CN103656643A - Application of p38-STAT1 (Signal Transducer and Activator of Transcription 1) signal path conditioning agent to preparation of product for controlling HTRA1 (High Temperature Requirement A1) expression - Google Patents

Abstract

The invention provides an application of a p38-STAT1 (Signal Transducer and Activator of Transcription 1) signal path conditioning agent to the preparation of a product for controlling HTRA1 (High Temperature Requirement A1) expression. The p38-STAT1 signal path conditioning agent comprises an agonist and an inhibitor, wherein the agonist is an interferon-gamma and/or anisomycin and an analogue thereof, and the inhibitor is a phosphokinase p38 inhibitor or STAT1 inhibitor; the phosphokinase p38 inhibitor is SB203580 and an analogue thereof, or the STAT1 inhibitor is an MTA (Methylthioadenosine) and an analogue thereof; and a serine protease HTRA1 is related to inflammatory diseases and related diseases thereof or diseases related to the formation or migration of tumors. Therefore, the invention develops a novel HTRA1 gene expression control method to treat HTRA1 related diseases and provides a novel application of the p38-STAT1 signal path conditioning agent, and the p38-STAT1 signal path agonist can be used for inhibiting the expression of the serine protease HTRA1 so that the HTRA1 related diseases are treated.

Description

Application in the product that p38-STAT1 signal path regulator is expressed at preparation regulation and control HTRA1

Technical field

The invention belongs to biomedicine field.The present invention is specifically related to a kind of application of signal path regulator, relates in particular to the application of a kind of p38-STAT1 signal path regulator in the product of preparation regulation and control HTRA1 expression, and described product is medicine or test kit.

Background technology

Serine protease HTRA1(High temperature requirement A1) be a kind of secreted protein enzyme.The serine protease HTRA1 multiple extracellular matrix proteins such as fibronectin, aggrecan, decorin, fibromodulin of can degrading, and participate in to regulate TGF-signal beta path, so its stable state for cell adhesion, motion and extracellular matrix and metabolism has important regulating action.In addition, there are many human diseasess such as studies confirm that in a large number serine protease HTRA1 and osteoarthritis (OA), rheumatic arthritis (RA), senile degeneration of macula (AMD) and tumor formation and migration closely related.

Injection recombinant human interferon gamma is a kind of clinical application, is mainly used in the diseases such as rheumatic arthritis (RA), myelodysplastic syndrome and atoipc dermatitis and condyloma acuminatum.

Up to the present, not having does not anyly have about how regulating the report of serine protease HTRA1 gene expression the clinical treatment that any HTRA1 of take is target spot yet, more by interferon gamma, does not suppress the report that serine protease HTRA1 expresses.

Summary of the invention

Therefore, the object of the invention is the deficiency of carrying out the treatment of serine protease HTRA1 relevant disease from serine protease HTRA1 gene expression regulation for failing at present, the application of a kind of p38-STAT1 signal path regulator in the product of preparation adjusting serine protease HTRA1 expression is provided, described product is medicine or test kit, is serine protease HTRA1 treating correlative diseases drug provision important molecule target spot.

For above-mentioned purpose, technical scheme provided by the invention is as follows:

Unless specialized, " IFN-γ " herein all refers to " interferon gamma ".

Unless specialized, " HTRA1 " herein all refers to " serine protease HTRA1 ".

On the one hand, the invention provides a kind of p38-STAT1 signal path regulator in the medicine of preparation regulation and control serine protease HTRA1 expression or the application in test kit.

Preferably, described p38-STAT1 signal path regulator comprises agonist and inhibitor.

Preferably, described agonist is interferon gamma and/or anisomycin and analog thereof, for suppressing serine protease HTRA1, expresses.

Preferably, described inhibitor is SB203580 and/or MTA and analog thereof, for raising serine protease HTRA1, expresses.

Preferably, described serine protease HTRA1 and diseases associated with inflammation and relevant disease thereof or form or move relevant disease with tumor relevant.

Preferably, described diseases associated with inflammation and relevant disease thereof are selected from one or more in osteoarthritis, rheumatic arthritis and senile degeneration of macula.

Preferably, described diseases associated with inflammation is rheumatic arthritis.

Preferably, to form or move relevant disease be melanoma for described and tumor.

Again on the one hand, the invention provides a kind of p38-STAT1 signal path regulator and activating the medicine of the HTRA1 relevant disease causing or the application in test kit for the preparation for the treatment of serine protease HTRA1 and upstream passages thereof.

Preferably, described p38-STAT1 signal path regulator comprises agonist and inhibitor.

Preferably, described agonist is selected from interferon gamma and/or anisomycin and derivant thereof.

Preferably, described inhibitor is phosphokinase p38 inhibitor or STAT1 inhibitor.

Preferably, described phosphokinase p38 inhibitor is SB203580 and analog thereof, or described STAT1 inhibitor is MTA and analog thereof.

Preferably, described serine protease HTRA1 relevant disease comprises diseases associated with inflammation and relevant disease thereof or forms or move relevant disease with tumor.

Preferably, described diseases associated with inflammation and relevant disease thereof are selected from one or more in osteoarthritis, rheumatic arthritis and senile degeneration of macula.

Preferably, described diseases associated with inflammation is rheumatic arthritis.

Preferably, to form or move relevant disease be melanoma for described tumor.

