CN107982278A - A kind of medicine and application for being used to treat angiogenesis-mediated disease - Google Patents
A kind of medicine and application for being used to treat angiogenesis-mediated disease Download PDFInfo
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Abstract
The invention belongs to medicine and pharmacology technical field, discloses a kind of medicine and application for being used to treat angiogenesis-mediated disease, there is provided a kind of medicine for being used to treat angiogenesis-mediated disease prepared using arsenic trioxide；The method for the curative effect of medication that assessment is used to treat angiogenesis-mediated disease includes：Assess inhibitory action of the arsenic trioxide to 1 TGF β CTGF VEGF vascular proliferation function modules of arthropathy synovial cell TSP；Arsenic trioxide is assessed by suppressing 1 TGF β CTGF VEGF vascular proliferations function modules of TSP and then the inhibitory action to proliferation of microvascular endothelial cells in lesion joint.The arsenic trioxide of the present invention can be by suppressing 1 TGF β of TSP, 1 CTGF VEGF function modules, and then suppresses vascular proliferation, plays the purpose for the treatment of vascular proliferative diseases.
Description
Technical field
The invention belongs to medicine and pharmacology technical field, more particularly to a kind of medicine for being used to treat angiogenesis-mediated disease and
Using.
Background technology
Blood vessel forms extensive network in human body, and oxygen and nutrition, abnormal blood are provided for each position of body
Pipe is grown and function is the mark of cancer and diseases associated with inflammation, result in the progress of disease.Vascular proliferation associated joint disease
Middle local patholoic change depends on neovascularization to ensure sustainable supply sick cell and inflammatory cell oxygen and nutrition.Physiological conditions
Under, synovium of joint backing layer is only made of 1-2 confluent monolayer cells.Under the pathologic condition of the arthropathies such as RA, the inflammatory such as TNF-α and IL-1
Cell factor promotes synovial cell's quantity showed increased, and usual cell can reach 4-10 layers.It is thin into fiber-like synovial membrane in synovial membrane
Main proliferative cell of the born of the same parents (fibroblast-like synoviocytes, FLS) as synovial membrane, can in merokinesis, and with
The monocyte and new vessels of derived from peripheral blood further promote the infiltration of inflammatory cell, can also further induce synovial membrane
Cell Proliferation, and the synovial tissue for propagation provides nutriment, shows the biological behaviour of local tumor sample, attacks and break
Bad articular cartilage, subchondral bone, tendon and ligament, ultimately cause joint deformity and deformity.RA synovial tissues are cut into slices and used interior
Chrotoplast specific antibody dyes, it is possible to find synovial membrane lower floor has abundant capillary network, and small quiet after visible capillary
Arteries and veins, angiogenesis are the joint diseases such as RA one tissue pathologies changes of early stage, are the important morbidity links of such disease, suppress
New vessels forms a kind of brand-new treatment method for being likely to become the vascular proliferation associated joint disease such as RA.By acting on blood
The different links of pipe generation, including the suppression, the connection of angiogenic factors that are produced to growth factors such as VEGF (use antibody
Or solvable receptor), block the transduction of downstream signal, block the degraded etc. of matrix to be all likely to become the potential target for the treatment of RA.
The major target of current anti-angiogenic agent is a kind of albumen for being referred to as vascular endothelial growth factor (VEGF), it
Vital effect is played in angiogenesis.Although for many years it is known that VEGF inhibitor and cytotoxic drug combinations
Treatment, but there are the clinic that the adverse reaction of cardiovascular and cerebrovascular and its topical type of curative effect limit VEGF inhibitor for VEGF inhibitor
Using.In addition, the preclinical and data of clinical analysis, which disclose anti-angiogenic therapy, can produce tolerance.
