CN103757024B - A kind of tiny RNA and application thereof disturbing TLR4 acceptor - Google Patents
A kind of tiny RNA and application thereof disturbing TLR4 acceptor Download PDFInfo
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- CN103757024B CN103757024B CN201410043633.0A CN201410043633A CN103757024B CN 103757024 B CN103757024 B CN 103757024B CN 201410043633 A CN201410043633 A CN 201410043633A CN 103757024 B CN103757024 B CN 103757024B
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Abstract
The invention belongs to biomedicine technical field.Chronic pain is one of manifestation of neuron plasticity change, and its physiologic characteristic is that the reactivity of the pain sensation increases, and treatment is difficult and mechanism is not clear, and in treatment, multiplex opioid receptor retarding agent, exists more side effect.The invention provides a kind of tiny RNA disturbing TLR4 acceptor, is its sequence as SEQ? ID? shown in NO:5.Present invention also offers the application of this tiny RNA in treatment chronic pain medicine.Tiny RNA of the present invention can adopt the mode of direct injection, or adopts the mode of vector expression, can suppress the expression of TLR4 in vivo and in vitro, reaches the effect for the treatment of chronic pain, and route of administration is convenient, and there is not the reaction such as habituation, tolerance, can life-time service.
Description
Technical field
The invention belongs to biomedicine technical field, be specifically related to a kind of suppress TLR4 acceptor tiny RNA and application at preparation treatment chronic pain medicine.
Background technology
Chronic pain is one of manifestation of neuron plasticity change, and its physiologic characteristic is that the reactivity of the pain sensation increases, and main manifestations is hyperpathia (hyperalgesia) and allergy (allodynia).This type of pain therapy difficulty, mainly because its mechanism is not clear.At present in treatment, be mainly the opioid receptor retarding agent of representative with morphine, but there is numerous side effects and the reaction such as habituation, tolerance, thus limit its life-time service.Therefore, need that a kind of result for the treatment of is good badly, novel method that long action time, side effect are light, reach long-term, effectively control the object of chronic pain.
Toll-like receptor 4 (Toll like receptor4, TLR4) be one of Toll family, bacteria lipopolysaccharide (lipopolysaccharide, LPS) the important path of intracellular signal transduction is the main cause of disease pattern recognition receptors of transmembrane receptor on LPS target cell membrane and natural immune system.TLR4 has extracellular leucine repeat region and Intracellular signals region, extracellular signal activates TLR4 by CD14, and be combined with the TLR4 receptor domain of adaptor protein MyD88 in the TLR4 born of the same parents of activation, by MyD88 dead space (death domain) again with IL-1 receptor-associated kinase (IL-1receptor associated kinase, IRAK) combine, cause the autophosphorylation of IRAK, thus activate tumor necrosis factor receptor-associated factor-6 (TNF-α receptorassociated factor6, TRAF-6), then make I κ B family α by activating IKK, beta kinase activates, cause the extensive phosphorylation of I κ B family and degrade, NF-κ B is made to insert to karyon, its active dimeric active cell factor is (as IL-1, IL-6, IL-8, IL-12 etc.) and the transcribing of costimulatory molecules CD80 and CD86 gene, translation, inflammatory factor is finally caused to discharge (see document: Takeuchi O and S akira.Toll-like receptors:their physiological role and signal transduction system.IntImmunopharmacol, 2001, 1 (4): 625-635).
TLR4 acceptor plays a significant role in the generation of pain, development and maintenance.Find the high expression level of spinal cord spongiocyte TLR4 and nerve injury in L5 nerve injury model male rat model after, allodynia is closely related.After L5 neural cutting, the hot threshold of pain of TLR4-knockout and TLR4 mutant mouse, the mechanical threshold of pain are apparently higher than wild mouse, the expression of spinal cord microglia activation correlating markings and proinflammatory factor can be reduced to male L5 neural cutting rat intrathecal injection TLR4 inhibition oligonucleotide (ODN), obviously alleviate the susceptibility of rat to the pain sensation, the effect of TLR4 in microglia activation is confirmed mouse and rat model, diagnosis and treatment for clinical neurogenic pain provide novel targets (see document: Tanga FY, NutileMcMenemy N, DeLeo JA.The CNS role of Toll-like receptor4in innateneuroimmunity and painful neuropathy.Proc Natl Acad Sci USA, 2005, 102 (16): 5856-5861).
