CN111020024A - Application of TLR9 - Google Patents
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- CN111020024A CN111020024A CN201911233661.8A CN201911233661A CN111020024A CN 111020024 A CN111020024 A CN 111020024A CN 201911233661 A CN201911233661 A CN 201911233661A CN 111020024 A CN111020024 A CN 111020024A
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Abstract
The invention belongs to the field of molecular biology, and relates to application of TLR 9. The TLR9 is used as a target or marker for monitoring, and/or diagnosing, and/or treating thyroid-associated eye disease, a detection reagent of TLR9 or TLR9 is used for preparing a diagnostic agent for thyroid-associated eye disease, a blocker or inhibitor of TLR9 is also used for preparing a medicament for treating thyroid-associated eye disease, and a detection reagent of TLR9 or TLR9 is also used for screening the medicament for treating thyroid-associated eye disease. The siRNA is used as a specific interfering molecule for blocking and/or inhibiting TLR9, and the siRNA, the derivative and the modifier thereof are used for preparing a medicament for preventing and/or treating thyroid-related eye disease, a kit containing the medicament and biological materials. The invention provides a potential intervention target point for thyroid-associated ophthalmopathy gene therapy and provides a theoretical basis for thyroid-associated ophthalmopathy gene therapy.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to application of TLR 9.
Background
Thyroid-associated eye disease (TAO) is an autoimmune disease associated with Grave's disease, and patients with TAO may have hyperthyroidism, hypothyroidism or normality. The volume of the orbital contents is increased due to inflammatory reaction and fibrosis of extraocular muscles and orbital connective tissues, which causes symptoms such as eyeball herniation, eyelid recession, bulbar conjunctival edema, eyeball movement disorder, double vision, optic nerve compression and the like. TAO can be divided into two types: type I is primarily characterized by retrobulbar adipose and connective tissue infiltration, and type II is primarily extraocular myositis. Both types may occur concurrently or separately.
The main methods at present comprise glucocorticoid, orbital radiotherapy and orbital decompression, the research on biological treatment of thyroid-related eye diseases is less, the research is late, and the researched target molecules are limited.
The existing research finds that the Clathrin (Clathrin) and a signal path mediated by the Clathrin may participate in inflammatory signal transduction of thyroid-associated eye diseases, and siRNA for inhibiting the heavy chain of the Clathrin (Clathrin) can slow down the proliferation of orbital fibroblasts, reduce the generation of hyaluronic acid and oxygen free radicals, and further may become potential molecules for inhibiting orbital local inflammation. Targeting Sp1 and UDP-glucose dehydrogenase (UGDH) with specific siRNAs reduced the levels of hyaluronic acid synthase-1 (HAS-1) and HAS-2, and reduced the accumulation of hyaluronic acid in orbital fibroblasts.
The research of the invention proves that the expression of the TLR9 signal path is positively correlated with the molecules of local inflammation of eye sockets, and the siRNA designed by the invention can be used for regulation, thereby providing a new gene therapy approach for thyroid-related eye diseases.
Disclosure of Invention
The siRNA provided by the invention can be used for down-regulating the expression of TLR9 in orbital fibroblasts of thyroid-associated eye diseases so as to down-regulate the expression of inflammatory factors IL-6, TNF- α and ICAM-1, so that the expression of TLR9 in the orbital fibroblasts is related to the thyroid-associated eye diseases, a new target or marker is provided for monitoring, diagnosing and treating the thyroid-associated eye diseases, the siRNA is used as a specific interference molecule for blocking a TLR9 signal channel, and a new potential molecule and/or path is provided for the intervention and/or treatment of the thyroid-associated eye diseases.
The TLR9 is used as a target or marker for monitoring, and/or diagnosing, and/or treating thyroid-associated eye disease, a detection reagent of TLR9 or TLR9 can be used for preparing a diagnostic agent for thyroid-associated eye disease, a blocking agent or an inhibitor of TLR9 can be used for preparing a medicine for treating thyroid-associated eye disease, and a detection reagent of TLR9 or TLR9 can be used for screening the medicine for treating thyroid-associated eye disease.