Also on the one hand, the invention provides a kind of pharmaceutical composition of expressing for regulating and controlling serine protease HTRA1, the p38-STAT1 signal path regulator that described pharmaceutical composition contains effective dose and pharmaceutically acceptable carrier.

Preferably, described p38-STAT1 signal path regulator comprises agonist and inhibitor.

More preferably, described agonist is interferon gamma and/or anisomycin and analog thereof.

Preferably, described inhibitor is phosphokinase p38 inhibitor or STAT1 inhibitor.

Preferably, described phosphokinase p38 inhibitor is SB203580 and analog thereof.

Preferably, described STAT1 inhibitor is MTA and analog thereof.

Preferably,, the interferon gamma of described effective dose and the concentration of analog thereof are 0-100ng/ml.

Preferably, the concentration of interferon gamma and analog thereof is 100ng/ml;

Preferably, the concentration of the anisomycin of described effective dose is 1-10 μ M.

Preferably, the valid density of described SB203580 is 10 μ M; Or the concentration of MTA is 10 μ M.

The present invention has confirmed that by test p38-STAT1 signal path regulator can regulate and control the gene expression of HTRA1, wherein agonist can be by suppressing the gene expression of HTRA1, thereby effectively treat RA, disclose in addition p38-STAT1 signal path agonist and can be used in treatment osteoarthritis (OA), the HTRA1 relevant diseases such as senile degeneration of macula (AMD) and melanoma, widened the range of application of recombinant interferon γ, and p38-STAT1 signal pathway inhibitor can raise the gene expression of HTRA1, thereby the novel drugs target molecule that has been HTRA1 treating correlative diseases drug provision.

Accompanying drawing explanation

Below, describe by reference to the accompanying drawings embodiment of the present invention in detail, wherein:

Fig. 1 shows that interferon gamma can significantly reduce HTRA1 gene expression in l cell and macrophage, wherein, Fig. 1 a is that variable concentrations is respectively 0, 1, 10, 50, the result of the test of HTRA1 gene expression in fibroblast after the interferon gamma of 100ng/ml is processed, Fig. 1 b processes the result of the test of HTRA1 gene expression in the fibroblast after different time for take interferon gamma that concentration is 100ng/ml, Fig. 1 c is that variable concentrations is respectively 0, 1, 10, 50, the result of the test of HTRA1 gene expression in macrophage after the interferon gamma of 100ng/ml is processed, Fig. 1 d processes the result of the test of HTRA1 gene expression in the macrophage after different time for take interferon gamma that concentration is 100ng/ml, in the cell that vertical coordinate represents take not process with interferon gamma, HTRA1 mrna expression level is benchmark, HTRA1 mrna expression level in cell in interferon gamma processing procedure,

Fig. 2 shows that interferon gamma suppresses mice joint and the HTRA1 gene expression of large intestine position, vertical coordinate represents take by HTRA1 mrna expression level in the sample of any mass treatment, to be not benchmark, HTRA1 mrna expression level in sample in interferon gamma processing procedure, in figure, LPS represents lipopolysaccharide, wherein, Fig. 2 a shows that interferon gamma suppresses the HTRA1 gene expression of mice joint part, 1 processes the HTRA1 mrna expression of rear mice joint part for adding interferon gamma, 2 process the HTRA1 protein expression of rear mice joint part for adding interferon gamma, in figure, LPS represents lipopolysaccharide, GAPDH represents glyceraldehyde phosphate dehydrogenase, as internal reference,

Fig. 2 b shows that interferon gamma suppresses the HTRA1 gene expression at mice large intestine position, 1 processes the HTRA1 mrna expression at rear mice large intestine position for adding interferon gamma, 2 process the HTRA1 protein expression at rear large intestine position for adding interferon gamma, in figure, LPS represents lipopolysaccharide, GAPDH represents glyceraldehyde phosphate dehydrogenase, as internal reference;

Fig. 2 c shows that interferon gamma knock-out mice joint part HTRA1 gene expression raises, 1 processes the HTRA1 mrna expression of rear mice joint part for adding interferon gamma, 2 process the HTRA1 protein expression of rear mice joint part for adding interferon gamma, in figure, LPS represents lipopolysaccharide, GAPDH represents glyceraldehyde phosphate dehydrogenase, IFN-γ KO or KO represent the mice of IFN-γ gene knockout, and B6 represents wild type B6 mice, and contrast represents the mice (IFN-γ KO and wild type B6 mice) of not injecting LPS;

Fig. 2 d shows that the HTRA1 gene expression of interferon gamma knock-out mice large intestine position raises, 1 processes the HTRA1 mrna expression at rear mice large intestine position for adding interferon gamma, 2 process the HTRA1 protein expression at rear large intestine position for adding interferon gamma, GAPDH represents glyceraldehyde phosphate dehydrogenase, IFN-γ KO represents the mice of IFN-γ gene knockout, B6 represents wild type B6 mice, and contrast represents the mice (IFN-γ KO and wild type B6 mice) of not injecting LPS;

Fig. 3 shows that interferon gamma suppresses mice joint HTRA1 and expresses and arthritis symptom, wherein, Fig. 3 a demonstration wild type and Interferon-gamma gene knock-out mice are induced arthritic prevalence, in figure, B6 represents to induce arthritic wild type B6 mice, and IFN-γ KO represents to induce arthritic IFN-γ knock out mice;