RNA disturbs (RNA interference, RNAi) refer to when importing the double-stranded RNA with endogenous mRNA coding region homology in cell, there is degraded and cause the phenomenon of gene silencing in this mRNA, is a kind of phenomenon suppressing specific gene to be expressed in organism.External source import or the double-stranded RNA introduced by the various mode such as transgenosis, virus infection by can the enzyme Dicer of specific recognition double-stranded RNA by RNase III family, the mode relied on ATP is progressively cut into the small molecules interference RNA fragment (small interfering RNAs, siRNA) of 19 ~ 23nt.SiRNA double-strand double-strand under RISC effect is untied, and the target RNA of homology combines, and under the effect of endonuclease, is cut off by target mRNA, thus has blocked genetic expression.RNA interference is current most efficient gene " silence " technology, and theoretically, the gene that excitability increases in pain pathway all may become RNA interference target position, thus reduces its excitability, reaches therapeutic purpose.But, jamming effectiveness is high, and the acquisition of the tiny RNA of high specificity is more difficult, and not homotactic difference can cause the forfeiture of validity, thus affecting its pharmacological value, we adopt rat model to find to disturb the tiny RNA of rat TLR4 acceptor to have the effect for the treatment of pain.(see document: Wu Feixiang, Miao Xuerong, Xu Xuewu, Sun Yuming, Yu Weifeng. intrathecal injection is for the neurogenic pain of the siRNA alleviation sciatic nerve ligation rat of Toll-like receptor 4 gene. The 2nd Army Medical College journal .2011,32(4): 377-381).But the treatment for human body still needs the tiny RNA effectively disturbing humanized TLR4 acceptor.
There is no at present about disturbing the tiny RNA of humanized TLR4 acceptor and being used for the treatment of the bibliographical information of chronic pain.
Summary of the invention
The object of the present invention is to provide a kind of tiny RNA disturbing TLR4 acceptor, another object of the present invention is to provide the application of this tiny RNA in preparation treatment chronic pain medicine.
The pain that the present inventor's intrathecal injection in early stage TLR4-siRNA obviously can reduce the rat models of neuropathic pain that nerve injury causes is quick, illustrate that TLR4 is a target of effectively treating chronic pain, also illustrate that RNA perturbation technique is used for the treatment of the feasibility of chronic pain simultaneously.(siRNA sequence be the sequence of rat: 5 '-GUCUCAGAUAUCUAGAUCU-3; see document: Wu Feixiang; Miao Xuerong; Xu Xuewu; Sun Yuming; Yu Weifeng. intrathecal injection is for the neurogenic pain of the siRNA alleviation sciatic nerve ligation rat of Toll-like receptor 4 gene. The 2nd Army Medical College journal .2011,32(4): 377-381; And Wu FX, Bian JJ, Miao XR, Huang SD, Xu XW, Gong DJ, Sun YM, Lu ZJ, YuWF.Intrathecal siRNA against Toll-like receptor4reduces nociception in a ratmodel of neuropathic pain.Int J Med Sci, 2010,7 (5): 251-259.).
Therefore, the present inventor screens the tiny RNA of the TLR4 of interference people further, obtains the tiny RNA of a kind of efficient interference TLR4, can degrade to interference TLR4mRNA, thus suppressing the expression of interference TLR4 albumen, this tiny RNA can become a kind of new medicine for the treatment of chronic pain.
Concrete technical scheme of the present invention is as follows:
TLR4 sequence (GI:2459627) is obtained by Gene bank, the RNAi design software that open reading frame part input http://i.cs.hku.hk/ ~ sirna/software/sirna.php website in its sequence provides is screened online, GC is then selected to compare between 40%-60%, be Blast one by one to analyze and snp analysis, choose the sequence that 3 of being positioned at open reading frame are different, as follows:
Ⅰ.GCATGGAGCTGAATTTCTA（SEQ ID NO:1）
Ⅱ.GGATTTATCCAGGTGTGAA（SEQ ID NO:2）
Ⅲ.CCTGAACCCTATGAACTTT（SEQ ID NO:3）
The RNA sequence of its correspondence is:
Ⅰ.GCAUGGAGCUGAAUUUCUA（SEQ ID NO:4）
Ⅱ.GGAUUUAUCCAGGUGUGAA（SEQ ID NO:5）
Ⅲ.CCUGAACCCUAUGAACUUU（SEQ ID NO:6）
The pEGFPC1 reporter plasmid that the present invention will build containing people TLR4 gene, RNA and pEGFPC1-TLR4 cotransfection 293 cell of above-mentioned sequence pair being answered by liposome method, carry out the detection of RNA jamming effectiveness, and then the sequence filtering out RNA jamming effectiveness the highest is: GGAUUUAUCCAGGUGUGAA(SEQ ID NO:5).
A first aspect of the present invention, provide a kind of tiny RNA disturbing TLR4 acceptor, its sequence is as shown in SEQID NO:5.
