CN108096579A - Using application of the c-Rel genes as target spot in the drug for preparing treatment rheumatoid arthritis - Google Patents
Using application of the c-Rel genes as target spot in the drug for preparing treatment rheumatoid arthritis Download PDFInfo
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Abstract
This application discloses using application of the c Rel genes as target spot in the drug for preparing treatment rheumatoid arthritis.The application is had found by research by inhibiting c Rel gene expressions, it can inhibit the expression of TNF α, IL 1 β, IL 6, IL 12, IL 23 etc., and then weaken adaptive immunity reaction, the effect for the treatment of rheumatoid arthritis is played, and has developed a kind of c Rel gene specifics siRNA of new treatment rheumatoid arthritis accordingly.For the application using c Rel genes as the drug of target treatment rheumatoid arthritis, toxic side effect is small, safe, can be as the long-term administration for the treatment of rheumatoid arthritis.
Description
Technical field
This application involves antirheumatic fields, are being made more particularly to by target spot of c-Rel genes
Application in the drug of standby treatment rheumatoid arthritis.
Background technology
Rheumatoid arthritis is a kind of autoimmune disease, is known as " first of disabling disease ", is claimed by world medical circle
For not dead cancer, one of four big chronic diseases of 21 century threat human health are nowadays become.China's rheumatoid arthritis hair
Sick rate is high, and patient numbers are 5,000,000 or so.Since this sick cause of disease and pathogenesis are staggeredly complicated, clinically lack root at present
It controls and prevents this sick effective measures.
The pathological characteristics of rheumatoid arthritis are mainly shown as the persistent hyperplastic, cartilage and osteoclasia of synovial membrane, clinical table
Now lost for chronic inflammation, multiple arthroncus, pain and its caused anchylosis or even function.The whole world is about
The people of 0.5%-0.8% suffers from arthritis, incidence and age positive correlation, and women incidence is higher than male.Although joint
The scorching cause of disease is complicated, but nearest research finds arthritis and tumor susceptibility gene, environmental risk factors invasion and attack, apparent modification and translation
Modification is related afterwards.Main pathologic process is：The Dendritic Cells and macrophage of activation migrate into articular cavity, and secrete inflammation
Inflammation factor and chemotactic factor (CF), and then recruitment B cell and T cell enter articular cavity.The T cell of activation, B cell, macrophage, list
The fibroblast of nucleus and local joint activation promotes osteoclast to generate simultaneously eventually by approach such as RANKL/RANK
Cause bone erosion with destroying.Current data shows TNF-α, IL-6, IL-1 β, IL-12, IL23, IL-17, IFN-γ etc.
Inflammatory factor plays key effect in the inflammatory reaction of articular cavity.What is more important, these inflammatory factors are in difference
By NF- κ B transcription factor regulating and expressings in degree.
The drug for the treatment of of arthritis is broadly divided into two major classes at present, and one kind is chemical synthetic drug, including glucocorticoid,
Methotrexate (MTX), sulfasalazine, leflunomide, hydroxychloroquine and JAK enzyme inhibitor tropsch imatinibs etc.；It is another kind of, it is biology
Preparation is lived including the various monoclonal antibody drugs and inhibition T cell using TNF-α, IL-6R, B cell surface C D20 as target spot
The soluble fusion protein Orencia of change.Clinically, when patient early diagnosis for arthritis when, it is necessary to which first is used in combination immediately
Aminopterin and glucocorticoid control arthritic development.When this Regimen Chemotherapy is ineffective, it is necessary to replace other chemistry
Drug carrys out combination with glucocorticoids treatment or directly increases biological agent to treat.It can be seen that arthritic needs to grow
Phase takes glucocorticoid.Glucocorticoid may act on entire Rel/NF- κ B families, and Rel/NF- κ B families and organism
The maintenance of normal physiological function is closely related, therefore glucocorticoid side effect is big, unsuitable long-time service.
