CN105617377A - Product for inhibiting and/or preventing influenza virus secondary bacterial infection - Google Patents
Product for inhibiting and/or preventing influenza virus secondary bacterial infection Download PDFInfo
- Publication number
- CN105617377A CN105617377A CN201410594422.6A CN201410594422A CN105617377A CN 105617377 A CN105617377 A CN 105617377A CN 201410594422 A CN201410594422 A CN 201410594422A CN 105617377 A CN105617377 A CN 105617377A
- Authority
- CN
- China
- Prior art keywords
- tgf
- inhibitor
- beta
- receptor
- type
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a product for inhibiting and/or preventing influenza virus secondary bacterial infection. The invention provides an application of a TGF-beta signaling pathway inhibitor, namely an application of a TGF-beta signaling pathway inhibitor in preparation of a product for inhibiting and/or preventing influenza virus secondary bacterial infection. The TGF-beta signaling pathway inhibitor is at least one of the following inhibitors: an anti-TGF-beta monoclonal antibody, a soluble TGF-beta receptor, a TGF-beta antisense oligonucleotide, an inhibitor for inhibiting TGF-beta expression or inhibiting activity, a small-molecule inhibitor of a TGF-beta receptor and a small-molecule inhibitor of a TGF-beta signaling pathway downstream factor. By research results of a key role of TGF-beta in A-type influenza virus secondary bacterial infection, an impact factor for inhibiting TGF-beta signaling pathway or regulating TGF-beta is brought forward, and the effect of preventing settling and infection of bacteria at respiratory tract due to influenza virus infection is achieved. The product of the invention has the advantage of early-stage, high-efficiency and wide-spectrum blocking of bacterial adhesion.
Description
Technical field
The invention belongs to biomedicine field, relate to the product of a kind of suppression and/or flu-prevention virus secondary bacterial infection.
Background technology
The infection that influenza A (influenzaAvirus, IAV) causes is to cause one of M & M is the highest in the world disease, causes about 5,000,000 people to infect every year, and 25-50 ten thousand people is dead. Secondary bacterial pneumonia is the main cause of influenza patient death. National sanitary institutes in 2008 detect the Lungs section dying from 1918 influenzas from 58 examples again, it was demonstrated that major lesions is serious bacterial pulmonary infection. Scientists retrieved 1919-1929 all dead about 1918 influenzas postmortem research totally 8000 many cases delivered, from 15 countries, the Pleural fluid finding 80% influenza death patient is positive bacterial culture, is wherein mainly streptococcus pneumoniae (S.pneumococcus), staphylococcus aureus (S.aureus) and A type streptococcus (S.pyogenes). It follows that: world's flu outbreak of 1918 causes 50,000,000 people's main causes of death not to be influenza virus itself but influenza secondary bacterial pneumonia. Similar to it, streptococcus pneumoniae and streptococcic secondary infection are also major complications popular for H1N1 in 2009 and the cause of death.
Currently for the mechanism of influenza virus secondary bacterial infection indefinite, the prevention of lacking of property and Therapeutic Method. The prevention to influenza serious symptom and death and control are had a strong impact on. Therefore, in the urgent need to exploitation safely, effectively, the prevention of wide spectrum and application easy to spread and/or treatment new method.
TGF-�� belongs to TGF-�� superfamily member. TGF-�� and other member of TGF-�� superfamily have 30��40% homologys. In mammal, find have TGF-�� 1, TGF-�� 2, TGF-�� 3,2 four hypotypes of TGF-�� 1 �� at present. Between each hypotype, homology is significantly high: TGF-�� 1 and TGF-�� 2 have 71% amino acid identity, TGF-�� 1 and TGF-�� 3 to have 77% homology, and TGF-�� 2 and TGF-�� 3 have 80% homology. The each hypotype functional similarity of TGF-��, in conjunction with the receptor (TGF-�� R) that cell surface is identical. Have now been found that TGF-�� R also exists three kinds of forms of I, II, III type, be distributed in almost all of histiocyte surface. Wherein, I type and II receptor participate in the transduction of signal, and III receptor does not directly participate in signal transduction. TGF-�� is a kind of polytropism, polyphenic cytokine, by cell surface receptor signal transduction pathway in the way of autocrine or paracrine, regulating the increment of cell, differentiation, apoptosis, the synthesis of extracellular matrix albumen, the reparation of wound, immunologic function etc. have important adjustment effect. TGF-�� exists with the dormancy complex form of inactive: by the homodimer TGF-�� (biologically-active moiety) of 25KD, and activity associated protein (LAP) of diving and potential TGF-�� associated proteins (LTBP) form. Under the splitting action of protease or other molecules, active TGF-�� will be had to discharge from complex, be combined (I receptor can activate Smads, and II receptor is combined with TGF-��) with film surface receptor heterodimer; And then regulation type SMAD albumen (R-Smad) in activation downstream, including Smad2 and Smad3, by signal conduction to endochylema; R-Smad is together with adjustment type SMAD albumen (CommonmediatorSmad, Co-Smad; Have now been found that only have Smad4) combine after, complex is transferred to nucleus, with target gene (including integrin (integrin) and FN (Fibronectin, Fn)) combine, promotion target gene expression. Additionally, TGF-�� is also by the signal transduction pathway activation target gene of Smad non-dependent, such as JNK signal path.
Summary of the invention
It is an object of the present invention to provide the new application of a kind of TGF-�� signal pathway inhibitor.
New application provided by the present invention is the application suppressing transforming growth factor �� (transforminggrowthfactor-beta, TGF-��) signal pathway inhibitor in the product of preparation suppression and/or flu-prevention virus secondary bacterial infection.
In the present invention, this new application is embodied as: TGF-�� signal pathway inhibitor is the application in the product of at least one function in preparation has (1)-(4) as follows:
(1) up-regulated of stromatin that the pneumonocyte surface that causes of influenza infection is relevant to bacterial adhesion is suppressed;
(2) the respiratory system pathogenic bacterium that suppression influenza infection the causes adhesion to pneumonocyte;
(3) respiratory system pathogenic bacterium the settling down and/or infecting in pulmonary that influenza infection causes is suppressed;
(4) bacterial pneumonia of prevention and/or treatment influenza virus secondary.