A diagnostic agent for thyroid-associated eye disease contains a detection reagent for TLR9 and/or TLR 9.
A medicine for treating thyroid-related eye diseases contains a blocking agent or an inhibitor of TLR 9.
A screening agent for the medicines for treating thyroid-associated ophthalmopathy contains the detection reagent of TLR9 and/or TLR 9.
The invention provides a specific interfering molecule siRNA for blocking and/or inhibiting TLR9, which is shown as follows:
(1) siRNA formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand shown in SEQ ID No. 2; or
(2) siRNA formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand which has more than 50 percent of homology with the RNA single strand shown in SEQ ID No. 2; preferably siRNA formed by complementing an RNA single strand shown in SEQ ID No.1 and an RNA single strand which has homology of more than 70% with the RNA single strand shown in SEQ ID No. 2; more preferably siRNA formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand which has more than 90% homology with the RNA single strand shown in SEQ ID No. 2; or
(3) siRNA formed by complementing the RNA single strand shown in SEQ ID No.3 and the RNA single strand shown in SEQ ID No. 4; or
(4) siRNA formed by complementing the RNA single strand shown in SEQ ID No.3 and the RNA single strand which has more than 50 percent of homology with the RNA single strand shown in SEQ ID No. 4; preferably siRNA formed by complementing an RNA single strand shown in SEQ ID No.3 and an RNA single strand which has more than 70% of homology with the RNA single strand shown in SEQ ID No. 4; more preferably siRNA formed by complementing the RNA single strand shown in SEQ ID No.3 and the RNA single strand which has more than 90% homology with the RNA single strand shown in SEQ ID No. 4; or
(5) siRNA formed by complementing the RNA single strand shown in SEQ ID No.5 and the RNA single strand shown in SEQ ID No. 6; or
(6) siRNA formed by complementing the RNA single strand shown in SEQ ID No.5 and the RNA single strand which has more than 50 percent of homology with the RNA single strand shown in SEQ ID No. 6; preferably siRNA formed by complementing an RNA single strand shown in SEQ ID No.5 and an RNA single strand which has more than 70% of homology with the RNA single strand shown in SEQ ID No. 6; more preferably, the siRNA is formed by complementing the RNA single strand shown in SEQ ID No.5 and the RNA single strand which has more than 90% homology with the RNA single strand shown in SEQ ID No. 6.
As one embodiment of the present invention, the siRNA that blocks the TLR9 signaling pathway is:
(1) siRNA formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand shown in SEQ ID No. 2; or
(2) siRNA formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand which has more than 50 percent of homology with the RNA single strand shown in SEQ ID No. 2; preferably siRNA formed by complementing an RNA single strand shown in SEQ ID No.1 and an RNA single strand which has homology of more than 70% with the RNA single strand shown in SEQ ID No. 2; more preferably, the siRNA is formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand which has more than 90% homology with the RNA single strand shown in SEQ ID No. 2.
More specifically, sirnas that block the TLR9 signaling pathway are: siRNA formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand shown in SEQ ID No. 2.
The invention also provides a DNA sequence for encoding the siRNA provided by the invention.
The invention also provides a nucleic acid construct containing the DNA sequence for encoding the siRNA provided by the invention. The nucleic acid construct is operably linked to one or more regulatory sequences to perform expression-encoding actions in a host cell including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
The invention also provides a recombinant vector, which contains the siRNA provided by the invention, or contains a DNA sequence encoding the siRNA provided by the invention, or contains a nucleic acid construct encoding the DNA sequence of the siRNA provided by the invention.
The invention also provides a recombinant cell, a nucleic acid construct containing the siRNA provided by the invention, or the DNA sequence encoding the siRNA provided by the invention, or a recombinant vector containing the siRNA provided by the invention, or the DNA sequence encoding the siRNA provided by the invention, or the nucleic acid construct containing the DNA sequence encoding the siRNA provided by the invention.
The invention also provides a chemical modifier of the siRNA, which has the same or similar action or effect as the siRNA provided by the invention.