Rel/NF- κ B family proteins be it is a kind of have in immune, inflammation, development and the occurrence and development of tumour it is most important
The transcription factor of effect.Mammal Rel/NF- κ B families are by five member compositions：c-Rel、RelA(p65)、RelB、NF-κ
B1 (p50/p105) and NF- κ B2 (p52/p100).The function of each family members of Rel/NF- κ B is similar but not exactly the same, certain
The afunction of a member can not be completely by other members of the family Lai complementary, this may be from Rel/NF- κ B different thin
Differential expression in born of the same parents' type and regulation and control it is different target gene it is related.Most Rel/NF- κ B family members are thin in the prominent colloid of widow
It is expressed in the various kinds of cell type such as born of the same parents, neuron, astrocytes, endothelial cell, lymphocyte and myeloid cell, and c-Rel master
It to be expressed in the lymphocyte and myeloid cell of lymphoid tissue.The NF- κ B of activation enter after nucleus with promoter region
Fixed nucleotide sequence with reference to and promotor gene transcribe.NF- κ B regulate and control the table of a variety of and inflammation and the relevant gene of immune response
It reaches, such as IL-6, TNF-α, IFN-γ, IL-2 and IL-17, and the pathogenic process of various autoimmune disease is participated in, therefore
Rel/NF- κ B families are the molecular targets of an effective treatment autoimmune disease and diseases associated with inflammation, block Rel/NF-
The expression of the κ B families factor will effectively control inflammation reaction.The proteasome inhibitor listed at present, such as U.S. FDA batch
Accurate PS-341, has inhibitory action to Rel/NF- κ B.In addition, the more glucocorticoid of current applying clinical, also in that can
Inflammatory reaction is efficiently controlled to inhibit the expression of Rel/NF- κ B families.But because these drugs not only inhibit Rel/NF-
The κ B families factor, and other many incoherent molecules are acted on, therefore the common drawback of these drugs is that side effect is big, only
Energy short-period used, controls immediate allergic symptom.On the other hand, since Rel/NF- κ B family proteins are widely present in eukaryocyte
Interior, important pathophysiological process is closely related with inflammatory reaction, immune response etc., according to drug be directed to entire Rel/
NF- κ B families will generate apparent side effect.
Theoretically, drug using entire NF- κ B families as target spot, which can inhibit inflammatory reaction and reach, improves arthritic symptom
Effect, such as methotrexate (MTX) and glucocorticoid.But most of NF- κ B family proteins are universal in many cells of body
Expression, it is and related to the maintenance of body normal physiological function, as in course of infection it is congenital and adaptive immunity regulate and control, inflammation
Disease reaction, anti-apoptotic, cell Proliferation etc., cause that these drug side-effects are big, poor specificity, can only use to control in a short time
Acute allergic reaction.It is thus impossible to long-time service treats chronic inflammation disease using entire NF- κ B families as the drug of target spot
Disease, such as rheumatoid arthritis.Therefore, there is an urgent need for research and development are a kind of not only can be with long-time service, but also have no toxic side effect or toxic side effect
Low antirheumatic.
The content of the invention
The purpose of the application is to provide a kind of specific target spot of new treatment rheumatoid arthritis, i.e. c-Rel genes,
And application of the gene target in the drug for preparing treatment rheumatoid arthritis.
To achieve these goals, the application employs following technical scheme：
The one side of the application disclose using c-Rel genes be target spot prepare treatment rheumatoid arthritis drug in
Application.
Wherein, refer in particular to subtract using gene knockout, clpp gene as target spot using c-Rel genes or chemicals reduces c-
The expression of Rel genes makes c-Rel gene silencings.
It should be noted that the application by the study found that by inhibiting c-Rel gene expressions, can inhibit TNF-α,
The expression of IL-1 β, IL-6, IL-12, IL-23, and then weaken adaptive immunity reaction, improve arthritic symptom, reach treatment
The purpose of rheumatoid arthritis.Also, carry out rheumatoid arthritis treatment, high specificity, poison by target spot of c-Rel genes
Small side effects.
The another side of the application discloses a kind of siRNA of c-Rel gene specifics as treatment rheumatoid arthritis
Drug application.
The another side of the application discloses a kind of siRNA of c-Rel gene specifics and is preparing treatment rheumatoid joint
Application in scorching drug.
It should be noted that the inhibition c-Rel gene expressions of specificity, one of means are exactly siRNA, also, this
Application is confirmed by research, and effective siRNA, specific inhibition c-Rel gene tables can be designed based on c-Rel genes
It reaches, achievees the purpose that treat rheumatoid arthritis.