The phenotype of mouse hind leg after Fig. 3 b demonstration wild type and Interferon-gamma gene knock-out mice induction arthritis, in figure, WT refers to wild type B6 mice, WT CIA refers to the arthritic wild type B6 mice of induction, IFN-γ KO represents IFN-γ knock out mice, and IFN-γ KO CIA refers to the arthritic IFN-γ knock out mice of induction;

The photo of hind leg joint HE dyeing before and after mice after Fig. 3 c demonstration wild type and Interferon-gamma gene knock-out mice induction arthritis, in figure, WT refers to wild type B6 mice, WT CIA refers to the arthritic wild type B6 mice of induction, IFN-γ KO represents IFN-γ knock out mice, and IFN-γ CIA refers to the arthritic IFN-γ knock out mice of induction;

Fig. 3 d shows the inhibition that wild type and Interferon-gamma gene knock-out mice induce arthritic HTRA1 to express, 1 result of expressing for HTRA1 wherein, in figure, WT refers to wild type B6 mice, WT CIA refers to the arthritic wild type B6 mice of induction, IFN-γ KO represents IFN-γ knock out mice, and IFN-γ KO CIA refers to the arthritic IFN-γ knock out mice of induction; 2 is Western blot result, in figure, contrast is not for inducing arthritic mice, CIA represents to induce meta-arthritic mice, wherein, B6 represents B6 mice, and KO represents IFN-γ knock out mice, vertical coordinate represents take that in wild type B6 mice, HTRA1 mrna expression level is benchmark, HTRA1 mrna expression level in the arthritic process small mouse of induction;

Fig. 3 e demonstration is to inducing the prevalence after arthritic wild-type mice injection of interferon γ, and in figure, CIA contrast refers to the wild type B6 mice of induction arthritis (CIA); CIA+IFN-γ refers to induction arthritis (CIA), the wild type B6 mice of while injection of interferon, CIA+LPS refers to induction arthritis (CIA), inject the wild type B6 mice of LPS simultaneously, CIA+IFN-γ+LPS refers to induction arthritis (CIA), simultaneously the wild type B6 mice of injection of interferon and LPS;

Fig. 3 f shows inducing the phenotype of the mouse hind leg after arthritic wild-type mice injection of interferon γ, in figure, Con refers to the wild type B6 mice of induction arthritis (CIA), IFN-γ refers to induction arthritis (CIA), the wild type B6 mice of injection of interferon treatment simultaneously, LPS refers to induction arthritis (CIA), the wild type B6 mice of simultaneously injecting LPS, IFN-γ+LPS refers to induction arthritis (CIA), simultaneously the wild type B6 mice of injection of interferon and LPS;

Fig. 3 g shows inducing the photo of the mice front and back hind leg joint HE dyeing after arthritic wild-type mice injection of interferon γ, in figure, Con refers to the wild type B6 mice of induction arthritis (CIA), IFN-γ refers to, the wild type B6 mice of while injection of interferon, LPS refers to induction arthritis (CIA), the wild type B6 mice of simultaneously injecting LPS, IFN-γ+LPS refers to induction arthritis (CIA), simultaneously the wild type B6 mice of injection of interferon and LPS;

Fig. 3 h shows inducing the HTRA1 in mice after arthritic wild-type mice injection of interferon γ to express, wherein 1 is the result of HTRA1 mrna expression, 2 is Western blot result, in figure, contrast refers to wild type B6 mice (not inducing arthritis), CIA refers to the wild type B6 mice of induction arthritis (CIA), CIA+IFN-γ+LPS refers to induction arthritis (CIA), the wild type B6 mice of while injection of interferon and LPS, CIA+LPS refers to induction arthritis (CIA), inject the wild type B6 mice of LPS simultaneously, CIA+IFN-γ+LPS refers to induction arthritis (CIA), the wild type B6 mice vertical coordinate of injection of interferon and LPS represents the standard that is expressed as with the HTRA1 mRNA in wild type B6 mice (not inducing arthritis) simultaneously, the expression of HTRA1 mRNA in the process small mouse of induction arthritis and injection of interferon γ, GAPDH represents glyceraldehyde phosphate dehydrogenase, as internal reference,

Fig. 4 shows that interferon gamma passes through activated protein kinase p38 STAT1 path inhibition HTRA1 and expresses, wherein, the expression of HTRA1 mRNA in the macrophage system RAW264.7 that Fig. 4 a demonstration phosphokinase p38 inhibitor (SB203580) is processed, in figure, DMSO represents that volume by volume concentration is the macrophage system RAW264.7 that 1 ‰ DMSO processes, in contrast, SB203580 represents the macrophage system RAW264.7 that SB203580 processes, IFN-γ represents the macrophage system RAW264.7 that IFN-γ processes, IFN-γ+SB203580 represents the macrophage system RAW264.7 that IFN-γ and SB203580 process, the standard that is expressed as of HTRA1 mRNA in the macrophage system RAW264.7 that vertical coordinate represents to process with DMSO, the expression of HTRA1 mRNA in the process that adds interferon gamma or SB203580 processing in RAW264.7,