The present invention is detected carrying out RNA jamming effectiveness by the pEGFPC1 reporter plasmid containing TLR4 gene, obtains the highest tiny RNA of jamming effectiveness; Acted on again the neural microglia of people by tiny RNA, find that tiny RNA has the rise of the TLR4 of good suppression endotaxin induction.The mechanism of action of tiny RNA of the present invention: TLR4 plays a significant role in the generation of chronic pain, development and maintenance, express in chronic pain and roll up, easily can change the release of the incite inflammation factor, cause cascade reaction, cause the vicious cycle of the pain sensation, cause hyperalgesia.Therefore lower TLR4 expression of receptor and there is the effect suppressing chronic pain.RNA perturbation technique degradable mRNA thus the expression of arrestin, so, adopt this technology to suppress the expression of TLR4 just can reach the object for the treatment of pain.
A second aspect of the present invention, provides the application of tiny RNA in preparation treatment chronic pain medicine of above-mentioned interference TLR4 acceptor.
The application of tiny RNA in preparation treatment chronic pain medicine of described interference TLR4 acceptor, described medicine can be injection.
Chronic pain of the present invention, biological value that what it referred to do not have, the pain of paranormal tissue injury healing time (the usual >3 month), comprise inflammatory pain, neurogenic pain and pain caused by cancer.
The application of tiny RNA of the present invention in preparation treatment chronic pain medicine, tiny RNA direct injection can be adopted, maybe will comprise the recombinant vectors of this tiny RNA through subarachnoid space direct injection, as slow virus, adenovirus or plasmid etc., the object for the treatment of chronic pain can be reached, and route of administration is convenient, respond well.
Meanwhile, because the present invention is not traditional opioid receptor retarding agent, there is not the reaction such as habituation, tolerance, can life-time service.The present invention is that treatment chronic pain provides new treatment means.
Accompanying drawing explanation
Fig. 1 is the expression of flow cytometry display report carrier pEGFG-TLR4 Green fluorescence;
Fig. 2 is the expression of the neural microglia nmda receptor mRNA of real-time quantitative PCR detecting human;
Fig. 3 is the expression that western blotting detects the neural microglia nmda receptor albumen of people;
In figure, siRNA group refers to siRNA-TLR4 II; MM group refers to the RNA group (MismathchRNA) of mispairing; NS group refers to physiological saline group; Control group refers to blank group.
Embodiment
Describe the present invention below in conjunction with drawings and Examples, but enforcement of the present invention is not limited only to this.
Embodiment 1: the screening process of the tiny RNA sequence of interference TLR4
One, containing structure and the qualification of the pEGFPC1 reporter plasmid of people TLR4 acceptor gene
According to people TLR4 receptor sequence, design primer:
Upstream primer: 5 '-CGGAGCGTTTCAGACTCCGGAGCCTCAGCTGTT-3 ' (SEQ ID NO:7);
Downstream primer: 5 '-CGCGTCTTGCCCAGCTGGGTCCAATAAATT-3 ' (SEQ ID NO:8).
Primer two ends are respectively containing Sac I and Sal I restriction enzyme site.Adopt RT-PCR, obtain the partial sequence in the people TLR4 sequence expression cassette containing interference target sequence.
This fragment and pEGFPC1 plasmid (purchased from American Invitrogen company) are cut with Sac I and Sal I enzyme respectively, then spending the night with T4DNA ligase enzyme 16 DEG C connects into pEGFPC1-hNMDA.Transformed E .coli.DH5 α competent cell, chooses clone's extracting plasmid DNA, carries out pcr amplification and goes out the laggard pacing sequence qualification of correct band.
1, PCR identification system:
94 DEG C of 5min unwind; 94 DEG C of 50s, 55 DEG C of 50s, 72 DEG C of 90s totally 30 circulations; 72 DEG C of 10min extend.Get 5 μ l, 1% agarose gel electrophoresis qualification PCR primer.Reclaim test kit with the PCR of QIAgen company to reclaim, carry out next step Sac I enzyme and cut.
2, PCR primer Sac I enzyme is cut
After mixing, of short duration centrifugal, 37 DEG C of incubations 6 hours.1% agarose gel electrophoresis, reclaim with the Gel Extraction test kit of QIAgen company, product is dissolved in 30 μ l ddH
2in O, carry out next step Sal I enzyme and cut.
3, PCR primer Sal I enzyme is cut
After mixing, of short duration centrifugal, 37 DEG C of incubations 6 hours.1% agarose gel electrophoresis, reclaim with the Gel Extraction test kit of QIAgen company, product is dissolved in 30 μ l ddH
2in O ,-20 DEG C save backup.
