CN104004762B - TGF-R antisense sequences and the application in preparation anti-airways inflammatory reaction medicine thereof - Google Patents
TGF-R antisense sequences and the application in preparation anti-airways inflammatory reaction medicine thereof Download PDFInfo
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- CN104004762B CN104004762B CN201410214424.8A CN201410214424A CN104004762B CN 104004762 B CN104004762 B CN 104004762B CN 201410214424 A CN201410214424 A CN 201410214424A CN 104004762 B CN104004762 B CN 104004762B
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Abstract
The invention belongs to genetically engineered and pharmacy field, disclose TGF-R antisense sequences and the application in preparation anti-airways inflammatory reaction medicine thereof.Is the nucleotide sequence of this TGF-R antisense sequences as SEQ? ID? shown in NO.1.This antisense sequences can make airway inflammation cellular infiltration obviously reduce, and can be used for preparation anti-airways inflammatory reaction medicine.
Description
Technical field
The invention belongs to genetically engineered and pharmacy field, relate to TGF-R antisense sequences and the application in preparation anti-airways inflammatory reaction medicine thereof.
Background technology
Bronchial asthma is common children chronic disease, and morbidity is remarkable ascendant trend in recent years.Nineteen ninety, the morbidity of Children in China bronchial asthma was rise to 1.5% in 0.91%, 2000.Infer according to the World Health Organization, the current whole world has 300,000,000 people to suffer from bronchial asthma.2005, the whole world was about 25.5 ten thousand because of the number of bronchial asthma death, wherein 80% occurs in developing country.The economical load that bronchial asthma is brought has exceeded the summation of pulmonary tuberculosis and acquired immune deficiency syndrome (AIDS).Therefore the pathogenesis studying bronchial asthma further has urgent current demand.
TGF-signal β path is one of signal path of key in cell, have impact on the various vital movements of cell, comprises cell proliferation, differentiation, apoptosis etc.In airway epithelial cell, the blocking-up of TGF-signal β path may inhibit the communication in immune response between epithelial cell and immunocyte, then causes the reduction of inflammatory reaction.
TGF-β is one and has multiple bioactive cytokine, plays multiple effect in Bronchial Asthma.First the adjustable lymphocytic many reactions of TGF-β are the immune responses of the conditioning signal of immunne response, adjustable respiratory tract, regulate Rheology.Secondly TGF-β stimulates some cell type grow, as stimulation fibroblastic growth, is the important cytokine of fibrosis, in close relations with bronchial asthma Airway Remodeling.
High-caliber TGF-β can cause Airway Remodeling, its mechanism of action is: 1, TGF-β promotes SMA and goblet cell hyperplasia hypertrophy, increase collagen and fibronectin synthesis, and promote that it is at extrtacellular matrix deposition, cause luminal stenosis and irreversible pulmonary function to change.2, TGF-β reduces the generation of cell surface plasma enzymes, rolls up the synthesis of plasminogen activator thing inhibitors I type antigen, reduces its cracking cellulose, participates in the effect of respiratory tract repair and reconstruction.3, TGF-β is indirectly by some composition in arrestin kinases network, reduces the degraded of extracellular matrix.
When TGF-β plays biological function, first combine with the TGF-R (TGF-beta receptor) of surface of cell membrane, after causing acceptor, reaction produces a series of biologically.Therefore by intervening TGF-R, the adjustment to TGF-β path can be realized, thus intervene the morbidity of bronchial asthma.
Antisense sequences refer to can with specific mRNA exact complementarity, specific blockage its RNA translated or DNA molecular.Utilize antisense sequences to close some genetic expression specifically, make it low expression or do not express.Utilize the short and small antisense oligonucleotide of solid phase phosphoramidite method synthetic, this is the most general application mode of antisense sequences.Antisense sequences, as one of gene therapy medicament, has plurality of advantages compared with conventional medicament.1, high degree of specificity; 2, high biological activity, abundant quantity of information; 3, high efficiency; 4, low toxicity, safety.
Although antisense technology is the technology of comparative maturity, antisense sequences deposits functioning efficiency in problem in actual applications, and the antisense sequences of not all has obvious functioning efficiency.The crucial part of antisense sequences intervention is to search out high efficiency sequence.But there is no the report of efficient TGF-R antisense sequences at present.
Summary of the invention
Object of the present invention provides a kind of TGF-R antisense sequences.This antisense sequences efficiently can suppress airway inflammation.
Another object of the present invention is the application of above-mentioned TGF-R antisense sequences in preparation anti-airways inflammatory reaction medicine.
The object of the invention is to be realized by following technical measures:
A kind of gene, this gene is the antisense sequences of TGF-R, and its nucleotide sequence is as shown in SEQIDNO.1.Namely described TGF-R antisense sequences is SEQ5 '-AGCAGCCCCCGACCCATGGC-3 '.
The application of described gene in preparation anti-airways inflammatory reaction medicine.
Beneficial effect of the present invention:
We are by the antisense sequences of information biology design TGF-RmRNA, nucleotide sequence: 5 '-AGCAGCCCCCGACCCATGGC-3 '.This antisense sequences can suppress the airway inflammation of bronchial asthma efficiently.This sequence is not the sequence naturally existed in organism, has no the function report of this sequence in existing document.
The present invention designs the non-existent TGF-R antisense sequences of nature by information biology, and functional study is carried out to it, after the application TGF-R antisense sequences of the mice with asthma model of inducing at OVA (ovalbumin), in BALF (bronchoalveolar lavage fluid), total cellular score and eosinophilic granulocyte all decline.After application TGF-R antisense sequences, the airway inflammation cellular infiltration of the mice with asthma model group of OVA induction obviously reduces.Therefore, TGF-R antisense sequences can suppress the airway inflammation of bronchial asthma, can be used for the medicine preparing anti-airways inflammatory reaction.