In the present invention, described TGF-�� signal pathway inhibitor can be following middle at least one: the monoclonal antibody of anti-TGF-beta, soluble TGF-p receptor, TGF-�� antisense oligonucleotide, suppression TGF-�� are expressed or suppress the micromolecular inhibitor of the inhibitor of its activity, the micromolecular inhibitor of TGF-�� receptor, TGF-�� signal path downstream elements.
Described TGF-�� can be: TGF-�� 1, TGF-�� 2, each hypotype such as TGF-�� 3 or TGF-�� 1 �� 2.
The monoclonal antibody of described anti-TGF-beta can be: the monoclonal antibodies such as the Mus of people source TGF-��, rabbit or sheep.
The TGF-�� receptor of described solubility can be: TGF-�� I type and/or TGF-�� II type and/or TGF-�� III receptor soluble protein; Or through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and have identical activity by its derivative protein.
Described TGF-�� antisense oligonucleotide can be: the antisense DNA of TGF-��, TGF-�� RNA disturb (RNAi) fragment or carry the antisense DNA of described TGF-�� or the expression vector of the RNA interference fragment of described TGF-��.
The expression of described suppression TGF-�� or the inhibitor suppressing its activity can be Pirfenidone.
Described TGF-�� receptor can be: TGF-�� I type and/or TGF-�� II type and/or TGF-�� III receptor; Micromolecular inhibitor concretely SB431542, LDN-193189, LY2157299, LY2109761, SB525334, DMH1, LDN-212854, ML347, LDN-193189HCl, K02288, SB505124, GW788388, LY364947 or RepSox of described TGF-�� receptor.
Described TGF-�� signal path downstream elements is Smad3, JNK or AP-1; Micromolecular inhibitor concretely SIS3, SP600125, JNK-IN-8, JNKInhibitorIX or SR11302 of described TGF-�� signal path downstream elements.
Described respiratory system pathogenic bacterium can be the A type streptococcus of various serotype, streptococcus pneumoniae, staphylococcus aureus, hemophilus influenza or moraxelle catarrhalis.
The described pneumonocyte surface stromatin relevant to bacterial adhesion specifically can such as fibronectin (Fibronectin, Fn) or the various integrin (integrin) relevant to bacterial adhesion, such as Integrin ��_5.
In the present invention, described pneumonocyte is specially alveolar epithelial cells (such as A549 cell) or takes from the lung cells of mice.
Described product can be medicine.
It is a further object to provide a kind of product.
Product provided by the present invention, its active component is TGF-�� signal pathway inhibitor; The purposes of described product be following at least one:
A () suppresses and/or flu-prevention virus secondary bacterial infection;
B () suppresses the up-regulated of the stromatin that the pneumonocyte surface that causes of influenza infection is relevant to bacterial adhesion;
The adhesion to pneumonocyte of the c respiratory system pathogenic bacterium that () suppression influenza infection causes;
D () suppresses respiratory system pathogenic bacterium the settling down and/or infecting in pulmonary that influenza infection causes;
The bacterial pneumonia of (e) prevention and/or treatment influenza virus secondary.
In the present invention, described TGF-�� signal pathway inhibitor can be following middle at least one: the monoclonal antibody of anti-TGF-beta, soluble TGF-p receptor, TGF-�� antisense oligonucleotide, suppression TGF-�� are expressed or suppress the micromolecular inhibitor of the inhibitor of its activity, the micromolecular inhibitor of TGF-�� receptor, TGF-�� signal path downstream elements.
Described TGF-�� can be: TGF-�� 1, TGF-�� 2, each hypotype such as TGF-�� 3 or TGF-�� 1 �� 2.
The monoclonal antibody of described anti-TGF-beta can be: the monoclonal antibodies such as the Mus of people source TGF-��, rabbit or sheep.
The TGF-�� receptor of described solubility can be: TGF-�� I type and/or TGF-�� II type and/or TGF-�� III receptor soluble protein; Or through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and have identical activity by its derivative protein.
Described TGF-�� antisense oligonucleotide can be: the antisense DNA of TGF-��, TGF-�� RNA disturb (RNAi) fragment or carry the antisense DNA of described TGF-�� or the expression vector of the RNA interference fragment of described TGF-��.
The expression of described suppression TGF-�� or the inhibitor suppressing its activity can be Pirfenidone.
Described TGF-�� receptor can be: TGF-�� I type and/or TGF-�� II type and/or TGF-�� III receptor; Micromolecular inhibitor concretely SB431542, LDN-193189, LY2157299, LY2109761, SB525334, DMH1, LDN-212854, ML347, LDN-193189HCl, K02288, SB505124, GW788388, LY364947 or RepSox of described TGF-�� receptor.
Described TGF-�� signal path downstream elements is Smad3, JNK or AP-1; Micromolecular inhibitor concretely SIS3, SP600125, JNK-IN-8, JNKInhibitorIX or SR11302 of described TGF-�� signal path downstream elements.
Described respiratory system pathogenic bacterium can be the A type streptococcus of various serotype, streptococcus pneumoniae, staphylococcus aureus, hemophilus influenza or moraxelle catarrhalis.
Stromatin concretely fibronectin (Fibronectin, Fn) that described pneumonocyte surface is relevant to bacterial adhesion or the integrin (integrin) of various different �� or �� chain of being correlated with from bacterial adhesion, such as Integrin ��_5.
In the present invention, described pneumonocyte is specially alveolar epithelial cells (such as A549 cell) or takes from the lung cells of mice.
Described product can be medicine.
The occupation mode of described medicine includes nasal cavity suction, oral, subcutaneous injection or intradermal injection.
In an embodiment of the present invention, above all of described influenza virus is all specially influenza A virus; Described influenza A virus is specially H1 type influenza virus, such as H1N1 type influenza virus, concrete such as strain A/PuertoRico/08/1934 (PR8).
In an embodiment of the present invention, above all of described A type streptococcus (S.pyogenes) is all specially A type streptococcus (S.pyogenes) M1 type; Above all of described streptococcus pneumoniae (S.pneumococcus) is all specially streptococcus pneumoniae (S.pneumococcus) T19F; Above all of described staphylococcus aureus (S.aureus) is all specially staphylococcus aureus (S.aureus) RN6390 (FnBPA+B+).