Nearly ten years, TNF-α antagonist has become one of most strong weapon for the treatment of rheumatic disease, at present Clinical practice
TNF-α antagonist mainly have infliximab, Etanercept and adalimumab, they can alleviate and prevent facing for RA
Bed and Advances in Imaging, significantly mitigate the symptom of RA patient, improve function and improve the quality of living.However, TNF-α antagonist
Also there is its limitation.TNF-α antagonist can cause potential tuberculosis infection or cause potential tuberculosis resume combustion, Britain's rheumatism
One of disease association analysis shows that, the incidence using tuberculosis after adalimumab is about 144/10000 (people times/year), Ying Fu
Sharp former times monoclonal antibody is about 136/10000 (people times/year), and Etanercept is about 39/10000 (people times/year), is controlled using tnf inhibitor
The appearance of the autoantibody including anti-ds-DNA antibody is can also result in after treatment, for the hepatitis B or third using TNF antagonists
Hepatopath, also needs to keep a close eye on the change of the transaminase, Viral Quantification of patient.In addition, the heterogeneity of RA patient causes many trouble
Person reacts bad to biological agent treatment method, some patients are almost difficult to touch this treatment because of reasons such as economy, regions
Or the diseases such as tumour are suffered from advance and are excluded from outside medication.Thus, still demand is used to treat rheumatoid arthritis etc.
The improved method or preparation of continuation vascular proliferative diseases.According to disclosure, these demands can be by applying three oxidations two
Arsenic is realized.
As at present2O3Treatment to disease is confined to disease in the blood system and such as pernicious reality of stomach cancer, the cancer of the esophagus, liver cancer more
The treatment of body knurl, its curative mechanism focus on more：
Induce pathogenic cell apoptosis：1. activate mitogen-activated protein kinases signal path；2. suppress Telomerase
The expression of activity and telomerase reverse transcriptase gene；3. reduce mitochondrial transmembrane potentials；4. lower the expression of proto-oncogene：Lower
Oncogene expresses such as Bcl-2, Bcl-XL, or up-regulation expression of tumor suppressor gene such as Bax, P53, Bcl-xs etc., also adjusts thin
Born of the same parents' cycle relies on the expression of gene, causes cell metabolism abnormal, ability of cell proliferation is weakened and apoptosis or differentiation.
Induce tumor cell differentiation：Mainly expressed by reducing c myc etc., and up-regulation cell differentiation antigen
CD11b is expressed, so as to promote leukaemia's maturation, differentiation.
Suppress tumor cell proliferation：As2O3Make cell-cycle arrest in a dose-dependent manner.Cell cycle, there are the G1/S phases
Point is limited with G2/M phases transferase 12, the cell in the G2/M phases is easy to apoptosis.As2O3By suppressing cell cycle related proteins,
CDK6 is such as raised, lowers the expression of CDK1 and cdc-2, and dramatically increases the combination of P21 and cdc-2, CDK4 etc., causes cell
Cycle Arrest finally prevents cell from into mitosis telophase and apoptosis in G1 the and G2/M phases.
Coup injury DNA：As2O3CHOK1 cell chromosomes deformity and sister chromosome monomer can be caused to exchange, and DNA
Fracture and micronucleus produce, and suppress DNA and repair.
Suppress Nasopharyngeal neoplasms：CD44 gene encoding productions are a kind of transmembrane glycoproteins, are one of cell adhesion molecules,
Often appear in the tumour shifted, it is believed that it is a kind of marker of tumour diffusion transfer.Researches show that As2O3It can show
Writing reduces the expression of stomach cancer cell Adhesion molecule CD44 gene coded protein.
As2O3Influence to tumor mice immune function：The reduction of immune function is the reason for causing tumour growth, development
One of.With immunocyte that is antitumor, antiviral, playing immunologic surveillance function in body.Research shows As2O3Liver can be improved
Cancer mouse body's immunity, suppresses the growth of tumour.As2O3This effect may show with it lethality of immunocyte
It is related to write enhancing.
Anti-angiogenesis：The major target of current anti-angiogenic agent is a kind of referred to as vascular endothelial growth factor
(VEGF) albumen, it plays vital effect in angiogenesis.Although for many years it is known that VEGF inhibitor with
Cytotoxic drug combinations are treated, but there are the adverse reaction of cardiovascular and cerebrovascular and its limitation of curative effect to limit for VEGF inhibitor
The clinical practice of VEGF inhibitor.In addition, the preclinical and data of clinical analysis disclose VEGF inhibitor anti-angiogenic therapy
Tolerance can be produced.