The siRNA is used as a specific interfering molecule for blocking and/or inhibiting TLR9, and the siRNA and derivatives and modifiers thereof can be used for preparing a medicament for preventing and/or treating thyroid-related eye diseases, can also be used for preparing a kit containing the medicament, and can also be used for preparing biological materials.
The invention provides a medicine/medicine box/kit for preparing a medicine/medicine box/kit for preventing and/or treating thyroid-related eye diseases, which contains the siRNA, the derivative and the modifier thereof provided by the invention. The effective dose of siRNA in the medicine is 0-80 nM, preferably 10-60 nM, and more preferably 40 nM.
A biomaterial containing the siRNA provided by the invention and derivatives and modifiers thereof. The effective dose of siRNA in the medicine is 0-80 nM, preferably 10-60 nM, and more preferably 40 nM.
The siRNA provided by the invention can be used for down-regulating the expression of TLR9 molecules in the orbital fibroblast, down-regulating the activation degree of a TLR9 pathway of the orbital fibroblast, inhibiting the generation and development of thyroid-related eye diseases, providing a very potential intervention target point for thyroid-related eye disease gene therapy, and providing a theoretical basis for thyroid-related eye disease gene therapy. The siRNA and the derivatives and the modifiers thereof, and the medicine, the medicine box, the reagent or the kit prepared based on the siRNA and the derivatives and the modifiers thereof provide a new medical approach for treating, monitoring and preventing thyroid-related eye diseases.
Drawings
FIG. 1 shows the morphology of cells in each group, FIG. 1A. tissue blocks derived from TAO in active phase were cultured for 5 days; FIG. 1B. tissue blocks from normal control group were cultured for 7 days; FIG. 1C active-phase TAO-derived tissue blocks cultured for 9 days; FIG. 1D. tissue blocks derived from TAO in stationary phase were cultured for 10 days; FIG. 1E. tissue blocks from TAO source in stationary phase were cultured for 12 days; FIG. 1F tissue Block derived from TAO in active phase cultured for 14 days
FIG. 2 shows the results of cell identification, A.Visentin staining (+) (. times.400), B.CK staining (-) (. times.400), C.Desmin staining (-) (. times.400), and D.S-100 staining (-) (. times.400).
Figure 3 is the level of TLR9 mRNA expression from each set of orbital fibroblasts.
FIG. 4 shows the expression of various inflammatory factors after siRNA down-regulates the expression level of TLR9 of active TAO cells.
Detailed Description
Study on the inhibitory Effect of siRNA on the expression of TLR9, IL-6, TNF- α and ICAM-1mRNA in orbital fibroblasts
1. Experimental Material
(1) RFect small nucleic acid transfection reagent: in the experiment, RFect small nucleic acid transfection reagent provided by Bai Dynasty biology company is used as a transfection carrier to carry out siRNA transfection. RFect is a novel small nucleic acid transfection reagent, adopts novel animal-derived nano materials, has low toxicity and high transfection performance, can be used for transfecting micromolecule RNA and DNA with the length less than 200bp, such as microRNA, siRNA, antisense RNA and the like, and is suitable for the transfection of most adherent cells. Antibiotics are not added throughout the transfection process as they can lead to cell death.