Through long-term further investigation, the present inventor finds that TGF-�� infects the key factor in secondary bacterial infection in influenza A. Experiment in vitro proves that influenza A can promote the activity of the TGF-�� of alveolar epithelial cells system A549 emiocytosis, and then raise stromatin relevant to bacterial adhesion downstream, such as fibronectin (Fibronectin, and the expression of cell surface molecule integrin (integrin) Fn), the adhesion causing antibacterial (A type streptococcus, streptococcus pneumoniae and staphylococcus aureus) increases. And by the monoclonal antibody of TGF-��, the inhibitor SIS3 of the inhibitor SB431542 or its signal path downstream gene Smad3 of TGF-�� receptor can significantly inhibit the adhesion of above-mentioned antibacterial. Zoopery proves, after the mouse infection H1N1 type influenza virus A/PuertoRico/08/1934 (PR8) of TGF-�� signal path downstream gene Smad3 disappearance, the amount of settling down of the fibronectin (Fn) on its lung cells surface and the expression of Integrin ��_5 and A type streptococcus (M1) is below wild-type mice. More than prove to suppress TGF-�� activity and/or its signal path, and/or regulating and controlling effect can effectively reduce or prevent influenza virus secondary respiratory tract pathogenic bacterium settling down and infection rate at people's respiratory system in the factor of influence of TGF-��. The preparation of new method of the present invention can be used for preventing and treating the respiratory system infection caused by various bacteria relying on stromatin and integrin of influenza virus secondary.
The present invention utilizes the result of study of TGF-�� pivotal role in influenza A infection secondary bacterial infection. Propose to suppress TGF-�� signal path or regulating and controlling effect in the factor of influence of TGF-��, reach the effect of respiratory tract bacterial colonisation and the infection stoping influenza infection secondary, have early stage, efficiently, wide spectrum block the superiority of bacterial adhesion.
Accompanying drawing explanation
Fig. 1 is the embodiment 1 result activity of TGF-�� of human squamous lung cancer A549 emiocytosis (IAV can promote). * represents significant difference in P < 0.01 level.
Fig. 2 is embodiment 2 result (expression of IAV promotion A549 cell surface integrin �� 5 and fibronectin, can be suppressed by TGF-�� signal pathway inhibitor SB431542 or SIS3). A is the IAV expression promoting A549 cell surface integrin �� 5, can be suppressed by TGF-�� signal pathway inhibitor SB431542; B is that IAV promotes that A549 cell surface fibrillar connects the expression of albumen, can be suppressed by TGF-�� signal pathway inhibitor SB431542; C is the IAV expression promoting A549 cell surface integrin �� 5, can be suppressed by TGF-�� signal pathway inhibitor SIS3; D is that IAV promotes that A549 cell surface fibrillar connects the expression of albumen, can be suppressed by TGF-�� signal pathway inhibitor SIS3. In figure, namely SB represents SB431542. * represents significant difference in P < 0.01 level; * * represents significant difference in P < 0.001 level.
Fig. 3 is embodiment 3 result (IAV promotes A type streptococcus, and streptococcus pneumoniae and staphylococcus aureus adhere to A549 cell, and this facilitation can be suppressed by TGF-�� signal pathway inhibitor SB431542 or SIS3). A is A type streptococcus (GAS) result; B is streptococcus pneumoniae (Sp) result; C is staphylococcus aureus (Sa) result. In figure, vertical coordinate unit is %. * significant difference in P < 0.05 level is represented; * represents significant difference in P < 0.01 level; * * represents significant difference in P < 0.001 level.
Fig. 4 is embodiment 4 result (IAV can promote the expression of mouse lung cell surface integrin �� 5 and fibronectin and A type streptococcus m 1 type antibacterial settling down in pulmonary). A is that IAV can promote A type streptococcus m 1 type antibacterial settling down at mouse lung; B is the IAV expression that can promote mouse lung cell surface integrin �� 5 and fibronectin. * represents significant difference in P < 0.01 level; * * represents significant difference in P < 0.001 level.
Fig. 5 is for identifying smad3 knock out mice genome agarose gel electrophoresis and judging genotype schematic diagram. A is smad3 gene schematic diagram; B is for building smad3 knock out mice genotype schematic diagram; C is smad3 knock out mice genome agarose gel electrophoresis qualification result.
Fig. 6 is that the result of embodiment 5 is (after IAV infection TGF-�� signal path downstream gene smad3 knock out mice, the expression of mouse lung cell surface integrin �� 5 and fibronectin, and A type streptococcus m 1 type antibacterial is below the IAV wild-type mice infected in the amount of settling down of pulmonary). A is that IAV infects after TGF-�� signal path downstream gene smad3 knock out mice, A type streptococcus m 1 type antibacterial in the amount of settling down of pulmonary lower than the IAV wild-type mice infected; B is that after IAV infects TGF-�� signal path downstream gene smad3 knock out mice, the expression of mouse lung cell surface integrin �� 5 and fibronectin is lower than the IAV wild-type mice infected. * significant difference in P < 0.05 level is represented.
Detailed description of the invention
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all commercially obtain.
Quantitative test in following example, is respectively provided with three times and repeats experiment, results averaged.
smad3+/-Mice: come from animal institute of Chinese Academy of Sciences teacher Zhao Yong. List of references: WuT, SunC, ChenZ, ZhenY, PengJ, QiZ, YangX, ZhaoY.Smad3-deficientCD11b (+) Gr1 (+) myeloid-derivedsuppressorcellspreventallograftrejectionv iathenitricoxidepathway.JImmunol.2012Nov15; 189 (10): the 4989-5000. public can obtain from Institute of Microorganism, Academia Sinica.
H1N1 type influenza virus A/PuertoRico/08/1934 (PR8): derive from American Type Culture institute (AmericanTypeCultureCollection, ATCC). ATCC numbers: VR-1469.
A549 cell (people source alveolar epithelial cells): derive from American Type Culture institute (AmericanTypeCultureCollection, ATCC). ATCC numbers: CRM-CCL-185.
MLEC cell: derive from American Type Culture institute (AmericanTypeCultureCollection, ATCC). List of references: LacmannA, HessD, GohlaG, RoussaE, KrieglsteinK (2007) the Activity-dependentreleaseoftransforminggrowthfactor-beta inaneuronalnetworkinvitro.Neuroscience150:647-657. public can obtain from Institute of Microorganism, Academia Sinica.