The another side of the application discloses a kind of siRNA, the siRNA be capable of specificity act on c-Rel genes,
The positive-sense strand of siRNA is sequence shown in Seq ID No.1, and antisense strand is sequence shown in Seq ID No.2；
Seq ID No.1：5’-CAACCGGACAUACCCGUCU-3’
Seq ID No.2：5’-AGACGGGUAUGUCCGGUUG-3’
Also, 3 ' ends of positive-sense strand and antisense strand are respectively provided with beyond " dTdT " end of double-strand complementary structure.
It should be noted that sequence antisense strand shown in sequence positive-sense strand shown in Seq ID No.1 and Seq ID No.2
SiRNA is that the application researches and develops and passes through confirmation and can inhibit inflammatory reaction, and the c-Rel genes for treating rheumatoid arthritis are special
Different in nature siRNA.It is appreciated that on the basis of the application confirms that c-Rel genes design siRNA can be based on, the application's
Under guidance, however not excluded that other similary siRNA with c-Rel gene specifics can also be developed.
The another side of the application discloses a kind of drug for treating rheumatoid arthritis, and the active ingredient of the drug includes
The siRNA of c-Rel gene specifics.
Preferably, drug further includes nano-carrier, and the nano-carrier is for conveying c-Rel gene specifics in vivo
siRNA。
It should be noted that siRNA is easily digested in vivo, half-life short, and therefore, in the drug of the application,
It is preferred to be conveyed in vivo using the form of nano-carrier.It is appreciated that in addition, however not excluded that other manner can also be used
Conveying siRNA in vivo.
It should also be noted that, the drug of the application can also add other pharmaceutically acceptable auxiliary materials or addition
Agent to form different dosage forms, is not specifically limited herein.
Preferably, nano-carrier is PEG-PLL-PLLeu triblock copolymer nano-micelles.
It should be noted that PEG-PLL-PLLeu triblock copolymer nano-micelles act not only as carrier in vivo
SiRNA is conveyed, and it is mainly absorbed by antigen presenting cell, particularly can effectively be taken the photograph by Dendritic Cells and macrophage
It takes.
In a kind of realization method of the application, the c-Rel gene specifics of the drug of rheumatoid arthritis are treated
SiRNA is exactly the application with sequence antisense strand shown in the positive-sense strand of sequence shown in Seq ID No.1 and Seq ID No.2
siRNA。
The application disclose on one side again the siRNA of the application or the drug of the application prepare treatment graft rejection or
Application in the drug of inflammation.
It should be noted that the siRNA or drug of the application can inhibit TNF-α, IL-1 β, IL-6, IL-12, IL-23
The expression of the inflammations factor is waited, therefore, can be not only used for for example treating this kind of autoimmune disease of rheumatoid arthritis
Disease can be equally used for treating the relevant other inflammation of these inflammatory factors or related to these inflammatory factors for treating
Other diseases or symptom, such as graft rejection, be not specifically limited herein.
Due to using the technology described above, the advantageous effect of the application is：
The application has found that, by inhibiting c-Rel gene expressions, TNF-α, IL-1 β, IL-6, IL- can be inhibited by research
12nd, the expression of IL-23 etc., and then weaken adaptive immunity reaction, the effect for the treatment of rheumatoid arthritis is played, and is ground accordingly
A kind of c-Rel gene specifics siRNA of new treatment rheumatoid arthritis is sent out.The application is using c-Rel genes as target spot
The drug of rheumatoid arthritis is treated, toxic side effect is small, safe, can be as the long-term for the treatment of rheumatoid arthritis
Medication.