(2) TLR 9-siRNA: the TLR9-siRNA adopted in the experiment is designed and synthesized by Gima gene biology company, and provides 3 siRNA, 1 negative control siRNA and 1 FAM fluorescence labeled siRNA in total, and the sequence is as follows:
(3) primary culture orbital fibroblast cell culture
And (4) disinfecting the super clean bench and related articles by ultraviolet rays half an hour in advance. Placing the obtained orbit fatty fiber tissue into a sterile culture dish in a super clean bench, washing the tissue block for 3 times by using PBS buffer solution, adding 3ml of DMEM culture medium into the culture dish after completely sucking the washing solution, and soaking the sample so as to prevent the tissue block from being dried and losing activity. Using an ophthalmic scissors to cut the tissue blocks into small blocks of about 1mm × 1mm × 1mm, and cutting the small blocks as much as possible to destroy the tissue connection so that the cells can climb out of the tissue blocks). Transferring the cut small blocks into a 25ml cell culture bottle by using a disposable sterile pasteur pipette, evenly spreading the small blocks on the bottom of the bottle, slightly turning the culture bottle to enable the bottom of the bottle to be upward, then adding 3ml of DMEM culture solution containing 20% FBS into the bottle, and tightly covering the bottle cap. Placing the culture flask into a container at 37 deg.C with 5% CO by volume fraction2Culturing in incubator, slightly sticking the tissue block to the wall of the culture bottle after about 8-10 hours, carefully turning the culture bottle over to make the bottom of the bottle downward, soaking the tissue block in culture solution, gently moving to prevent the tissue block from floating, continuously placing the culture bottle at 37 ℃ with 5% CO by volume fraction2Culturing in an incubator. And observing the cell climbing condition in the culture bottle under an inverted microscope every day, and replacing the culture solution according to the color of the culture solution in the culture bottle and the cell growth speed, wherein the solution is generally replaced once every 2-3 days. The cells can be observed to climb out from the periphery of the tissue block under a microscope for 3-5 days, the growth condition, the cell morphology and the like of the cells are observed every day, the record is well made, the cell fusion is basically paved at the bottom of the bottle after about 2 weeks, and at the moment, the cell passage can be carried out.
(4) Identification of primary culture orbital fibroblasts
And (5) observing the condition that the cells climb out from the edges of the tissue blocks under an inverted microscope, and observing the cell morphology and the growth condition after passage.
① transferred to third generation OFs (where cells are in logarithmic growth phase) and seeded in 6-well plates pre-mounted with coverslips at approximately 1X 105cell/hole, cross mixing, placing in 37 ℃, 5% CO2 incubator to culture;
② observing the growth condition of cells periodically, taking out 6-well plate when the cells are fully covered with the cover glass about 50-60%, washing with sterile PBS buffer solution for 5min × 3 times, removing culture solution and metabolic waste, and air drying;
③ adding 4% formaldehyde 2ml into each hole, standing for 30 min, fixing cells, washing with sterile PBS buffer solution for 5min × 3 times, air drying, adding sterile fetal calf serum dropwise, and incubating in incubator at 37 deg.C for 15 min;
④ sucking fetal calf serum with Pasteur pipette, dripping mouse anti-human Vimentin mAb, mouse anti-human CKmAb, mouse anti-human S-100mAb and mouse anti-human Desmin mAb into different wells, and incubating in refrigerator at 4 deg.C overnight;
⑤ washing with PBS buffer solution for 3 min, adding biotin-labeled goat anti-mouse IgG dropwise, and incubating at 37 deg.C for 20 min;
⑥ washing with PBS buffer solution for 3 min, adding SP compound dropwise, and incubating at 37 deg.C for 20 min;
⑦ PBS buffer solution is washed for 3 minutes for 3 times, DAB color developing agent is developed, THB is washed for 1 time, and the DAB color developing agent is immersed in substrate solution and is placed in the dark for 10 minutes;
⑧ washed with tap water for 5min, counterstained with hematoxylin, mounted, and observed by microscope photography.
2. Experimental methods
(1) siRNA transfection efficiency assay
① to select the siRNA sequence with the highest transfection efficiency, OFs derived from active-phase TAO was selected, 5X 10 per well in a six-well plate4After the cell is cultured for 24 hours, the 3 siRNAs are transfected respectively, after 12 hours, the three groups of cells are subjected to RT-PCR experiment to determine the expression condition of TLR9, and the result shows that the transfection efficiency of siRNA1 is highest, so that the siRNA1 is selected for subsequent experiments.
② to select the optimal concentration for siRNA transfection, OFs from active TAO was selected, 5X 10 per well in a six well plate4After the cell is cultured for 24h, the cell is divided into 5 groups, negative control siRNA with FAM fluorescent label and 10 mu l RFect are respectively added according to final concentration of 0nM, 20nM, 40nM, 60nM and 80nM, the cell is cultured in a incubator with 5% CO2 at 37 ℃ for 6h, and then the intensity of the fluorescence signal in each group of cells is observed under a fluorescence microscope, and the result shows thatThe 40nM group showed the strongest fluorescence signal, so all subsequent experiments were performed with siRNA at a final concentration of 40 nM.