Fig. 4 b represents that phosphokinase p38 inhibitor suppresses the effect in HTRA1 gene expression at interferon gamma, in figure, contrast is macrophage system RAW264.7, p38 shRNA is the RAW264.7 cell that p38 gene RNA disturbs, the 1 macrophage system RAW264.7 processing for not adding interferon gamma, the 2 macrophage system RAW264.7 that process for adding interferon gamma, 3 is the RAW264.7 cell that p38 gene RNA disturbs for not adding the p38 shRNA of interferon gamma processing, 4 is the RAW264.7 cell that p38 gene RNA disturbs for adding the p38 shRNA of interferon gamma processing, vertical coordinate represents the standard that is expressed as with the HTRA1 mRNA in macrophage system RAW264.7, the expression of HTRA1 mRNA in the process that adds interferon gamma processing in RAW264.7 or p38 shRNA,

Fig. 4 c represents the expression of HTRA1 mRNA in macrophage system RAW264.7 that anisomycin processes, in figure, DMSO represents that volume by volume concentration is the macrophage system RAW264.7 that 1 ‰ DMSO processes, in contrast, 1 μ M represents the macrophage system RAW264.7 that 1 μ M anisomycin is processed, 10 μ M represent the macrophage system RAW264.7 that 10 μ M anisomycin are processed, + SP600125 refers to the macrophage system RAW264.7 processing at 10 μ M anisomycin+SP600125, the standard that is expressed as of HTRA1 mRNA in the macrophage system RAW264.7 that vertical coordinate represents to process with DMSO, the expression of HTRA1 mRNA in the process that adds anisomycin or SP600125 processing in RAW264.7,

Fig. 4 d represents to show the expression of HTRA1 mRNA in the macrophage system RAW264.7 that STAT1 inhibitor (MTA) processes, and in figure, contrast represents the macrophage system RAW264.7 that DMSO that volume by volume concentration is 1 ‰ processes; IFN-γ represents the macrophage system RAW264.7 that IFN-γ processes; IFN-γ+MTA represents the macrophage system RAW264.7 that IFN-γ and MTA process; MTA represents the macrophage system RAW264.7 that MTA processes, and during vertical coordinate represents to contrast, HTRA1 mRNA's is expressed as benchmark, the expression of HTRA1 mRNA in macrophage system RAW264.7 in adding the process that interferon gamma or MTA process;

Fig. 4 e represents that STAT1 suppresses the effect in HTRA1 gene expression at interferon gamma, in figure, contrast is macrophage system RAW264.7, STAT1 shRNA is the RAW264.7 cell that STAT1 gene RNA disturbs, the 1 macrophage system RAW264.7 processing for not adding interferon gamma, the 2 macrophage system RAW264.7 that process for adding interferon gamma, the 3 RAW264.7 cells that disturb for not adding the STAT1 gene RNA of interferon gamma processing, the 4 RAW264.7 cells that disturb for adding the STAT1 gene RNA of interferon gamma processing, vertical coordinate represents not add the benchmark that is expressed as of HTRA1 mRNA in the macrophage system RAW264.7 that interferon gamma processes, the expression of HTRA1 mRNA in macrophage system RAW264.7 or STAT1 shRNA in the process that adds interferon gamma processing,

The result of Fig. 5 for by selected by flow cytometry apoptosis p38 RNA interference cell system and STAT1 RNA interference cell being, wherein, Fig. 5 a is the selected by flow cytometry apoptosis result of selected by flow cytometry apoptosis p38 interference cell system, Fig. 5 b is the selected by flow cytometry apoptosis result of STAT1 RNA interference cell system, in figure, positive (GFP+) cell line of GFP that M1 is selected by flow cytometry apoptosis;

Fig. 6 is the HTRA1 expression that interferon gamma reduces rheumatic arthritis patient synovial fluid cell.Wherein, Fig. 6 a represents that interferon gamma processes synovial fluid cell from 8 arthritics after 24 hours, the expression of HTRA1 mRNA, in figure, the point on the left side represents contrast, the point on the right represents the expression of the HTRA1 mRNA of the patient after interferon gamma is processed, during vertical coordinate represents to contrast, the expression of HTRA1 mRNA is benchmark, the expression of the HTRA1 mRNA of the patient after interferon gamma is processed;

The expression of HTRA1 mRNA in the synovial fluid cell that Fig. 6 b demonstration phosphokinase p38 inhibitor (SB203580) is processed, in figure, DMSO represents that volume by volume concentration is the synovial fluid cell that 1 ‰ DMSO processes, in contrast, SB203580 represents the synovial fluid cell that SB203580 processes, IFN-γ represents the synovial fluid cell that IFN-γ processes, IFN-γ+SB203580 represents the synovial fluid cell that IFN-γ and SB203580 process, the standard that is expressed as of HTRA1 mRNA in the synovial fluid cell that vertical coordinate represents to process with DMSO, the expression of HTRA1 mRNA in the process that adds interferon gamma or SB203580 processing in synovial fluid cell,

The expression of HTRA1 mRNA in the synovial fluid cell that Fig. 6 c demonstration STAT1 inhibitor (MTA) is processed, in figure, DMSO represents that volume by volume concentration is the synovial fluid cell that 1 ‰ DMSO processes, in contrast, MTA represents the synovial fluid cell that MTA processes, IFN-γ represents the synovial fluid cell that IFN-γ processes, IFN-γ+MTA represents the synovial fluid cell that IFN-γ and MTA process, the standard that is expressed as of HTRA1 mRNA in the synovial fluid cell that vertical coordinate represents to process with DMSO, the expression of HTRA1 mRNA in the process that adds interferon gamma or MTA processing in synovial fluid cell,

Fig. 7 is for the interferon gamma by evidence is by activating P 38 kinases, and then the signal path schematic diagram of transcriptional factors STAT1;

Fig. 8 is p38-STAT1 signal path figure.