Description of the drawings
Fig. 1 is that the swelling situation of mouse claw in the embodiment of the present application carries out appraisal result figure；
Fig. 2 is joint section testing result figure in the embodiment of the present application；
Fig. 3 is the ratio statistical results chart of mouse spleen and weight in the embodiment of the present application；
Fig. 4 is the testing result statistical chart of IFN-γ in mice serum in the embodiment of the present application；
Fig. 5 is the testing result statistical chart of IL-17A in mice serum in the embodiment of the present application；
Fig. 6 is the testing result statistical chart of TNF-α in mice serum in the embodiment of the present application；
Fig. 7 is the testing result statistical chart of IL-1 β in mice serum in the embodiment of the present application；
Fig. 8 is the testing result statistical chart of IgG1 in mice serum in the embodiment of the present application；
Fig. 9 is the testing result statistical chart of IgG2a in mice serum in the embodiment of the present application；
Figure 10 is the coloration result of Turnover of Mouse Peritoneal Macrophages intracellular dye c-Rel in the embodiment of the present application；
Figure 11 is the coloration result of mouse spleen dyeing CD11c and c-Rel in the embodiment of the present application；
Figure 12 is the mRNA detection of expression results of TNF-α in Turnover of Mouse Peritoneal Macrophages in the embodiment of the present application；
Figure 13 is the mRNA detection of expression results of IL-1 β in Turnover of Mouse Peritoneal Macrophages in the embodiment of the present application；
Figure 14 is the mRNA detection of expression results of IL-6 in Turnover of Mouse Peritoneal Macrophages in the embodiment of the present application；
Figure 15 is the mRNA detection of expression results of IL-12 in Turnover of Mouse Peritoneal Macrophages in the embodiment of the present application；
Figure 16 is the mRNA detection of expression results of IL-23 in Turnover of Mouse Peritoneal Macrophages in the embodiment of the present application；
Figure 17 is the mRNA detection of expression results of TNF-α in mice spleen total cell in the embodiment of the present application；
Figure 18 is the mRNA detection of expression results of IL-1 β in mice spleen total cell in the embodiment of the present application；
Figure 19 is the mRNA detection of expression results of IL-6 in mice spleen total cell in the embodiment of the present application；
Figure 20 is the mRNA detection of expression results of IL-12 in mice spleen total cell in the embodiment of the present application；
Figure 21 is the mRNA detection of expression results of IL-23 in mice spleen total cell in the embodiment of the present application；
Figure 22 is the expression of IFN-γ in mouse SPL in the embodiment of the present application；
Figure 23 is the expression of IL-17A in mouse SPL in the embodiment of the present application；
Figure 24 is the expression of IFN-γ in mouse PLN in the embodiment of the present application；
Figure 25 is the expression of IL-17A in mouse PLN in the embodiment of the present application；
Figure 26 is the mRNA detection of expression results of TNF-α in mouse rear solid end in the embodiment of the present application；
Figure 27 is the mRNA detection of expression results of IL-6 in mouse rear solid end in the embodiment of the present application；
Figure 28 is the mRNA detection of expression results of IL-1 β in mouse rear solid end in the embodiment of the present application；
Figure 29 is the mRNA detection of expression results of IFN-γ in mouse rear solid end in the embodiment of the present application；
Figure 30 is the mRNA detection of expression results of IL-17A in mouse rear solid end in the embodiment of the present application；
Figure 31 is the mRNA detection of expression results of IL-23 in mouse rear solid end in the embodiment of the present application；
Figure 32 is the percentage of CD3+ cells on total cells in mouse rear solid end in the embodiment of the present application；
Figure 33 is the absolute number of CD3+ cells in mouse rear solid end in the embodiment of the present application；
Figure 34 is the percentage of F4/80+ cells on total cells in mouse rear solid end in the embodiment of the present application；
Figure 35 is the absolute number of F4/80+ cells in mouse rear solid end in the embodiment of the present application；
Figure 36 is total cell number in mouse rear solid end in the embodiment of the present application.
Specific embodiment
The study found that c-Rel gene preferentially expresseds are in inflammatory cell, and it is only involved in the immune response of part, c-Rel missings
Mouse growth during will not be subject to developing and communicable disease is influenced, therefore target c-Rel and can be substantially reduced drug
Side effect.Present inventor proposes a new hypothesis, i.e. a member as NF- κ B families, c-Rel genes is not only one
Kind oncogene, and be a kind of tumor susceptibility gene of autoimmune disease.Because the study found that c-Rel depleted mices are to device
Official's specific autoimmune disease such as rheumatoid arthritis and type-1 diabetes mellitus have tolerance.More than hypothesis is based on, and is reflected
It is big in the side effect for targeting entire NF-kB families inhibition inflammatory reaction, and c-Rel priority expressions can simultaneously regulate and control bag in inflammatory cell
A series of expression of inflammatory factors including IL-6, IL1- β, IFN-γ, IL-12, IL-23, IL-17 etc. is included, the application creates
Property advance an idea, only using c-Rel as target spot come inhibit inflammatory reaction, treatment rheumatoid arthritis.Also, eventually by examination
It tests and confirms and can treat rheumatoid arthritis by target spot of c-Rel genes, and confirm that can be directed to c-Rel genes designs
Go out effective siRNA, specific inhibition c-Rel gene expressions achieve the purpose that treat rheumatoid arthritis.