A type streptococcus (S.pyogenes) M1 type: derive from the state university in Minnesota department of microbiology, list of references: " WangB; DileepanT; BriscoeS; HylandKA; KangJ, KhorutsA, ClearyPP.InductionofTGF-beta1andTGF-beta1-dependentpredo minantTh17differentiationbygroupAstreptococcalinfection. ProcNatlAcadSciUSA.2010; 107 (13): 5937-42. ". The public can obtain from Institute of Microorganism, Academia Sinica.
Streptococcus pneumoniae (S.pneumococcus) T19F: derive from Tsing-Hua University's medical college infectious disease research center, Beijing, list of references: " GuilingLi; FenZ.Hu; XianweiYang; YujunCui; JunYang; FenQu, GeorgeF.Gao, andJing-RenZhang. (2012) CompleteGenomeSequenceofStreptococcuspneumoniaeStrainST5 56, aMultidrug-ResistantIsolatefromanOtitisMediaPatient.J.Ba cteriol.vol.194 (12): 3294-3295. ". The public can obtain from Institute of Microorganism, Academia Sinica.
Staphylococcus aureus (S.aureus) RN6390 (FnBPA+B+): derive from the state university in Minnesota department of microbiology, list of references: " WangB; YureckoRS; DedharS �� &ClearyPP (2006) Integrin-linkedkinaseisanessentiallinkbetweenintegrinsan duptakeofbacterialpathogensbyepithelialcells.Cellularmic robiology8 (2): 257-266. ". The public can obtain from Institute of Microorganism, Academia Sinica.
SB431542 (cat.13031), for the micromolecular inhibitor of TGF-�� receptor, purchase is from CaymanChemical (AnnArbor, MI). SIS3 (cat.S0447), for the micromolecular inhibitor of TGF-�� signal path downstream elements, purchase is from Sigma (St.Louis, MO). The monoclonal antibody (cat.ab55988) of anti-Integrin ��_5 (�� 5integrin) and the monoclonal antibody (cat.sc-9068) of anti-fibronectin (Fn) are bought from R&D (Minneapolis respectively, and SantaCruzBiotechnology (SantaCruz, CA) MN). Common rabbit igg (cat.AB-105-C) is bought from R&D (Minneapolis, MN). The goat anti-rabbit igg (cat.F0382) of FITC labelling is bought from Sigma. TGF-�� STD (cat.MB100B): buy from R&D (Minneapolis, MN), use with reference to R&Dkit explanation. Luciferase substrate: LuciferaseAssaySystem (cat.E1500) buys from promega. Culture medium constituent (Neopeptone (cat.211681); ToddHewittBroth (cat.249240)) buy from Bi Di medical apparatus and instruments (Shanghai) Co., Ltd.. Culture medium constituent (BloodAgarBase (cat.CM905); Aseptic de-fiber Sanguis caprae seu ovis (cat.P62)) buy from Beijing Luqiao Technology Co., Ltd.. From Beijing Suo Laibao Science and Technology Ltd., (content of penicillin is 10kU/ml, and the content of streptomycin is 10mg/ml in cell cultivation mycillin mixed liquor (cat.P1400) purchase. The working concentration of the penicillin in cell culture fluid is 100U/ml, and the working concentration of streptomycin is 0.1mg/ml). G418 (cat.11811023) buys from the Shanghai past that (1gG418 is dissolved in 2ml1MHEPES (pH7.3), final concentration of 500mg/ml ,-20 DEG C of preservations in bio tech ltd. Working concentration is 500ug/ml)
Embodiment 1, IAV can promote that human squamous lung cancer A549 secretes the activity of TGF-��
One, the preparation of sample is detected
1, cultivate A549 cell, inoculate 48 orifice plates, 0.5-1 �� 105The every hole of/0.5ml. It is placed in 37 DEG C, 5%CO2Incubator overnight incubation;
2, Growth of Cells is to 90-100% degrees of fusion, and PBS washes 1-2 time;
3, the DMEM culture medium of 270 �� l/ hole serum-frees is added;
4, virus liquid (0.5-1 �� 10 of 30 �� l/ hole H1N1 type influenza virus PR8 are added4TCID50) or PBS control (30 �� l/ hole PBS), after mixing. It is placed in 37 DEG C, 5%CO2Incubator is cultivated 45min;
5, wash cell 1-2 time with PBS, remove virus liquid;
6, adding the DMEM complete culture solution of 300 �� l, cultivate different time points and collect supernatant, time point is 2h respectively, 4h, 6h;
7, the cell conditioned medium after collecting is through 1000g, and 4 DEG C, after centrifugal 10min removes cell debris ,-20 DEG C or-80 DEG C of placements save backup, or continue luciferase detection test (luciferaseassay).
Two, luciferase detection test (luciferaseassay)
1, the MLEC cell that will cultivate, by 4-5 �� 10496 orifice plates are inoculated in/100 every holes of �� l; It is placed in 37 DEG C of 5%CO2Incubator hatches 3h, prepares detection sample simultaneously;
2, prepared by TGF-�� STD: with DMEM (1%FBS+ streptomycin and the dual anti-+ G418 of penicillin) culture fluid by 1:2 gradient dilution TGF-�� (the initial concentration 2000pg/ml of TGF-��, finally it is diluted to 7.8pg/ml), 80 DEG C, (TGF-�� dormancy complex is under strong acid, highly basic, high temperature, fibrinolysin, protease effect to hatch 5min, slough LAP, play biological effect);
3, after 3h, taking out 96 orifice plate MLEC cells, careful suction abandons cell conditioned medium;
The detection sample (100 �� l) of the TGF-�� STD (100 �� l) 4, step 2 prepared or step one preparation is separately added in 96 orifice plate cells, notes not stirring cell. It is placed in 37 DEG C of 5%CO2Incubator overnight incubation;
5, taking out 96 orifice plate cells of overnight incubation, careful suction abandons supernatant; PBS washes cell 1-2 time, notes not stirring cell;
6, every hole adds the lysis buffer (formula: 0.25MTris-HCL, pH7.4 of 50 �� l; 1mMDTT; Volume fraction 0.1%TritonX-100), room temperature concussion cracks completely to cell.