The specific embodiment

Unless specialized, in following examples, mice kind used is B6 wild-type mice (purchased from Peking University's Experimental Animal Center), IFN-γ knock out mice is B6 background, purchased from U.S. JACKSON laboratory (The Jackson Laboratory, company's network address: http://www.jax.org/), IFN-γ knock out mice article No. is 002287; Mice details please refer to http://jaxmice.jax.org/strain/002287.html.

Unless specialized, in following examples, reagent used is analytical pure level reagent, and can be commercially available from regular channel.

embodiment 1 interferon gamma can reduce the expression of serine protease HTRA1 in vitro

By conventional method, obtain mice (purchased from Peking University's Experimental Animal Center, kind is B6 mice) fibroblast and macrophage, be specially conventional method and put to death experiment mice, cut mice stomach wall open, with the phosphate buffer (PBS) of pre-cooling, rinse mouse peritoneal, and collect flushing liquor, and centrifugal 5 minutes of 1700rpm, precipitation is Turnover of Mouse Peritoneal Macrophages.Under pregnant Mus aseptic condition, take out uterus by 12.5 days-14.5 days, and take out mice embryonic, deduct head and extremity, PBS washing 3 times; Mice trunk is cut into approximately 1 cubic millimeter of fritter, adds pancreatin, in incubator, hatch 15 minutes, piping and druming repeatedly, adds cell culture medium to stop digestion reaction, centrifugal 5 minutes of 1700rpm, abandon supernatant, add fresh culture, will sink to the bottom thing resuspended, and move in culture dish, in incubator, cultivate 3-7 days, PBS rinses twice, and pancreas enzyme-EDTA digests, goes down to posterity, and this cell is mouse embryo fibroblasts (MEF).

Above-mentioned two kinds of cells are moved in culture plate and cultivated; after adherent purification; working concentration gradient is respectively 0,1,10,50,100ng/ml interferon gamma (purchased from U.S. Peprotech company) processes cell 24 hours and concentration is that 100ng/ml interferon gamma is processed after different time; use respectively TRIzol test kit (purchased from American I nvitrogen company); according to the operation of test kit description, extract cell total rna (list of references: Olson J H; Xiang X; Et al.Allurin, a 21-kDa sperm chemoattractant from Xenopus egg jelly, is related to mammalian sperm-binding proteins.Proc Natl Acad Sci USA.2001 Sep25; 98 (20): 11205-10.); Then, according to the operation of test kit description, by its reverse transcription, be cDNA again, be specially the total RNA of 1 microgram that gets said extracted, add 0.5 microlitre Oligo dT(purchased from Japanese TAKARA company), 1 microlitre RNA enzyme inhibitor, AMV reverse transcriptase and AMV buffer (all purchased from Japanese TAKARA company), water mends volume to 25 microlitres.Reactant is placed in to 42 ℃ of water-baths and hatches 1 hour, gained product is cDNA.

Then according to test kit description operation, use Realtime-PCR test kit (purchased from Japanese TAKARA company), be detected as HTRA1 gene expression in fibrocyte and macrophage.The cDNA that is specially to extract is template, PCR response procedures be 95 ℃ 3 minutes; 95 ℃ 30 seconds; 58 ℃ 30 seconds; 72 ℃ 30 seconds, period is 35.(CFX96 is carried out in PCR reaction in real-time PCR, purchased from U.S. Biorad company), a part of sequence (SEQ ID NO:2) in the gene coded sequence (SEQ ID NO:1) of amplification HTRA1 mRNA, experimental result be take the phosphoribosyl transferase (HPRT, phosphoribosyltransferase) of each sample and is compared and draw as internal reference.

HTRA1 detects primer and amplified production sequence:

Forward primer (Htra1 Sense): 5 '-CAAGGATGTGGATGAAAAGGC-3 ' (SEQ IDNO:3)

Downstream primer (Htra1 Reverse): 5 '-ATGATAGCGTCTGTCTGAATGTAGTC-3 ' (SEQ ID NO:4)

Amplified production is: caagga tgtggatgaa aaggcggaca ttgcgcttat caagattgac caccagggaaagctgccagt cctgctgctc ggccgctcct cagagctgag acctggagaa tttgtagttg ccattggaagccccttttct cttcaaaaca cagtcaccac tgggatcgtc agcaccaccc agcgaggcgg caaagagctgggacttcgga actccgatat g gactacatt cagacagacg ctatcat(SEQ ID NO:2)

HPRT detects primer and amplified production sequence:

Forward primer (HPRT Sense): 5 '-AGTACAGCCCCAAAATGGTTAAG-3 ' (SEQID NO:7)

Downstream primer (HPRT Reverse): 5 '-CTTAGGCTTTGTATTTGGCTTTTC-3 ' (SEQ ID NO:8)

Amplified production is the partial sequence in the gene coded sequence (SEQ ID NO:10) of HPRT mRNA: agtacagccc caaaatggtt aaggttgcaa gcttgctggt gaaaaggacc tctcgaagtg ttggatacaggccagacttt gttggatttg aaattccaga caagtttgtt gttggatatg cccttgacta taatgagtac ttcagggatt tgaatcacgt ttgtgtcatt agtgaaactg gaaaagccaa atacaaagcc taag(SEQID NO:9)