The application is on the basis of above research, it is proposed that the new application of c-Rel gene targets, and based on such a
New discovery has developed new targeted drug, is rheumatoid arthritis and other and TNF-α, IL-1 β, IL-6, IL-12, IL-23
Etc. the treatment of the relevant symptom of inflammatory factors provide new scheme and strategy.
The application is described in further detail below by specific embodiments and the drawings.Following embodiment is only to the application
It is further described, should not be construed as the limitation to the application.
Embodiment
This example uses the DBA/1 mouse arthritis that 2 Collagen Type VI of chicken induces as disease model, this model and mankind's rheumatoid
Property arthritic pathologic process it is similar, be carry out drug development classical model.It is as follows in detail：
First, mouse is immunized using 2 Collagen Type VI of chicken and Freund's complete adjuvant in we, using 2 Collagen Type VI of chicken and not after 14 days
Non-fully adjuvant carries out secondary immunity to family name.It observes and scores every other day after secondary immunity, when mouse disease index is 1, random point
For control group and experimental group, siNC and siRel are given in abdominal cavity respectively, i.e. siNC is given in control group abdominal cavity, and experimental group abdominal cavity is given
siRel。
Wherein, siNC is purchased from Suzhou Ji Ma Co., Ltds, is and general negative control of the objective gene sequence without homology
Sequence.
SiRel is the specific siRNA that this example is designed according to c-Rel genes, has sequence shown in Seq ID No.1 just
The siRNA of sequence antisense strand shown in adopted chain and Seq ID No.2, also, 3 ' ends of positive-sense strand and antisense strand be respectively provided with it is super
Go out " dTdT " end of double-strand complementary structure,
Seq ID No.1：5’-CAACCGGACAUACCCGUCU-3’
Seq ID No.2：5’-AGACGGGUAUGUCCGGUUG-3’.
Abdominal cavity is given siNC or siRel and is carried out after treating 22 days, mouse claw swelling situation is observed, to four pawls of mouse
The swelling situation of son carries out scoring respectively and summation score：0 point, normally；1 point, a toe swelling；2 points, more than one toe swells
It is swollen, but the entire non-swelling of claw or the slight swelling of entire claw；3 points, entire claw swelling；4 points, entire claw serious swelling
Or anchylosis deformity.
Mouse claw swelling situation bibliography：Collagen-Induced Arthritis in Mice,Lisette
Bevaart,Margriet J.Vervoordeldonk,and Paul P.Tak。
Appraisal result draws analysis using GrapdhPad Prism5, and analysis result is as shown in Figure 1, in Fig. 1, abscissa is
Abdominal cavity give after number of days, ordinate be mouse claw swelling situation scoring, "●" curve be siNC control groups scoring analysis
Curve, " ■ " curve are the analysis curves of siRel experimental groups scoring, and " * * " represents P<0.01, " * * * " represents P<0.001, numerical value
It is shown as means standard deviation (n=3).Fig. 1's the results show that the abdominal cavity experimental mice claw swelling of giving siRel is apparent
It is controlled.
All analysis charts of this example are drawn using GrapdhPad Prism5, and disease severity uses Mann-Whitney
U is examined.
At the same time, the 22nd day spinal cord detachment is put to death into mouse, takes the knee joint of siNC, siRel group mouse same area,
Formalin fix 48 it is small when after, be placed in decalcification seven days in 10% EDTA solution, the EDTA during which more renewed every three days is molten
Liquid.After decalcification, routine paraffin wax embedding, section, dewaxing, dyeing are carried out, to the progress such as synovium of joint, articular cavity and structure
Observation.Coloration result cut into slices as shown in Fig. 2, in Fig. 2, siNC is the slice map of control group, and siRel is the slice map of experimental group.
Fig. 2's the results show that inflammatory cell infiltration significantly reduces in siRel experimental mices joint, joint structure is complete, only slight
Synovial hyperplasia.
In addition, this example weighs to the spleen and weight of the mouse of experimental group and control group, analyze mouse spleen with
The ratio of weight, and analysis chart is drawn using GrapdhPad Prism5, which can reflect inflammation whole in Mice Body
React severity.The results are shown in Figure 3, and in Fig. 3, ordinate is mouse spleen and the ratio of weight, and abscissa is respectively
SiNC control groups and siRel experimental groups, " * " represent P<0.05, numerical value is shown as means standard deviation (n=3).The result of Fig. 3
It has been shown that, siRel experimental mices spleen are substantially reduced with weight ratio, and it is in vivo that this shows that siRel processing can significantly reduce mouse
Inflammatory reaction severity.