(2) Cell preparation
Culturing cells according to the above method, collecting cells in several groups of growth periods, and diluting the cell concentration to 2 × 10 with DMEM culture solution containing 20% fetal calf serum but no double antibody4cell/ml. The siRNA is divided into four groups of 0h, 12h, 24h and 36h according to the time after the siRNA acts, each group comprises 1 six-hole plate, each six-hole plate is divided into 2 groups according to the active period TAO source OFs, the stationary period TAO source OFs and the normal control OFs, and each group comprises 2 holes which are marked. The day before transfection, cells were seeded and 2500. mu.l of the corresponding cell suspension was added to each well to give cells at 30-50% density at transfection.
(3) siRNA-RFect mixture preparation
The procedure was performed according to the formulation recommended by the protocol for RFect small nucleic acid transfection reagent (dose per well in six well plates): (1)120pmol siRNA with 250 u L DMEM medium (serum and double antibody free) dilution; (2) mu.l RFect was diluted with 250. mu.l DMEM medium (without serum and double antibody). Mixing, and incubating at room temperature for 5 min; (ensure the third step is performed within 25min, not too late) (3) after 5min incubation, mix RFect and siRNA dilutions (total volume 500. mu.l), mix gently, incubate at room temperature for 20 min. In actual preparation, according to experimental design, TLR9-siRNA transfection is carried out on three groups of 6h, 12h and 24h, and the total 18 wells need to be loaded, so the preparation is carried out according to 20 times of the volume of the above dosage (in order to ensure that the reagent dosage is enough, the amount of two wells is increased).
(4) Add siRNA-RFect mix and CpG-ODN to wells
Adding 500 mu l of siRNA-RFect mixture into each hole of three groups of six-hole plates of 12h, 24h and 36h, wherein each hole contains 3000 mu l of mixed culture solution, and adding 500 mu l of DMEM medium (without serum and double antibody) into each hole of 0h group of six-hole plates to eliminate sample adding errors; adding 6 μ l of CpG-ODN solution into each well at corresponding time (0h, 12h, 24h, 36h) to make the final concentration 200nM, and activating TLR 9; gently shaking the culture plate to uniformly distribute the reagent and the cells, putting the culture plate into an incubator at 37 ℃ and 5% CO2 for culture for 6h, taking out a corresponding six-hole plate, extracting RNA for an RT-PCR experiment, detecting the expression levels of TLR9, IL-6, ICAM-1 and IFN-gamma mRNA, and observing the correlation between the TLR9 and related inflammatory factors.
(5) Statistical method
The data in the experiment are dose data, expressed as X +/-s, SPSS19.0 software is adopted to perform one-factor variance analysis on each group, Dunnett is used to perform difference analysis between two groups of data, and P <0.05 is used as the difference, so that the statistical significance is achieved.
3. Results of the experiment
(1) Culture and identification of primary orbital fibroblasts
Fat connective tissues are obtained from orbital surgeries of active-phase TAO patients, stationary-phase TAO patients and patients with benign orbital tumors with autoimmune diseases, and are separated and purified by a tissue mass culture method. OFs was isolated from each group, and OFs was more easily grown in vitro, with no apparent difference in cell morphology between groups.
After 3-5 days of inoculation, a small amount of cells climb out from the periphery of the tissue block, are in a slender spindle shape, and are connected into a net shape (fig. 1A and 1B). After 7-9 days, the cells swim out in a typical fusiform shape, and the cell bodies are full (fig. 1C and 1D). Around 2 weeks the cell fusions spread substantially to the bottom of the flask and partially overlapped, at which time the first passage (1E, 1F) was allowed. After passage, the cells grow faster, passage can be performed again after about 1 week, and relatively pure cells with stable biological characters can be obtained after repeated liquid change and passage.