Result as shown in Figure 1, shows by Realtime-PCR technology and confirms that interferon gamma can significantly reduce HTRA1 gene expression in mice and human fibroblasts and macrophage, and have time and dose dependent.

embodiment 2 interferon gammas can reduce the expression of serine protease HTRA1 in vivo

By conventional method to mice (wild type B6 mice, purchased from Peking University's Experimental Animal Center) and Interferon-gamma gene knock-out mice (this mice is wild type B6 mice background, purchased from U.S. JACKSON laboratory (The Jackson Laboratory, company's network address: http://www.jax.org/), IFN-γ knock out mice article No. is 002287, mice details please refer to http://jaxmice.jax.org/strain/002287.html) carry out respectively interferon gamma lumbar injection (5 μ g/ mice/skies, inject altogether 5 days), wherein LPS lumbar injection dosage is 100ng/ mice/5 day, within 5 days, perform the operation and obtain mice joint part and large intestine position afterwards, extraction organizes RNA reverse transcription to become cDNA(operational approach with embodiment 1), the cDNA extracting of take is again template, use Realtime-PCR test kit (purchased from Japanese TAKARA company), operation to specifications, HTRA1 gene expression in the sample at detection mice joint part and large intestine position.

The Mus joint part that extraction is obtained simultaneously and the tissue protein at large intestine position, adopt the immuning hybridization marking (Western-Blot) (primary antibodie is that HTRA1 specific antibody is purchased from U.S. Santa Cruz company) to detect HTRA1 protein level, concrete grammar is as follows:

By 200 microlitre pre-cooling cell pyrolysis liquid RIPA(purchased from Beijing Sai Chi biotechnology company) to being equipped with in 1.5 milliliters of centrifuge tubes of joint or Colorectal Tissues, piping and druming repeatedly, fully cell lysis, is placed in 15 minutes on ice; Centrifugal 15 minutes of 13000rpm; The protein concentration of the quantitative cell pyrolysis liquid of Beijing Sai Chi biotechnology BCA of company protein concentration detection kit for the protein sample of just collecting again, regulates the protein concentration of each sample consistent, then adds 6 * sample-loading buffer, boils 5-10 minute in boiling water bath; 2.13000rpm, 4 ℃ centrifugal 10 minutes, get supernatant; 3. every hole adds the sample (approximately 30 microgram) of equivalent, polyacrylamide gel electrophoresis (SDS-PAGE) isolated protein, and 95V voltage stabilizing electrophoresis 2-2.5 hour, then through electrotransfer, protein transduction is moved on pvdf membrane; 4. take a turn for the better pvdf membrane through 5% skim milk/TBST(TBS+0.05%Tween20) sealing (room temperature, 1 hour), add HTRA1 or internal reference GAPDH(glyceraldehyde phosphate dehydrogenase) specific antibody, 4 ℃ of gentle shaken overnight, topple over and remove after free primary antibodie, room temperature TBST washing three times, each 15 minutes, again with two anti-room temperature oscillation incubation of horseradish peroxidase (HRP) labelling 1 hour, finally again with PBST washing three times, in the chromogenic substrate of Millipore company, hatch 3 minutes, in darkroom exposure imaging.

Result as shown in Figure 2, shows that in animal body, experiment also confirms, lumbar injection recombinant interferon γ (5 μ g/ mice/sky) can significantly reduce the expression of mice large intestine and joint part HTRA1; In contrast, interferon gamma knocks out the corresponding rising of expression of the mice HTRA1 of HTRA1.

embodiment 3 interferon gammas reduce arthritis symptom

In order to confirm that interferon gamma is to the inhibiting clinical meaning of HTRA1 gene expression, we set up mouse arthritis model (Collagen-Induced Arthritis, CIA), below test in mice used be the B6 mice that wild type B6 mice and Interferon-gamma gene knock out.

The foundation of mouse arthritis model (CIA): chicken II Collagen Type VI (purchased from U.S. Sigma company) and complete Freund's adjuvant (purchased from U.S. Sigma company) is fully emulsified, making its concentration is 1mg/ml, re-use this emulsifying agent respectively at the 1st day and the 21st day, amount with 100 μ l/ mices is carried out subcutaneous injection to experiment mice, and starts to monitor mice extremities joint swelling degree at the 18th day; While injection of interferon (5 μ g/ mice/skies in inducing arthritic process with the B6 mice that wild type B6 mice and Interferon-gamma gene knock out, inject altogether 5 days) or LPS(lipopolysaccharide, it is gram negative bacteria wall composition, energy immune cell activated, injection volume is 100 μ g/ mices, only in first day injection once); At the 50th day, experiment mice is put to death, and carry out the HE staining examine (list of references: Damo Xu, Hui-Rong Jiang, et al.IL-33 Exacerbates Autoantibody-Induced Arthritis.J.Immunol.2010 of joint part; 184; 2620-2626) and carry out HTRA1 gene expression detection.HTRA1 gene expression detects real-time quantitative PCR (Realtime-PCR) (concrete operation method is with embodiment 1) and western hybridization trace (Western-Blot) technology (concrete operation method is with embodiment 2) of adopting respectively.