In addition, this example also has detected control group and the mice serum of experimental group, respectively to TNF-α therein, IL-1 β,
The IgG1 and IgG2a of IFN-γ, IL-17A and collagen specificity are detected, and are painted using GrapdhPad Prism5 statistics
Figure.It is specific as follows：
22nd day, mouse isoflurane is given, carries out plucking eyeball taking blood after mouse anesthesia, every mouse takes about 800 μ L.
The whole blood of taking-up is positioned in four degree of refrigerators, makes its natural coagulation, then 3000rpm is centrifuged 10 minutes, takes supernatant, is this
The mice serum of example.
IFN-γ, IL-17A, TNF-α, IL-1 β, the testing result of IgG1 and IgG2a are sequentially as shown in Fig. 4 to Fig. 9, Fig. 4
Into Fig. 9, ordinate is the content of each detection object, and abscissa is respectively siNC control groups and siRel experimental groups, and numerical value is shown
For means standard deviation (n=3), " * " represents P<0.05, " * * " represents P<0.01.Fig. 4 is to Fig. 9's the results show that siRel is real
The IgG1 and IgG2a for testing IL-1 β in group mice serum, IFN-γ, IL-17A and collagen specificity are significantly reduced, these show
SiRel effectively reduces the in vivo inflammatory reaction of mouse.
In addition, when treating the 22nd day, this example has separated Turnover of Mouse Peritoneal Macrophages and spleen total cell, carry out CD11c,
F4/80 and c-Rel dyeing, and drawn using GrapdhPad Prism5 statistics.Detailed step includes, and puts to death within the 22nd day small
Mouse is sterilized in being immersed in 75%, is fixed in super-clean bench, opens abdominal cavity, is filled using the ice-cold PBS solution containing 2% FBS
Abdominal cavity is washed, collects irrigating solution, 500g centrifugation 5min obtain the peritoneal macrophage that purity is more than 90%.Put to death mouse within 22nd day,
The spleen of mouse is taken, grinds and obtains single cell suspension, splitting erythrocyte terminates 500g centrifugation 5min after cracking, washes repeatedly twice,
Obtain leucocyte in spleen.
The coloration result of Turnover of Mouse Peritoneal Macrophages intracellular dye c-Rel is as shown in Figure 10, Tu10Zhong, and left figure is thin for F4/80+
The detection curve figure of born of the same parents, right figure are the statistical analysis figure of c-Rel；The coloration result of mouse spleen dyeing CD11c and c-Rel is as schemed
Shown in 11, Tu11Zhong, left figure is the detection curve figure of CD11c+ cells, and right figure is the statistical analysis figure of c-Rel.Figure 10 and Figure 11
The results show that siRel can effectively reduce the expression of c-Rel in Dendritic Cells in Turnover of Mouse Peritoneal Macrophages and spleen.
Further, after this example stimulates peritoneal macrophage and spleen total cell using LPS in vitro, examined by q-PCR
Measure by c-Rel regulate and control inflammatory factor, including TNF-α, IL-1 β, IL-6, IL-12, IL-23 mRNA expressions.Specifically
Method：It after peritoneal macrophage and spleen total cell count, is inoculated in 48 orifice plates with 2M/mL concentration, after cell settlement, added
Entering isometric culture medium containing LPS stimulates, the final concentration of 1 μ g/mL of LPS, stimulate 6 it is small when after collect cell, Trizol extractions are total
RNA carries out reverse transcription reaction according to the corresponding specification of TOYOBO reagents, obtains cDNA.Finally, qPCR reactions, qPCR are carried out
Specific reaction system is carried out with reference to TOYOBO reagents specification, the primer and qPCR reaction systems and conditioned reference of use：
Fan,T.et al.Treating psoriasis by targeting its susceptibility gene
Rel.Clinical immunology 165,47-54,doi:10.1016/j.clim.2016.03.009 (2016) inflammation because
Son quantifies testing result using after qPCR relative quantifications according to it, is counted and drawn using GrapdhPad Prism5.
The GrapdhPad Prism5 statistics of peritoneal macrophage q-PCR testing results is drawn as shown in Figure 12 to Figure 16,
12 to Figure 16 be sequentially the mRNA detection of expression results of TNF-α, IL-1 β, IL-6, IL-12, IL-23 in peritoneal macrophage.