4, pEGFPC1 plasmid Sac I enzyme is cut
After mixing, of short duration centrifugal, 37 DEG C of incubations 6 hours.
5,1% agarose gel electrophoresis, reclaim with the Gel Extraction test kit of QIAgen company, product is dissolved in 30 μ l ddH
2in O, carry out next step Sal I enzyme and cut.
6, reclaim product S al I enzyme to cut
After mixing, 37 DEG C of incubations 6 hours, 1% agarose gel electrophoresis, reclaims with the GelExtraction test kit of QIAgen company, and product is dissolved in 30 μ l ddH
2in O, be connected with RT-PCR product for next step.
7, ligation system
After mixing, 16 DEG C are incubated overnight.
8, recombinant plasmid transformed DH5 α bacterium
Get and connect product 5 μ l and add in E. coli .DH5 α competent cell 50 μ l prepared by Calcium Chloride Method, ice bath is after 1 hour, 42 DEG C of heat-shockeds 90 seconds, ice bath adds nonresistant LB nutrient solution 200 μ l after 4 minutes again, gentle jolting 1 hour in 37 DEG C of shaking tables, the kalamycin resistance (Kan of paving preheating
+) LB agar plate.Be inverted in the mono-clonal bacterial strain of the interior cultivation of 37 DEG C of biochemical cultivation cases after 12 hours on picking flat board to Kan
+in LB, logarithmic phase in 37 DEG C of violent joltings to bacterium, chooses clone's extracting plasmid DNA.Clone is called pEGFPC1-TLR4.
9, Hind III and the qualification of Nde I double digestion
37 DEG C of incubations 3 hours, 1% agarose gel electrophoresis.Clone's performing PCR qualification that electrophoretic band fulfills the expectation.
10, PCR identification system:
94 DEG C of 5min unwind; 94 DEG C of 50s, 55 DEG C of 50s, 72 DEG C of 90s totally 30 circulations; 72 DEG C of 10min extend.Get 5 μ l, 1% agarose gel electrophoresis qualification PCR primer.The clone that electrophoretic band fulfills the expectation serves the order-checking qualification of Hai Shenggong biotechnology company.
Two, the detection of RNA jamming effectiveness
With six orifice plate cellar culture 293 cells (purchased from Shanghai Inst. of Cytobiology, Chinese Academy of Sciences), by liposome method (see DNA transfection .J. Sha nurse Brooker work of: fat dye mediation, Huang Peitang translates. Molecular Cloning: A Laboratory guide. and the 3rd edition. Science Press publishes .2002,1276-1288.)
By three groups of siRNA-TLR4(I, II, III) and each 2 μ g cotransfection 293 cells of pEGFPC1-TLR4, and set blank group as negative control and pEGFPC1-pEGFPC1-TLR4 transfection group are positive control.Observe the intensity of green fluorescence next day.
RNA sequence is:
Ⅰ.GCAUGGAGCUGAAUUUCUA（SEQ ID NO:4）
Ⅱ.GGAUUUAUCCAGGUGUGAA（SEQ ID NO:5）
Ⅲ.CCUGAACCCUAUGAACUUU（SEQ ID NO:6）
Above-mentioned sequence entrusts the synthesis of Shanghai Polymorphism company.
1, siRNA-NMDA and pEGFPC1-TLR4 cotransfection HEK-293 cell strain
(1) 24h before transfection, by being in the cell of logarithmic phase with after the trysinization of 0.25%, is inoculated in 6 orifice plates by certain density, continues to cultivate 18-24h, when cell covers with floorage about 80%, carry out transfection.
(2) with the DMEM nutrient solution of serum-free antibiotic-free, cell is cleaned twice, the DMEM nutrient solution adding appropriate serum-free antibiotic-free is cultivated for subsequent use.
Each hole in (3) 6 orifice plates, three groups of siRNA-TLR4(I, II, III) and the consumption of pEGFPC1-TLR4 be respectively 2 μ g, positive control only adds 2 μ g pEGFPC1-TLR4 and does not add siRNA-TLR4, and blank group is negative control, neither adds.Jointly mixed with 200 μ lOpti-MEM I by siRNA-TLR4 and pEGFPC1-TLR4, be made into A liquid, room temperature places 5min.
(4) 5 μ l Lipofectamin2000 reagent are mixed at another Guan Zhongyu 200 μ l Opti-MEM I, be made into
B liquid, incubation at room temperature 5min.
(5) 200 μ l B mixed solutions are joined in 200 μ l A mixed solutions, fully mix, room temperature places 20min.