7, with Luminometer, (instrument used during detection luciferase, its model is GloMaxTM96MicroplateLuminometer) measure uciferase activity: take the cell pyrolysis liquid of 20 �� l steps 6 acquisitions and the luciferase substrate mixing of 50 �� l, measure fluorescence intensity (recording the flat light emission (RLU) that luminous value is Fireflyluciferase), TGF-�� content in sample is calculated according to standard substance, TGF-�� STD solution to add 100 �� l series concentration in step 4 carries out step 7 in the cell pyrolysis liquid 20 �� l that step 6 obtains and operates, obtain standard curve, utilize standard curve to obtain the content of TGF-�� in detection sample.
Result is shown in Fig. 1, it is seen that H1N1 type influenza virus PR8 can remarkably promote the content of the A549 emiocytosis activity TGF-�� of cultivation, and with PBS control ratio, activity improves about 4 times.
Embodiment 2, IAV promote A549 cell surface integrin �� 5 and fibronectin expression, can be suppressed by TGF-�� signal pathway inhibitor SB431542 or SIS3
One, the preparation of sample is detected
1, A549 cell is cultivated, by 1-2 �� 105/ 1ml inoculates 24 orifice plates in every hole, is placed in 37 DEG C of 5%CO2Incubator overnight incubation;
2, during cell 80-90% degrees of fusion, aseptic PBS washes 2 times, adds 0.5-1 �� 104The every hole of H1N1 type influenza virus PR8/500 �� l of TCID50, culture medium is serum-free DMEM (+4% (4g/100mL) BSA); TGF-�� signal path suppression group is simultaneously introduced 50 ��Ms of SB431542 or SIS3 (the storage concentration of SB431542 and SIS3 is 50mM, is dissolved in DMSO); Matched group only adds solvent (0.5 �� lDMSO). It is placed in 37 DEG C of 5%CO2Incubator hatches 18h. Experiment arranges the matched group through processing as follows simultaneously: infects without virus PR8 and only adds solvent (0.5 �� lDMSO), being designated as DMSO/PBS group.
Two, FCM analysis (FACS)
1,0.25% (0.25g/100ml) pancreatin 100 �� l/ hole, TGF-�� signal path suppression group that digestion step is got ready surely and the A549 cell of matched group so that it is in unicellular, DMEM complete medium terminates digestion, 800g, 5min centrifugal collecting cell, 106Cell/pipe;
2, FACSBuffer (formula: PBS+ volume fraction 0.2%FBS+0.01% (0.01g/100ml) Hydrazoic acid,sodium salt) washes cell 1-2 time;
3, it is separately added into an anti antibody (monoclonal antibody of anti-Integrin ��_5 or the monoclonal antibody of anti-fibronectin to be detected, it is rabbit source), compare antibody with common rabbit igg, the 0.5 �� g/10 each sample of �� l, fully mix with cell, hatch 30min for 4 DEG C;
4, FACSBuffer (formula is ibid) washes cell 1-2 time;
5, adding fluorescently-labeled two anti-(goat anti-rabbit iggs of FITC labelling), room temperature lucifuge hatches 30min;
6, FACSBuffer (formula is ibid) washes cell 1-2 time;
7, cell being resuspended in 500 �� lFACSbuffer (formula is ibid), flow cytomery, or is suspended from 500 �� l4% paraformaldehydes, 4 DEG C of lucifuges are placed maximum 7 days, to be measured.
Found that, for the normal A549 cell line (DMSO/PBS group) cultivated, the A549 cell line that PR8 the processes Fn of Flow cytometry cell surface or Integrin ��_5, its surface fluorescence is higher, show as peak to offset to the direction of high fluorescent, delimit the region (horizontal line delimited see the FCM analysis collection of illustrative plates upper right corner in Fig. 2 indicates region) of high expressed accordingly, calculate the relative amount of the cell of high expressed Fn or Integrin ��_5 in each group. The content of the cell of high expressed Fn or Integrin ��_5 is the cell ratio with tested total cellular score of high expressed Fn or Integrin ��_5. The content of the high expressed Fn of DMSO/PBS group or the cell of Integrin ��_5 is designated as 100%, the relative amount of the content of the cell of all the other group high expressed Fn or the Integrin ��_5 cell that ratio is each group of high expressed Fn or Integrin ��_5 by comparison.
Concrete outcome significantly promotes the A549 cell surface integrin �� 5 of cultivation and the expression of fibronectin referring to Fig. 2, H1N1 type influenza virus PR8, and this facilitation can be suppressed by TGF-�� signal pathway inhibitor SB431542 or SIS3.
Embodiment 3, IAV can promote that A type streptococcus, streptococcus pneumoniae and staphylococcus aureus adhere to A549 cell, and this facilitation can be suppressed by TGF-�� signal pathway inhibitor SB431542 or SIS3
One, PR8 virus infected cell
With embodiment 2 step one.
Two, bacterial adhesion experiment
For examination antibacterial: A type streptococcus (S.pyogenes) M1 type, streptococcus pneumoniae (S.pneumococcus) T19F, staphylococcus aureus (S.aureus) RN6390 (FnBPA+B+).
1, the cultivation of antibacterial prepares: inoculated bacteria, to corresponding culture medium (cultivating bacterium colony 10mlPBS to suspend, stand-by), is placed in 37 DEG C of 5%CO2Incubator cultivates 17-18h. 3000g10min is centrifuged, and removes culture medium (or PBS), adds 10mlPBS and wash once. Adding appropriate serum-free DMEM (+4% (4g/100mL) BSA), adjustment amount of bacteria is 0.5-1 �� 107CFU/ml��
Wherein, the culture medium of A type streptococcus (S.pyogenes) M1 type, streptococcus pneumoniae (S.pneumococcus) T19F and staphylococcus aureus (S.aureus) RN6390 (FnBPA+B+) is 10mlTHB-Neo.
THB-Neo:ToddHewittBroth30g; Neopeptone20g is dissolved in 1L distilled water, 121 DEG C of sterilization treatment 15min.
2, abandon step one H1N1 type influenza virus PR8 infect and cultivate the A549 cell culture medium of 18h, add bacteria suspension 200 �� l (1-2 �� 10 of above-mentioned preparation6CFU/ hole, MOI is 10:1), it is placed in 37 DEG C of 5%CO2Incubator cultivates 2h.
3, PBS washes cell and removes non-Adherent bacteria 4 times, add 0.25% (0.25g/100ml) pancreatin 100 �� l/ hole digestion A549 cell, be then directly added into 400 �� l/ holes aseptic 0.025% (volume fraction) TritonX-100 cell lysis.