The GrapdhPad Prism5 statistics of spleen total cell q-PCR testing results is drawn as shown in Figure 17 to Figure 21, and 17 to Figure 21 sequentially
The mRNA detection of expression results for being TNF-α, IL-1 β, IL-6, IL-12, IL-23 in spleen total cell.Figure 12 is indulged into Figure 21
Coordinate is the content of each detection object, and abscissa is respectively siNC control groups and siRel experimental groups, and numerical value is shown as mean value ± mark
Accurate poor (n=5), non-to be examined with t, " * " represents P<0.05, " * * * " represents P<0.001.
Figure 12 is to Figure 21's the results show that by the inflammation such as the TNF-α of c-Rel regulation and control, IL-1 β, IL-6, IL-12, IL-23
The mRNA expression of the factor is effectively lowered, and illustrates that siRel can effectively control the congenital inflammation of macrophage and Dendritic Cells
Reaction.
Further, this example separation spleen total cell and little Shu lymphonodi poplitei cells, by collecting supernatant after stimulated in vitro,
ELISA detects Th1 the and Th17 cell quantities of its collagen specificity.Specifically, the 22nd day, little Shu lymphonodi popliteis and spleen are taken, is ground
After prepare single cell suspension, splenocyte separately needs splitting erythrocyte.Jiang after lymphonodi poplitei cell and spleen total cell count, with 2M/mL
Concentration is inoculated in 48 orifice plates of pre-coated anti-CD3, after cell settlement, adds in isometric culture containing anti-CD28
Base stimulates, the final concentration of 1 μ g/mL of free anti-CD28, stimulate 48 it is small when after collect supernatant, 500g centrifugation 5min removals are thin
Born of the same parents detect IL-17A and the expression of IFN-γ using Ebioscience kits and according to its specification.
Testing result as shown in Figure 22 to Figure 25, wherein, Figure 22 and Figure 23 are sequentially the table of IFN-γ and IL-17A in SPL
Up to level, Figure 24 and Figure 25 are sequentially the expression of IFN-γ and IL-17A in PLN.Figure 22 to Figure 25's the results show that
ELISA detects that siRel substantially descends Th1 the and Th17 cell quantities of adjusting body and collagen specificity.As shown in the figure, at siRel
Reason can significantly reduce splenocyte He the IL-17A and IFN-γ of the secretion of lymphonodi poplitei cell, due to the main quilt of delivery system of this example
Dendritic Cells and macrophage passively absorb, without being absorbed by T cell, this illustrate reduce IL-17A and IFN-γ not by
In T cell secrete IL-17A or IFN-γ secretion capacity reduce caused by, but due to secretion IL-17A and IFN-γ
Caused by Th1 and Th17 percentages namely number reduce, illustrate that adaptive immunity reaction has also obtained effective control.
Finally, this example has detected the Infiltrating of the inflammatory factor and inflammatory cell in mouse rear solid end, specifically, the 22nd day
After putting to death mouse, the claws of siNC and siRel same areas is taken, the endogenous Rnase of rapid inactivation in about 20mg liquid nitrogen is placed in
In Trizol solution, homogenizer homogenate then extracts total serum IgE according to conventional steps.Reverse transcription is carried out according to TOYOBO specifications
And qPCR reactions, the primer and qPCR reaction systems and conditioned reference that this example uses：Fan,T.et al.Treating
psoriasis by targeting its susceptibility gene Rel.Clinical immunology 165,
47-54,doi:10.1016/j.clim.2016.03.009(2016)。
It treats the 22nd day, takes siNC and siRel group mouse rear solid ends, single cell suspension, streaming inspection are prepared after collagenase digesting
Survey the cell percentages and absolute number of CD3+ and F4/80+.
The mRNA detection of expression result of the inflammatory factors such as TNF-α, IL-6, IL-1 β, IFN-γ, IL-17A, IL-23 is sequentially
As shown in Figure 26 to Figure 31, for Figure 26 into Figure 31, ordinate is the relative quantity of each detection object, and abscissa is respectively siNC controls
Group and siRel experimental groups, numerical value are shown as means standard deviation (n=3), non-paired t test, *:P<0.05；**:P<
0.01；***:P<0.001.