Result as shown in Figure 3, shows that lumbar injection recombinant interferon γ can significantly reduce the expression of mice joint part HTRA1, and then reduces arthritis symptom; In contrast, the corresponding rising of expression of the mice HTRA1 that Interferon-gamma gene knocks out, arthritis symptom is even more serious.

embodiment 4 interferon gammas suppress the gene expression of HTRA1 by p38-STAT1 path

The molecular mechanism that suppresses HTRA1 gene expression in order to study interferon gamma, we have adopted the technology such as path agonist/inhibitor and RNA interference.

Molecular mechanism for research interferon gamma inhibition HTRA1 gene expression, adopts macrophage system RAW264.7(purchased from US mode culture collection warehousing, ATCC) is object of study.Use interferon gamma (injection concentration is 100ng/ml) and p38 inhibitor SB203580(purchased from German Merk company, article No. is 559389) (injection concentration is 10 μ M) or STAT1 inhibitor MTA(be purchased from U.S. Sigma company, article No. is D5011) (injection concentration is 10 μ M), and anisomycin (injection concentration is 1 μ M, 10 μ M) process RAW264.7 cell, after 24 hours, collect cell and extract RNA, adopt real-time quantitative PCR (Realtime-PCR) test kit (purchased from Japanese TAKARA company), operation to specifications, detect HTRA1 gene expression, concrete operation method is with embodiment 1.

In order further to confirm that p38 and STAT1 suppress the effect in HTRA1 gene expression at interferon gamma, we have prepared the RNA interference cell system (p38 shRNA and STAT1shRNA) of p38 and STAT1, and concrete grammar is: by the target jamming sequence of p38 and STAT1, (STAT1 interference sequence is 5 '-GCC GAG AAC ATA CCA GAGAAT-3 ' (SEQ ID NO:5); P38 interference sequence is 5 '-GAA CTT CGC AAA TGT ATT T-3 ' (SEQ ID NO:6)) by enzyme action method, embed in retroviral vector plasmid (vector plasmid using is LentiLox 3.7, purchased from U.S. Addgene company).Then by above-mentioned retroviral vector respectively with psPAX2 and pMD2.G(all purchased from Addgene company) cotransfection to 293 cell is (purchased from US mode culture collection warehousing, ATCC) in, after 48 hours, collect culture supernatant, and join in RAW264.7 cell culture fluid with this supernatant, cultivate after 48 hours, selected by flow cytometry apoptosis GFP positive cell, be p38 RNA interference and STAT1 RNA interference cell system (result as shown in Figure 5), add again interferon gamma and process respectively p38 and STAT1 RNA interference cell system, and be in contrast with the p38 and the STAT1 RNA interference cell that do not add interferon gamma processing.

As shown in Figure 4, Fig. 4 a shows that interferon gamma can suppress the expression of HTRA1 gene to result, and at the inhibitor of SB203580(phosphokinase p38) combined effect under, inhibitory action weakens; Fig. 4 b shows that the expression that interferon gamma suppresses HTRA1 gene in p38 RNA interference cell system weakens; Fig. 4 c shows that anisomycin also can suppress the expression of HTRA1 gene, and is phosphokinase jnk inhibitor at SP600125(SP600125, purchased from American I nvivogen company, article No. tlrl-sp60) effect under, inhibitory action is strengthened; Fig. 4 d shows that interferon gamma can suppress the expression of HTRA1 gene, and at MTA(transcription factor STAT1 inhibitor, can suppress its transcriptional activity, and purchased from U.S. SIGMA company, article No. is D5011) combined effect under, inhibitory action weakens; Fig. 4 e shows that the expression that interferon gamma suppresses HTRA1 gene in STAT1 RNA interference cell system weakens.

Thereby as shown in Figure 7, result confirmation interferon gamma is combined with cell surface interferon gamma receptor, activation signals is passed to intracytoplasmic phosphokinase p38, activating P 38 kinases, and then transcriptional factors STAT1.The STAT1 of activation can enter nucleus from Cytoplasm, and is combined in HTRA1 genetic transcription promoter region, thereby suppresses HTRA1 genetic transcription.

embodiment 5 interferon gammas suppress the gene expression of HTRA1 in people's synovial fluid cell

In order to determine that whether interferon gamma has the effect of same inhibition to rheumatic arthritis patient's HTRA1 gene expression, the interferon gamma that our working concentration is 100ng/ml has been processed 8 rheumatic arthritis patients' synovium of joint liquid cell and (has been taken from rheumatism immunity section of The People's Hospital of Peking University, and provide and signed Informed Consent Form by patient), then by Realtime-PCR method, detect HTRA1 mrna expression level in cell.The interferon gamma processing time is 24 hours, RNA extract and detection method with embodiment 1, result of the test be take the phosphoribosyl transferase (HPRT, phosphoribosyltransferase) of each sample and is compared and draw as internal reference.People HTRA1 and HPRT detection primer and amplified production sequence are as follows:

People HTRA1 detects primer and amplified production sequence:

Forward primer (Htra1 Sense): 5 '-CAAGGATGTGGATGAGAAAGCAGACA-3 ' (SEQ ID NO:11)

Downstream primer (Htra1 Reverse): 5 '-ATGATGGCGTCGGTCTGGATGTAGTC-3 ' (SEQ ID NO:12)

Amplified production is the partial sequence in the gene coded sequence (SEQ ID NO:13) of HTRA1 mRNA: caaggat gtggatgaga aagcagacat cgcactcatc aaaattgacc accagggcaagctgcctgtc ctgctgcttg gccgctcctc agagctgcgg ccgggagagt tcgtggtcgc catcggaagcccgttttccc ttcaaaacac agtcaccacc gggatcgtga gcaccaccca gcgaggcggcaaagagctgg ggctccgcaa ctcagacatg gactacatcc agaccgacgc catcat(SEQ IDNO:14)