4, by above-mentioned sample collection to 1.5mlEp pipe, whirlpool shakes 30s, takes appropriate dilution (ensureing that the clone's number on solid culture flat board is between 30-300) and coats on corresponding 90mm solid culture flat board.
Wherein, A type streptococcus (S.pyogenes) M1 type, streptococcus pneumoniae (S.pneumococcus) T19F and staphylococcus aureus (S.aureus) RN6390 (FnBPA+B+) are blood solids culture plate.
Blood solids culture plate: BloodAgarBase33g in 1L distilled water, 121 DEG C of autoclaving 15min, be cooled to 50 DEG C, add aseptic de-fiber Sanguis caprae seu ovis 60ml, pour plate after mixing, standby after cooling.
5,37 DEG C of 5%CO it are inverted in2Incubator is cultivated, and next day calculates CFU.
6, relative adhesion rate is calculated according to CFU. Adhesion rate is CFU and the ratio adding bacterium amount. The adhesion rate of DMSO/PBS group is designated as 100%, and relative adhesion rate is each group of adhesion rate ratio compared with DMSO/PBS group adhesion rate.
Result is shown in that Fig. 3, H1N1 type influenza virus PR8 can promote A type streptococcus, streptococcus pneumoniae, and staphylococcus aureus adheres to A549 cell, and this facilitation can be suppressed by TGF-�� signal pathway inhibitor SB431542 or SIS3.
Embodiment 4, IAV can promote the expression of mouse lung cell surface integrin �� 5 and fibronectin and A type streptococcus m 1 type settling down in pulmonary
One, the foundation of PR8 viral infection secondary A type streptococcus m 1 type antibacterial infecting mouse model
1, the Balb/C mice taking SPF level is randomly divided into 3 groups (matched group, PR8 low dose group and PR8 high dose group), 5-6/group; 2.5% (volume fraction) isoflurane inhalation anesthesia mice. PR8 low dose group adopts 500TCID50H1N1 type influenza virus PR8/40 �� lPBS collunarium infecting mouse; PR8 high dose group adopts 1 �� 104TCID50H1N1 type influenza virus PR8/40 �� lPBS collunarium infecting mouse; Matched group collunarium 40 �� lPBS;
2, after 3 days, 2.5% (volume fraction) isoflurane inhalation anesthesia mice, by 1-2 �� 107CFU/50ul collunarium infects A type streptococcus m 1 type antibacterial (A type streptococcus m 1 type antibacterial culturing and preparation, with reference to embodiment 3 step 21, are slightly made an amendment, and namely antibacterial is finally suspended in PBS);
3, CO after 24h2Method of suffocating puts to death mice, takes lung tissue, adds 1mlPBS and grinds homogenate, prepares sample.
Two, fluidic cell (FACS) detects the expression of pneumonocyte surface integrin �� 5 and fibronectin
1, sample 800 �� l, the 800g5min centrifugal segregation supernatant that above-mentioned steps one prepares is taken;
2,2ml erythrocyte cracked liquid (formula: 0.15MNH is added4Cl, 10nMKHCO3, 1nMNa4EDTA, pH7.2-7.4) room temperature effect 5min splitting erythrocyte;
3, add 2mlPBS to terminate, 800g5min centrifugal segregation supernatant;
4, fluidic cell (FACS) detection operation carries out with reference to embodiment 2 step 2.
Three, A type streptococcus m 1 type antibacterial settling down in pulmonary
1, take the sample 100 �� l that above-mentioned steps one prepares, doubling dilution, take appropriate dilution (ensureing that the clone's number on solid culture flat board is between 30-300) and coat on 90mm blood solids culture plate;
Blood solids culture plate: BloodAgarBase33g in 1L distilled water, 121 DEG C of autoclaving 15min, be cooled to 50 DEG C, add aseptic de-fiber Sanguis caprae seu ovis 60ml, pour plate after mixing, standby after cooling.
2,37 DEG C of 5%CO it are inverted in2Incubator is cultivated, and next day calculates CFU.
Result is shown in that Fig. 4, H1N1 type influenza virus PR8 can promote the expression of mouse lung cell surface integrin �� 5 and fibronectin and A type streptococcus m 1 type antibacterial settling down in pulmonary.
After embodiment 5, IAV infect TGF-�� signal path downstream gene smad3 knock out mice, mouse lung cell surface integrin �� 5 and fibronectin expression amount, and A type streptococcus m 1 type antibacterial is below the IAV wild-type mice infected in the amount of settling down of pulmonary
One, smad3-/-The acquisition of mice
1, by of the right age smad3+/-Mice mates by a hero two is female, and F2 is born the 3rd day for mice and cuts rat-tail a little, is placed in PCR pipe. Add 50 �� l lysates (formula: 10mMNaOH+0.1mMEDTA), hatch 30min for 95 DEG C. Tissue Lysis is smashed to pieces with rifle head after processing, and is placed in-80 DEG C in 7 days and can carry out PCR identified gene type.
2, F2 is for the qualification of murine genes type: with the rat-tail genomic DNA of above-mentioned steps 1 acquisition for template, carries out pcr amplification with F1 and R1 (or R2) primer pair formed, obtains pcr amplification product.
F1:5'-CCACCTCATTGCCATATGCCCTG-3';
R1:5'-CCCGAACAGTTGGATTCACACA-3';
R2:5'-CCAGACTGCCTTGGGAAAAGC-3'.
Fig. 5 is shown in by agarose gel electrophoresis and the judgement genotype schematic diagram of pcr amplification product. It can be seen that primer pair F1/R1 or F1/R2 can obtain PCR band for heterozygous mice, both smad3+/-Mice; Primer pair F1/R1 is passable, but F1/R2 can not obtain PCR band for wild-type mice, both smad3+/+Mice; Primer pair F1/R1 can not, but F1/R2 can obtain PCR band for knocking out type mice, both smad3-/-Mice.
Two, the foundation of PR8 viral infection secondary A type streptococcus m 1 type antibacterial infecting mouse model
1, the smad3 that SPF level is bred and identified is taken-/-Mice (PR8 group), 5-6/group; Breed with SPF level and identify and smad3-/-The smad3 that the features such as mice homology and age-sex are identical+/+Mice (matched group), 5-6/group.