The percentage of CD3+ cells on total cells is as shown in figure 32, and absolute number is as shown in figure 33, and F4/80+ cells account for total thin
The percentage of born of the same parents is as shown in figure 34, and absolute number is as shown in figure 35；Total cell number is as shown in figure 36.
Figure 26 is to Figure 31's the results show that siRel handles TNF-α, IL-6, IL-17A, IFN-γ table in mouse mouse claw
It is lowered up to apparent, Figure 32 is to Figure 36's the results show that the infiltration of T cell and macrophage significantly reduces.
The above result shows that siRel can be absorbed effectively by Dendritic Cells and macrophage, and inhibit TNF-α, IL-1 β,
The expression of IL-6, IL-12, IL-23, and then weaken adaptive immunity reaction, improve arthritic symptom, can be used in treating class wind
Wet arthritis.
The foregoing is a further detailed description of the present application in conjunction with specific implementation manners, it is impossible to assert this Shen
Specific implementation please is confined to these explanations.For those of ordinary skill in the art to which this application belongs, do not taking off
On the premise of conceiving from the application, several simple deduction or replace can also be made.
Claims (10)
1. using application of the c-Rel genes as target spot in the drug for preparing treatment rheumatoid arthritis.
2. application according to claim 1, it is characterised in that：It is described to include by target spot of c-Rel genes using clpp gene
It removes, clpp gene subtracts the either expression of chemicals reduction c-Rel genes or makes c-Rel gene silencings.
3. a kind of applications of siRNA of c-Rel gene specifics as the drug for the treatment of rheumatoid arthritis.
4. a kind of applications of siRNA of c-Rel gene specifics in the drug for preparing treatment rheumatoid arthritis.
5. a kind of siRNA, it is characterised in that：The siRNA be capable of specificity act on c-Rel genes, the siRNA is just
Adopted chain is sequence shown in Seq ID No.1, and antisense strand is sequence shown in Seq ID No.2；
Seq ID No.1：5’-CAACCGGACAUACCCGUCU-3’
Seq ID No.2：5’-AGACGGGUAUGUCCGGUUG-3’
Also, 3 ' ends of positive-sense strand and antisense strand are respectively provided with beyond " dTdT " end of double-strand complementary structure.
6. a kind of drug for treating rheumatoid arthritis, it is characterised in that：The active ingredient of the drug includes c-Rel genes
The siRNA of specificity.
7. drug according to claim 6, it is characterised in that：The drug further includes nano-carrier, the nano-carrier
For conveying the siRNA of the c-Rel gene specifics in vivo.
8. drug according to claim 7, it is characterised in that：The nano-carrier is total to for PEG-PLL-PLLeu three blocks
Polymers nano-micelle.
9. according to claim 6-8 any one of them drugs, it is characterised in that：The siRNA of the c-Rel gene specifics is
SiRNA described in claim 5.
10. siRNA according to claim 5 or claim 6-9 any one of them drug are preparing treatment transplanting
Application in the drug of repulsion or inflammation.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004027063A1 (en) * | 2002-09-19 | 2004-04-01 | Institut National De La Sante Et De La Recherche Medicale-Inserm | Use of sirnas for gene silencing in antigen presenting cells |
| CN101460634A (en) * | 2006-04-13 | 2009-06-17 | 康乃尔研究基金会有限公司 | Methods and compositions for targeting C-REL |
| CN105132429A (en) * | 2015-10-10 | 2015-12-09 | 华东理工大学 | SiRNA targeted to human KPNB1 genes and application thereof |
| CN106086021A (en) * | 2016-06-08 | 2016-11-09 | 深圳先进技术研究院 | The siRNA of specific antagonist c Rel application in treatment MS |
-
2017
- 2017-12-18 CN CN201711367608.8A patent/CN108096579A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004027063A1 (en) * | 2002-09-19 | 2004-04-01 | Institut National De La Sante Et De La Recherche Medicale-Inserm | Use of sirnas for gene silencing in antigen presenting cells |
| CN101460634A (en) * | 2006-04-13 | 2009-06-17 | 康乃尔研究基金会有限公司 | Methods and compositions for targeting C-REL |
| CN105132429A (en) * | 2015-10-10 | 2015-12-09 | 华东理工大学 | SiRNA targeted to human KPNB1 genes and application thereof |
| CN106086021A (en) * | 2016-06-08 | 2016-11-09 | 深圳先进技术研究院 | The siRNA of specific antagonist c Rel application in treatment MS |
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