(6) serum-free medium in six orifice plates is abandoned.
(7) every for AB mixed solution hole 1ml is added in six orifice plates, culturing cell, push away before and after soft and shake culture plate to mix liquid, make it uniform fold on cellular layer.
2, fluorescent phase-contrast inverted microscope is observed
The expression of EGFP in HEK-293 cell strain is observed at flow cytometer after transfection 24,48,72h.
Result is as Fig. 1, and compared with positive control, siRNA-TLR4 intervention group fluorescent weakening, 50% positive cell number obviously reduces, the most obvious with siRNA-TLR4 II.Illustrate that siRNA-TLR4 better can disturb the TLR4 fragment in pEGFPC1-TLR4, thus affect the EGFP simultaneously transcribed with it, cause the strength reduction of green fluorescence, positive cell number reduces, thus determines that following tiny RNA is the tiny RNA of high interference efficiency of the present invention:
Ⅱ.GGAUUUAUCCAGGUGUGAA（SEQ ID NO:5）。
Embodiment 2: tiny RNA of the present invention is to the restraining effect of nmda receptor NR2B in the neural microglia of people
Adopt neural microglia (the Human Microglia of cellar culture people, BH-HP1900, purchased from Bai Wo bio tech ltd, Shanghai), be divided into four groups: siRNA group (siRNA-TLR4 II), the RNA group (Mismathch RNA, MM group) of mispairing, physiological saline group (NS group) and Control group.
Employing liposome method (see: DNA transfection .J. Sha nurse Brooker work of fat dye mediation, Huang Peitang translates. Molecular Cloning: A Laboratory guide. and the 3rd edition. Science Press publishes .2002,1276-1288.) by each for RNA and siRNA-TLR4 II of mispairing 5 μ g(20 μ l) the neural microglia of transfected with human, physiological saline group adds normal saline.Adding intracellular toxin to final concentration in 12h, siRNA-TLR4 group, the RNA group (Mismathch RNA, MM group) of mispairing and physiological saline group (NS group) nutrient solution after after transfection is 3 μ g/ml, and Control group adds the physiological saline of equivalent.0,6,12,24, after 36h, row Real time-PCR and Western blotting detects the mRNA of TLR4 acceptor and the expression of albumen respectively.
Found that, after adding intracellular toxin, other three groups compared with Control group, the mrna expression of TLR4 acceptor increases greatly, compared with MM group and NS group, the expression of the TLR4 receptor mrna of siRNA group obviously declines, and illustrates that MNDA acceptor NR2B tiny RNA can alleviate the expression (Fig. 2) of the TLR4 receptor mrna of endotaxin induction.
Western blotting the results are shown in Figure 3, after adding intracellular toxin, other three groups compared with Control group, the protein expression of TLR4 acceptor increases greatly, compared with MM group and NS group, the expression of the TLR4 acceptor of siRNA obviously declines, and illustrates that interference TLR4 tiny RNA can alleviate the expression of the nmda receptor albumen of endotaxin induction, also illustrates that tiny RNA-TLR4 that we screen has the effect of good suppression TLR4 in Humanized cell system simultaneously.
More than show and describe ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (6)
1. disturb a tiny RNA for TLR4 acceptor, its sequence is as shown in SEQ ID NO:5.
2. one kind is disturbed the application of the tiny RNA of TLR4 acceptor in preparation treatment chronic pain medicine as claimed in claim 1.
3. the application of tiny RNA in preparation treatment chronic pain medicine of interference TLR4 acceptor according to claim 2, it is characterized in that, described medicine is injection.
4. the application of tiny RNA in preparation treatment chronic pain medicine of interference TLR4 acceptor according to claim 2, is characterized in that, the injection that described medicine is the such as tiny RNA shown in SEQ ID NO:5 for main active ingredient.
5. the application of tiny RNA in preparation treatment chronic pain medicine of interference TLR4 acceptor according to claim 2, is characterized in that, the injection that described medicine is made for the recombinant vectors comprising the tiny RNA as shown in SEQ ID NO:5.
6. the application of tiny RNA in preparation treatment chronic pain medicine of interference TLR4 acceptor according to claim 5, it is characterized in that, described recombinant vectors is lentiviral vectors, adenovirus carrier or plasmid.
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| The CNS role of Toll-like receptor 4 in innate;Flobert Y. Tanga;《PNAS》;20050419;第102卷(第16期);5856–5861 * |
| 鞘内注射针对Toll 样受体4基因的siRNA 缓解坐骨神经结扎大鼠的神经病理性疼痛;吴飞翔;《第二军医大学学报》;20110430;第32卷(第4期);摘要以及第378页 * |
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