2, the foundation of PR8 viral infection secondary A type streptococcus m 1 type antibacterial infecting mouse model
Carrying out with reference to embodiment 4 step one, PR8 group adopts high dose group, and both 1 �� 104TCID50H1N1 type influenza virus PR8/40 �� lPBS collunarium infecting mouse.
Three, FACS detects pneumonocyte surface integrin �� 5 and fibronectin expression
Carry out with reference to embodiment 4 step 2.
Four, A type streptococcus m 1 type antibacterial settling down in pulmonary
Carry out with reference to embodiment 4 step 3.
Result is shown in Fig. 6, after H1N1 type influenza virus PR8 infects TGF-�� signal path downstream gene smad3 knock out mice, mouse lung cell surface integrin �� 5 and fibronectin expression amount, and A type streptococcus m 1 type antibacterial is below the H1N1 type influenza virus PR8 smad3 infected in the amount of settling down of pulmonary+/+Wild-type mice.
Claims (10)
- The application in the product of preparation suppression and/or flu-prevention virus secondary bacterial infection of the 1.TGF-signal beta pathway inhibitor.
- 2.TGF-signal beta pathway inhibitor is the application in the product of at least one function in preparation has (1)-(4) as follows:(1) up-regulated of stromatin that the pneumonocyte surface that causes of influenza infection is relevant to bacterial adhesion is suppressed;(2) the respiratory system pathogenic bacterium that suppression influenza infection the causes adhesion to pneumonocyte;(3) respiratory system pathogenic bacterium the settling down and/or infecting in pulmonary that influenza infection causes is suppressed;(4) bacterial pneumonia of prevention and/or treatment influenza virus secondary.
- 3. application according to claim 1 and 2, it is characterised in that: described TGF-�� signal pathway inhibitor is following middle at least one: the monoclonal antibody of anti-TGF-beta, soluble TGF-p receptor, TGF-�� antisense oligonucleotide, suppression TGF-�� are expressed or suppress the micromolecular inhibitor of the inhibitor of its activity, the micromolecular inhibitor of TGF-�� receptor, TGF-�� signal path downstream elements.
- 4. application according to claim 3, it is characterised in that: the expression of described suppression TGF-�� or the inhibitor suppressing its activity are Pirfenidone;Described TGF-�� receptor is TGF-�� I type and/or TGF-�� II type and/or TGF-�� III receptor; The micromolecular inhibitor of described TGF-�� receptor is specially SB431542, LDN-193189, LY2157299, LY2109761, SB525334, DMH1, LDN-212854, ML347, LDN-193189HCl, K02288, SB505124, GW788388, LY364947 or RepSox;Described TGF-�� signal path downstream elements is Smad3, JNK or AP-1; The micromolecular inhibitor of described TGF-�� signal path downstream elements is specially SIS3, SP600125, JNK-IN-8, JNKInhibitorIX or SR11302.
- 5. according to described application arbitrary in claim 2-4, it is characterised in that: described respiratory system pathogenic bacterium be following in any one or several: A type streptococcus, streptococcus pneumoniae, staphylococcus aureus, hemophilus influenza and moraxelle catarrhalis; OrThe stromatin that described pneumonocyte surface is relevant to bacterial adhesion is fibronectin or integrin.
- 6. a product, its active component is TGF-�� signal pathway inhibitor; The purposes of described product be following at least one:A () suppresses and/or flu-prevention virus secondary bacterial infection;B () suppresses the up-regulated of the stromatin that the pneumonocyte surface that causes of influenza infection is relevant to bacterial adhesion;The adhesion to pneumonocyte of the c respiratory system pathogenic bacterium that () suppression influenza infection causes;D () suppresses respiratory system pathogenic bacterium the settling down and/or infecting in pulmonary that influenza infection causes;The bacterial pneumonia of (e) prevention and/or treatment influenza virus secondary.
- 7. product according to claim 6, it is characterised in that: described TGF-�� signal pathway inhibitor is following middle at least one: the monoclonal antibody of anti-TGF-beta, soluble TGF-p receptor, TGF-�� antisense oligonucleotide, suppression TGF-�� are expressed or suppress the micromolecular inhibitor of the inhibitor of its activity, the micromolecular inhibitor of TGF-�� receptor, TGF-�� signal path downstream elements.
- 8. product according to claim 7, it is characterised in that: the expression of described suppression TGF-�� or the inhibitor suppressing its activity are Pirfenidone;Described TGF-�� receptor is TGF-�� I type and/or TGF-�� II type and/or TGF-�� III receptor; The micromolecular inhibitor of described TGF-�� receptor is specially SB431542, LDN-193189, LY2157299, LY2109761, SB525334, DMH1, LDN-212854, ML347, LDN-193189HCl, K02288, SB505124, GW788388, LY364947 or RepSox;Described TGF-�� signal path downstream elements is Smad3, JNK or AP-1; The micromolecular inhibitor of described TGF-�� signal path downstream elements is specially SIS3, SP600125, JNK-IN-8, JNKInhibitorIX or SR11302.
- 9. according to described product arbitrary in claim 6-8, it is characterised in that: described respiratory system pathogenic bacterium be following in any one or several: A type streptococcus, streptococcus pneumoniae, staphylococcus aureus, hemophilus influenza and moraxelle catarrhalis; OrThe stromatin that described pneumonocyte surface is relevant to bacterial adhesion is fibronectin or integrin.
