ggcctgctcc acgagcccca agaccaagca ggattcctgc ccccagagcc cccagatgtg 480
ggctcctctg accccctgag catggtggga ccttcccagg gccgaagccc cagctacgct 540
tcctga 546
Claims (6)
1. the reforming technology and improved novel fibroblast growth factor of people source fibroblast growth factor-21 gene
Application of sub -21 analogs in treating and/or preventing the chromic fibrous pneumonopathy of diffusivity.
2. application as described in claim 1, it is characterised in that：The fibroblast growth factor-21 analog is as follows
（a）Or（b）：
（a）The protein that amino acid sequence forms shown in sequence in sequence table 1；
（b）By the amino acid sequence of sequence 1 by one or several amino acid residues substitution and/or lack and or add and
Have the function for the treatment of and/or preventing the protein derived from sequence 1 of the chromic fibrous pneumonopathy of diffusivity.
3. application as claimed in claim 1 or 2, it is characterised in that：The fibroblast growth factor-21 analog is to more
The therapeutic effect of the chromic fibrous pneumonopathy of unrestrained property is presented as（c1）Extremely（c7）At least one of：
（c1）The chromic fibrous pneumonopathy of diffusivity is inhibited to occur；
（c2）Inhibit the chromic fibrous pneumonopathy degree of diffusivity；
（c3）Reduce the level of collagenous fibres and/or muscle fibre in lungs；
（c4）Reduce the level of hydroxyproline in lungs；
（c5）Reduce I-type collagen and/or the level of α-smooth muscle actin in lungs；
（c6）It is horizontal to reduce TGF-β in lungs；
（c7）It is horizontal to improve Nrf2 in lungs, improves antioxidant ability of organism, inhibits TGF-β 1 horizontal.
4. the drug of a kind for the treatment of and/or the prevention chromic fibrous pneumonopathy of diffusivity, active constituent is fibroblastic growth
Factor-21 analog.
5. drug as claimed in claim 4, it is characterised in that：The fibroblast growth factor-21 analog is as follows
（a）Or（b）：
（a）The protein that amino acid sequence forms shown in sequence in sequence table 1；
（b）By the amino acid sequence of sequence 1 by one or several amino acid residues substitution and/or lack and or add and
Have the function for the treatment of and/or preventing the protein derived from sequence 1 of the chromic fibrous pneumonopathy of diffusivity.
6. drug as described in claim 4 or 5, it is characterised in that：The fibroblast growth factor-21 analog is to more
The therapeutic effect of the chromic fibrous pneumonopathy of unrestrained property is presented as（c1）Extremely（c7）At least one of：
（c1）Inhibit the chromic fibrous pneumonopathy degree of diffusivity；
（c2）The chromic fibrous pneumonopathy of diffusivity is inhibited to occur；
（c3）Reduce the level of collagenous fibres and/or muscle fibre in lungs；
（c4）Reduce the level of hydroxyproline in lungs；
（c5）Reduce I-type collagen and/or the level of α-smooth muscle actin in lungs；
（c6）It is horizontal to reduce TGF-β in lungs；
（c7）It is horizontal to improve Nrf2 in lungs, improves antioxidant ability of organism, inhibits TGF-β 1 horizontal.
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|---|---|---|---|
| CN201810342757.7A CN108434441A (en) | 2018-04-17 | 2018-04-17 | Application of novel fibroblast growth factor -21 analogs of son in treating the chromic fibrous pneumonopathy of diffusivity |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810342757.7A CN108434441A (en) | 2018-04-17 | 2018-04-17 | Application of novel fibroblast growth factor -21 analogs of son in treating the chromic fibrous pneumonopathy of diffusivity |
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| CN108434441A true CN108434441A (en) | 2018-08-24 |
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Effective date of registration: 20181226 Address after: 222001 No. 58 Haichang South Road, Haizhou District, Lianyungang City, Jiangsu Province Applicant after: Jiangsu Kangyuan Ruiao Biomedical Technology Co., Ltd. Address before: Room 314, Science Park, Northeast Agricultural University, 59 Wood Street, Xiangfang District, Harbin, Heilongjiang Province, 150030 Applicant before: Harbin Boao Bio-Medical Technology Development Company |
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Application publication date: 20180824 |