People HPRT detects primer and amplified production sequence:

Forward primer (HPRT Sense): 5 '-CAGTATAATCCAAAGATGGTCAA-3 ' (SEQ ID NO:15)

Downstream primer (HPRT Reverse): 5 '-TTAGGCTTTGTATTTTGCTTTTCC-3 ' (SEQ ID NO:16)

Amplified production is the partial sequence in the gene gene coded sequence (SEQ ID NO:17) of HPRT mRNA: cagtataatc caaagatggt caaggtcgca agcttgctgg tgaaaaggac cccacgaagt gttggatata agccagactt tgttggattt gaaattccag acaagtttgt tgtaggatat gcccttgactataatgaata cttcagggat ttgaatcatg tttgtgtcat tagtgaaact ggaaaagcaa aatacaaagcctaa(SEQ ID NO:18)

As shown in Figure 6 a, in 8 rheumatic arthritis patients' synovium of joint liquid cell, interferon gamma all can significantly reduce its HTRA1 gene expression dose to result.

In addition, the molecular mechanism that suppresses HTRA1 gene expression for studying interferon gamma in human cell, the interferon gamma that also working concentration is 100ng/ml and concentration are that the p38 inhibitor SB203580(of 10 μ M is purchased from German Merk company, article No. is 559389) or concentration be that the STAT1 inhibitor MTA(of 10 μ M is purchased from U.S. Sigma company, article No. is D5011) processing synovial fluid cell, after 24 hours, collect cell and extract RNA, adopt real-time quantitative PCR (Realtime-PCR) test kit (purchased from Japanese TAKARA company), operation to specifications, detect HTRA1 gene expression, concrete operation method is with embodiment 1, concrete design of primers is the same.

Result shows that interferon gamma has the effect of same inhibition to rheumatic arthritis patient's HTRA1 gene expression, and this inhibitory action realizes by activating P 38-STAT1 path.

Claims (13)

1. The medicine that 1.p38-STAT1 signal path regulator is expressed at preparation regulation and control serine protease HTRA1 or the application in test kit.
2. 2. application according to claim 1, is characterized in that, described p38-STAT1 signal path regulator comprises agonist and inhibitor, and preferably, described agonist is interferon gamma and/or anisomycin and analog thereof; Or preferably, described inhibitor is phosphokinase p38 inhibitor or STAT1 inhibitor and analog thereof;

   More preferably, described phosphokinase p38 inhibitor is SB203580, or described STAT1 inhibitor is MTA.
3. 3. application according to claim 1 and 2, is characterized in that, described serine protease HTRA1 and diseases associated with inflammation and relevant disease thereof or form or move relevant disease with tumor relevant.
4. 4. application according to claim 3, is characterized in that, described diseases associated with inflammation and relevant disease thereof are selected from one or more in osteoarthritis, rheumatic arthritis and senile degeneration of macula.
5. 5. application according to claim 4, is characterized in that, described diseases associated with inflammation is rheumatic arthritis.
6. 6. application according to claim 3, is characterized in that, described is melanoma with tumor formation or the relevant disease that moves forward.
7. 7.p38-STAT1 signal path agonist or inhibitor are activating the medicine of the HTRA1 relevant disease causing or the application in test kit for the preparation for the treatment of serine protease HTRA1 and upstream passages thereof.
8. 8. application according to claim 7, is characterized in that, described p38-STAT1 signal path regulator comprises agonist and inhibitor, and preferably, described regulator is selected from interferon gamma and/or anisomycin and analog thereof; Or preferably, described inhibitor is SB203580 and analog thereof.
9. 9. application according to claim 8, is characterized in that, described HTRA1 relevant disease comprises diseases associated with inflammation and relevant disease thereof or forms or move relevant disease with tumor.
10. 10. application according to claim 9, is characterized in that, described diseases associated with inflammation and relevant disease thereof are selected from one or more in osteoarthritis, rheumatic arthritis and senile degeneration of macula, and preferably, described diseases associated with inflammation is rheumatic arthritis.
11. 11. application according to claim 9, is characterized in that, described and tumor form or move relevant disease is melanoma.
12. A 12. pharmaceutical composition of expressing for regulating and controlling serine protease HTRA1, the p38-STAT1 signal path regulator that described pharmaceutical composition contains effective dose and pharmaceutically acceptable carrier, preferably, described p38-STAT1 signal path regulator comprises agonist and inhibitor, more preferably, described agonist is interferon gamma and/or anisomycin and analog thereof;

    Or preferably, described inhibitor is phosphokinase p38 inhibitor or STAT1 inhibitor; Also preferably, described phosphokinase p38 inhibitor is SB203580, or described STAT1 inhibitor is MTA.
13. 13. pharmaceutical compositions of expressing for regulating and controlling serine protease HTRA1 according to claim 12, it is characterized in that, the interferon gamma of described effective dose and the concentration of analog thereof are 0-100ng/ml, and preferably, the concentration of interferon gamma and analog thereof is 100ng/ml;

    Or preferably, the concentration of the anisomycin of described effective dose is 1-10 μ M; Or preferably, the valid density of described SB203580 is 10 μ M; Or the concentration of MTA is 10 μ M.

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