- 10. according to described application arbitrary in claim 1-9 or product, it is characterised in that: described product is medicine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410594422.6A CN105617377A (en) | 2014-10-29 | 2014-10-29 | Product for inhibiting and/or preventing influenza virus secondary bacterial infection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410594422.6A CN105617377A (en) | 2014-10-29 | 2014-10-29 | Product for inhibiting and/or preventing influenza virus secondary bacterial infection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105617377A true CN105617377A (en) | 2016-06-01 |
Family
ID=56032973
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410594422.6A Pending CN105617377A (en) | 2014-10-29 | 2014-10-29 | Product for inhibiting and/or preventing influenza virus secondary bacterial infection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105617377A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110769824A (en) * | 2017-05-30 | 2020-02-07 | 东佩制药股份公司 | IL-8 inhibitors for the treatment and/or prevention of bacterial secondary infections |
| CN115364103A (en) * | 2021-05-18 | 2022-11-22 | 深圳先进技术研究院 | Application of TGF (transforming growth factor) beta pathway inhibitor in preparation of medicine or health-care product for preventing or treating blood brain barrier damage |
| WO2023079080A1 (en) * | 2021-11-05 | 2023-05-11 | Frieslandcampina Nederland B.V. | Use of tgf in the prevention virus infection of the respiratory tract |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5730976A (en) * | 1992-03-06 | 1998-03-24 | Corixa Corporation | Method for treating macrophage pathogen infections by TGF-B antagonists |
| WO2007039151A1 (en) * | 2005-09-28 | 2007-04-12 | Universität Zürich | Blockers of transforming growth factor beta and its receptors for the treatment of infectious diseases |
| CN101678116A (en) * | 2006-05-11 | 2010-03-24 | 根特大学 | Sialoadhesin-related compositions and methods |
-
2014
- 2014-10-29 CN CN201410594422.6A patent/CN105617377A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5730976A (en) * | 1992-03-06 | 1998-03-24 | Corixa Corporation | Method for treating macrophage pathogen infections by TGF-B antagonists |
| WO2007039151A1 (en) * | 2005-09-28 | 2007-04-12 | Universität Zürich | Blockers of transforming growth factor beta and its receptors for the treatment of infectious diseases |
| CN101678116A (en) * | 2006-05-11 | 2010-03-24 | 根特大学 | Sialoadhesin-related compositions and methods |
Non-Patent Citations (2)
| Title |
|---|
| BEINAN WANG ET AL.: "Streptococcal modulation of cellular invasion via TGF-β1 signaling", 《PNAS》 * |
| 王剑: "流感病毒感染与粘膜免疫损伤", 《中国博士学位论文全文数据库医药卫生科技辑》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110769824A (en) * | 2017-05-30 | 2020-02-07 | 东佩制药股份公司 | IL-8 inhibitors for the treatment and/or prevention of bacterial secondary infections |
| CN110769824B (en) * | 2017-05-30 | 2023-05-23 | 东佩制药股份公司 | IL-8 inhibitors for the treatment and/or prophylaxis of bacterial secondary infections |
| CN115364103A (en) * | 2021-05-18 | 2022-11-22 | 深圳先进技术研究院 | Application of TGF (transforming growth factor) beta pathway inhibitor in preparation of medicine or health-care product for preventing or treating blood brain barrier damage |
| WO2022241724A1 (en) * | 2021-05-18 | 2022-11-24 | 深圳先进技术研究院 | APPLICATION OF TGFβ PATHWAY INHIBITOR IN PREPARATION OF DRUG OR HEALTHCARE PRODUCT FOR PREVENTING OR TREATING BLOOD BRAIN BARRIER DAMAGE |
| WO2023079080A1 (en) * | 2021-11-05 | 2023-05-11 | Frieslandcampina Nederland B.V. | Use of tgf in the prevention virus infection of the respiratory tract |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Seki et al. | Critical role of IL-1 receptor-associated kinase-M in regulating chemokine-dependent deleterious inflammation in murine influenza pneumonia | |
| Mao et al. | MicroRNA-23a is involved in tumor necrosis factor-α induced apoptosis in mesenchymal stem cells and myocardial infarction | |
| CN108359644A (en) | A kind of wide range salmonella bacteriophage and its application | |
| Lin et al. | The protective effect of naringenin on airway remodeling after Mycoplasma pneumoniae infection by inhibiting autophagy‐mediated lung inflammation and fibrosis | |
| Ouyang et al. | gga-miR-9* inhibits IFN production in antiviral innate immunity by targeting interferon regulatory factor 2 to promote IBDV replication | |
| Pena et al. | Strain-dependent effects of PB1-F2 of triple-reassortant H3N2 influenza viruses in swine | |
| CN107034197A (en) | canine influenza virus monoclonal antibody hybridoma cell strain F112 and application | |
| CN112384614B (en) | Oncolytic virus expressing interferon and uses thereof | |
| CN110022889A (en) | For improving growth, propagation and the oncolytic of oncolytic RNA virus and the composition and method of immunotherapeutic effects | |
| Choi et al. | Novel chimeric parapoxvirus CF189 as an oncolytic immunotherapy in triple-negative breast cancer | |
| CN105617377A (en) | Product for inhibiting and/or preventing influenza virus secondary bacterial infection | |
| Gao et al. | IL-10 knockdown with siRNA enhances the efficacy of Doxorubicin chemotherapy in EBV-positive tumors by inducing lytic cycle via PI3K/p38 MAPK/NF-kB pathway | |
| Dong et al. | Rutin reduces the pathogenicity of Streptococcus agalactiae to tilapia by inhibiting the activity of sortase A | |
| Thapa et al. | In ovo delivery of Toll-like receptor 2 ligand, lipoteichoic acid induces pro-inflammatory mediators reducing post-hatch infectious laryngotracheitis virus infection | |
| Huang et al. | Inhibitory effects of Lactobacillus plantarum metabolites on porcine epidemic diarrhea virus replication | |
| Salgueiro et al. | Generation of human lung organoid cultures from healthy and tumor tissue to study infectious diseases | |
| Golas et al. | Characterization of de novo lytic infection of dermal lymphatic microvascular endothelial cells by Kaposi's sarcoma-associated herpesvirus | |
| Periasamy et al. | CD140b (PDGFRβ) signaling in adipose-derived stem cells mediates angiogenic behavior of retinal endothelial cells | |
| Zhu et al. | Virus-encoded miR-155 ortholog in Marek’s disease virus promotes cell proliferation via suppressing apoptosis by targeting tumor suppressor WWOX | |
| CN106947763B (en) | The siRNA of the resisiting influenza virus infection of aerosolizable sucking and its application | |
| Jin et al. | Inhibiting pyrimidine biosynthesis impairs Peste des Petits Ruminants Virus replication through depletion of nucleoside pools and activation of cellular immunity | |
| Khan et al. | NF-кB pathway genes expression in chicken erythrocytes infected with avian influenza virus subtype H9N2 | |
| Fan et al. | β-catenin has potential effects on the expression, subcellular localization, and release of high mobility group box 1 during bovine herpesvirus 1 productive infection in MDBK cell culture | |
| Xu et al. | Thermal and environmental stability of Siniperca chuatsi Rhabdovirus | |
| Li et al. | Cellular protein GLTSCR2: a valuable target for the development of broad-spectrum antivirals |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160601 |
|
| RJ01 | Rejection of invention patent application after publication |