Accompanying drawing explanation
Fig. 1 is that TGF-R antisense sequences lowers total cellular score and eosinophilic granulocyte in BALF.
Fig. 2 is the airway inflammation cellular infiltration that TGF-R antisense sequences reduces the mice with asthma model group of OVA induction.
Embodiment
The invention will be further elaborated by the following examples, but do not limit the present invention.
Embodiment 1
One, the acquisition of TGF-R antisense sequences
Unstable position in TGF-RmRNA (GenBank:NC_000070.6) selected by application RNAstructure software.For the sphere of instability prediction target site of TGF-R, and avoid: four continuous print G; The palindrome of more than six.
TGF-R antisense sequences is SEQ5 '-AGCAGCCCCCGACCCATGGC-3 ' (SEQIDNO.1).
Two, the synthesis of TGF-R antisense sequences
TGF-R antisense sequences the SEQ5 '-AGCAGCCCCCGACCCATGGC-3 ' adopted in experiment is synthesized by Shanghai Sheng Gong biotech firm.
Three, the preparation of the mice with asthma model of OVA induction
At 0 day and 14 days two time points to BALB/c mouse abdominal injection sensitiser (the PBS damping fluid of 0.2ml is containing 20ugOVA+2mg aluminium adjuvant).Started to excite at the 27th day, mouse is placed in the 1%OVA (sigma, the U.S.) of encloses container Neulized inhalation PBS buffer, is atomized 30min at every turn.Continuous agitation 4 days, puts to death mouse, and it is to be checked to get lung tissue.
Four, aerosol transfection TGF-R antisense sequences and missense control sequence
At the 27th, 28,29,30 day of experiment, mouse is placed in encloses container Neulized inhalation PBS buffer TGF-R antisense sequences (3mgTGF-R antisense sequences is dissolved in the PBS damping fluid of 3ml, 0.1%w/v).Atomization flow 6ml/ divides.Every day Neulized inhalation once, each 30 minutes.Independent part uses antisense sequences amount to be 100mg/kg/ days.
At the 27th, 28,29,30 day of experiment, mouse is placed in encloses container Neulized inhalation PBS buffer missense control sequence (3mg missense control sequence is dissolved in the PBS damping fluid of 3ml, 0.1%w/v).Atomization flow 6ml/ divides.Every day Neulized inhalation once, each 30 minutes.Independent part uses missense control sequence amount to be 100mg/kg/ days.
In experiment, missense control sequence is in contrast SEQ5 '-CGGTACCCAGCCCCCGACGA-3 ' (SEQIDNO.2, the design consideration research convention of this sequence use the reverse sequence of TGF-R antisense sequences)
Five, group profile is divided
Control group: be normally raising group.
Asthmatic model group: be Neulized inhalation OVA group.
TGF-R antisense sequences: suck OVA and add TGF-R antisense sequences
Missense control sequence: suck OVA and add missense control sequence
Six, mouse alveolar wass and histopathology
After anesthetized mice, carry out neck dissection, expose tracheae, pour into PBS liquid 0.8ml from tracheae, lavation three times, aspirates 3 times during each lavation repeatedly.Get 20 μ L cell counting count boards to count.Treat that slide dries naturally after getting irrigating solution smear, be then placed in 10% neutral formalin solution and fix more than 10 minutes, carry out conventional H E dyeing subsequently.Just classified counting of leucocyte can be carried out after dyeing.Lung tissue fixes 24 hours in 4% paraformaldehyde, and routine paraffin wax embedding, section, HE dyes, om observation.Research finds, after application TGF-R antisense sequences in BALF total cellular score and eosinophilic granulocyte all comparatively bronchial asthma group significantly decline (Fig. 1).After application TGF-R antisense sequences, airway inflammation cellular infiltration is obviously less than bronchial asthma group (Fig. 2).Fig. 1: mice with asthma model group, in BALF, total cellular score and eosinophilic granulocyte digital display work increase.After adopting TGF-R antisense sequences, in BALF total cellular score and eosinophilic granulocyte all decline (n=6, * OVA group than control group, P<0.05; * OVA+TGF-R antisense sequences than OVA group, P<0.05)
Fig. 2: mice with asthma model group, airway inflammation cellular infiltration obviously increases.After application TGF-R antisense sequences, airway inflammation cellular infiltration is obviously less than model group.
Claims (2)
1. an antisense base sequences, it is characterized in that this antisense base sequences is the antisense sequences of TGF-R, its nucleotide sequence is as shown in SEQIDNO.1.
2. the application of antisense base sequences according to claim 1 in preparation anti-airways inflammatory reaction medicine.
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| EP3020813A1 (en) * | 2014-11-16 | 2016-05-18 | Neurovision Pharma GmbH | Antisense-oligonucleotides as inhibitors of TGF-R signaling |
| CN108524907A (en) * | 2018-07-11 | 2018-09-14 | 河南省南街村（集团）有限公司 | Soybean peptide is preparing the purposes in preventing Airway inflammatory response drug |
| CN110257500A (en) * | 2019-06-20 | 2019-09-20 | 南京市儿童医院 | LncRNA-AK000711 inhibits sequence and its application in preparation anti-airways remodeling drug |
| CN110295168A (en) * | 2019-06-20 | 2019-10-01 | 南京市儿童医院 | LncRNA-AK143701 inhibits sequence and its application in preparation anti-airways anti-inflammatory drugs |
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