And when each group of cells is transmitted to the third generation, taking partial cells to perform six-hole plate plating, and performing immunohistochemical identification when the cells are fully plated with a cover glass sheet about 50-60%. The results showed that each group of cells stained positive for Vimentin, and the positive brown reaction products were evenly distributed in the cytoplasm (fig. 2A); on the other hand, the staining of CK (FIG. 2B), Desmin (FIG. 2C), S-100 (FIG. 2D) was negative, which confirmed that the cells were mesoderm-derived cells and were identified as fibroblasts.
(2) Real-time fluorescent quantitative PCR (Real-time PCR) for determining TLR9 mRNA expression level of each group of orbital fibroblasts
Inoculating an active-phase TAO group, a stable-phase TAO group and a normal control group OFs in a logarithmic growth phase into a six-hole plate, culturing for 2 days at 37 ℃ in a 5% CO2 incubator, collecting cells, extracting total RNA, performing reverse transcription to form cDNA, performing RT-PCR experiments, repeating for 3 times in each group, obtaining TLR9 and GAPDH amplification curves and Ct values, and calculating to obtain the relative expression quantity of TLR9 mRNA. The results show that: the expression level of OFs TLR9 mRNA in the active period TAO group is obviously higher than that in the normal control group, the difference has statistical significance, and P is less than 0.001; the expression level of OFs TLR9 mRNA in the TAO group in the stationary phase is reduced compared with that in the normal control group, the difference has statistical significance, and P is less than 0.01. See table 1 for details.
TABLE 1 relative expression levels of different groups of OFs TLR9 mRNA
P <0.05, the mean difference in CT values of the three groups was statistically significant, indicating that the overall mean of the three groups was not all the same. Next, the normal control group was used as a control, and two-by-two comparison was performed using Dunnett t.
Note: taking a normal control group as a reference, the expression level of TLR9 mRNA in the TAO group in the active period is obviously higher than that in the normal control group, the difference has statistical significance, and P is less than 0.001; the expression level of TLR9 mRNA in the TAO group in the stationary phase is reduced compared with that in the normal control group, the difference has statistical significance, and P is less than 0.01.
(5) Effect of TLR9-siRNA on the expression levels of IL-6, ICAM-1 and IFN-. gamma.mRNA in orbital fibroblasts
The siRNA was transfected into active TAO group OFs as experimental group, and negative control siRNA was transfected into active TAO group OFs as control group, while stimulation with CpG-ODN was performed. Compared with the control group, the active-period TAO group transfected with siRNA has the advantage that the expression level of TLR9 mRNA is lowest at 24h, the expression levels of IL-6, ICAM-1 and IFN-gamma mRNA show the same change trend and are positively correlated with the expression level of TLR 9.
The expression level of TLR9 mRNA is reduced to the minimum when siRNA interferes for 24h, the difference has statistical significance, and P is less than 0.05; the expression levels of IL-6 and IFN-gamma mRNA are also reduced to the minimum level at 24h after the interference, the expression level of ICAM-1mRNA is in a continuous descending trend within 36h, the difference between each time point and 0h has statistical significance, and P is less than 0.05.
4. Conclusion of the experiment
The TLR9 specific interfering molecule siRNA designed in the research can obviously reduce the expression of TLR9 mRNA and the expression of products IL-6, ICAM-1 and IFN-gamma mRNA activated by a TLR9 channel, thereby proving the effectiveness of the TLR9 specific interfering molecule siRNA in the project and being expected to become an effective potential molecule for intervening and treating thyroid-related eye diseases.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention and not to limit it; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art will understand that: modifications to the specific embodiments of the invention or equivalent substitutions for parts of the technical features may be made; without departing from the spirit of the present invention, it is intended to cover all aspects of the invention as defined by the appended claims.
Claims (16)
- TLR9 is useful as a target or marker for monitoring, and/or diagnosing, and/or treating thyroid-related ocular disease.
- Use of a detection reagent for TLR9 and/or TLR9 in the preparation of a diagnostic agent for thyroid-associated eye disease.
- 3. A diagnostic agent for thyroid-associated eye disease, which comprises a detection reagent for TLR9 and/or TLR 9.
- A detection reagent for TLR9 and/or TLR9 is used for screening a medicine for treating thyroid-related eye diseases.
- 5. A screening agent for a drug for treating thyroid-associated eye disease, which is characterized by comprising a detection reagent for TLR9 and/or TLR 9.
- A blocker or inhibitor of TLR9 for use in the manufacture of a medicament for the treatment of thyroid-related eye disease.
- 7. A medicament for the treatment of thyroid-related eye disease comprising a blocking or inhibiting agent for TLR 9.
- 8. An siRNA that blocks and/or inhibits TLR9, as shown below:(1) siRNA formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand shown in SEQ ID No. 2; or(2) siRNA formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand which has more than 50 percent of homology with the RNA single strand shown in SEQ ID No. 2; preferably siRNA formed by complementing an RNA single strand shown in SEQ ID No.1 and an RNA single strand which has homology of more than 70% with the RNA single strand shown in SEQ ID No. 2; more preferably siRNA formed by complementing the RNA single strand shown in SEQ ID No.1 and the RNA single strand which has more than 90% homology with the RNA single strand shown in SEQ ID No. 2; or(3) siRNA formed by complementing the RNA single strand shown in SEQ ID No.3 and the RNA single strand shown in SEQ ID No. 4; or(4) siRNA formed by complementing the RNA single strand shown in SEQ ID No.3 and the RNA single strand which has more than 50 percent of homology with the RNA single strand shown in SEQ ID No. 4; preferably siRNA formed by complementing an RNA single strand shown in SEQ ID No.3 and an RNA single strand which has more than 70% of homology with the RNA single strand shown in SEQ ID No. 4; more preferably siRNA formed by complementing the RNA single strand shown in SEQ ID No.3 and the RNA single strand which has more than 90% homology with the RNA single strand shown in SEQ ID No. 4; or(5) siRNA formed by complementing the RNA single strand shown in SEQ ID No.5 and the RNA single strand shown in SEQ ID No. 6; or(6) siRNA formed by complementing the RNA single strand shown in SEQ ID No.5 and the RNA single strand which has more than 50 percent of homology with the RNA single strand shown in SEQ ID No. 6; preferably siRNA formed by complementing an RNA single strand shown in SEQ ID No.5 and an RNA single strand which has more than 70% of homology with the RNA single strand shown in SEQ ID No. 6; more preferably, the siRNA is formed by complementing the RNA single strand shown in SEQ ID No.5 and the RNA single strand which has more than 90% homology with the RNA single strand shown in SEQ ID No. 6.
- 9. A DNA sequence encoding the siRNA of claim 8.
- 10. A nucleic acid construct comprising the DNA sequence of claim 9.
- 11. A recombinant vector comprising the siRNA of claim 8, or comprising the DNA sequence of claim 9, or comprising the nucleic acid construct of claim 10.
- 12. A recombinant cell comprising the siRNA of claim 8, or comprising the DNA sequence of claim 9, or comprising the nucleic acid construct of claim 10, or comprising the recombinant vector of claim 11.
- 13. A chemical modification of the siRNA of claim 8.
- 14. The use of an siRNA according to claim 8, and/or a DNA sequence according to claim 9, and/or a nucleic acid construct according to claim 10, and/or a recombinant vector according to claim 11, and/or a recombinant cell according to claim 12 for the manufacture of a medicament/kit for the prevention and/or treatment of thyroid-related eye diseases, and/or for the manufacture of a biological material.
- 15. A medicament/kit for preparing a medicament/kit for preventing and/or treating thyroid-related eye disease, comprising the siRNA of claim 8, and/or the DNA sequence of claim 9, and/or the nucleic acid construct of claim 10, and/or the recombinant vector of claim 11, and/or the recombinant cell of claim 12.
- 16. A biological material comprising siRNA according to claim 8, and/or DNA sequence according to claim 9, and/or nucleic acid construct according to claim 10, and/or recombinant vector according to claim 11, and/or recombinant cell according to claim 12.
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