In short, although experimental results demonstrate As2O3Anti- blood disease and the mechanism of entity tumor are multipath, Mutiple Targets, but
It is still few by the research of anti-angiogenesis approach performance therapeutic effect, and is limited only to lower vascular endothelial growth factor
(VEGF) single-factor expression and function aspect.This patent basic research is with regard to As2O3By suppressing TSP-1-TGF- β-CTGF-
The effect of VEGF vascular proliferations function module and then performance treatment vascular proliferative diseases and molecular mechanism have been carried out compared with system
Demonstration, absolutely proves As2O3Anti-angiogenic proliferation function can also be extensively using a variety of vascular proliferative diseases such as rheumatoid arthritis
Treatment, is As2O3Using broader indication is provided, a kind of new medicine also is provided for vascular proliferative diseases,
At the same time a kind of new instrument is provided for Drug combination or multipath, Mutiple Targets targeted therapy.According to disclosure, these
Demand can be realized by applying arsenic trioxide.Vascular proliferation be rheumatoid arthritis (RA) early stage tissue pathologies change it
One, it is the necessary condition of pannus generation, but its mechanism is still not clear；Present analysis shows, thrombospondin-1 (TSP-
1), transforming growth factor-beta 1 (TGF-β 1), Connective Tissue Growth Factor (CTGF), vascular endothelial growth factor (VEGF) these
Angiogenesis factor expresses increase in RA patient synovial tissue, and may interact, to the vascular proliferation production of RA synovial tissues
Raw material impact.Although the treatment of biological agent anti-tnf-alpha is effective to RA, allow people's worry to be that this treatment method occurs more
Kind adverse reaction, or even the incidence probability of increase malignant tumour；Arsenic trioxide (As2O3) it has been used to the blood vessels such as treatment tumour
Proliferative diseases, cause global extensive concern；However, As2O3Effect and molecule machine to RA patient's synovial membrane angiogenesis
Make unclear.
During arsenic trioxide is cured the disease, anti-angiogenic propagation is played by suppressing TSP-1-TGF- β-CTGF-VEGF function modules
The specific associated proteins or action target spot of effect are still not clear, and need to be studied；Its difficulty is that human body is one complicated
System, there is thousands of protein molecular, therefore, search out the specific target protein molecule of arsenic trioxide, it is necessary to
Time and further a large amount of inputs of energy；The effect of arsenic trioxide and other treatment drug combination, adverse reaction, optimal agent
Amount is still not clear, and needs further to be studied.
In conclusion problem existing in the prior art is：TNF-α antagonist also has its limitation.TNF-α antagonist can
Cause potential tuberculosis infection or cause potential tuberculosis resume combustion.
The content of the invention
In view of the problems of the existing technology, the present invention provides a kind of medicine for being used to treat angiogenesis-mediated disease
And application.The present invention is arsenic trioxide clinical practice, there is provided more solid foundation.
The present invention be achieved in that it is a kind of using arsenic trioxide prepare be used for treat angiogenesis-mediated disease
Medicine；The medicine components for being used to treat angiogenesis-mediated disease prepared using arsenic trioxide are by mass by three oxygen
Change two arsenic 5mg and sodium chloride 50mg to form.
Another object of the present invention is to provide a kind of preparation method for the medicine for being used to treat angiogenesis-mediated disease.
The preparation method includes：
Using As (OR)3Pure As is made in hydrolysis2O3；Wherein, R CH3,C2H5；
AsCl3With methanol or ethanol synthesis generation As (OR)3, filtered-distillation-rectifying-As (OR)3Hydrolysis-filtering-dry
It is dry, obtain pure As2O3；
By As2O35ml is made with sodium chloride solution in powder, wherein, arsenic trioxide 5mg, sodium chloride 50mg.
Further, As (OR)3In hydrolysis, As in mass ratio (OR)3:H2O=1:1.6-3；The temperature of hydrolysis is 293 scholar 5K；
In drying steps, including：In vacuum drying chamber, to the As after filtering2O3It is heat-treated；Firing rate is 1.5
℃/min；Constant weight is heated under isothermal conditions；
AsCl3As (the OCH generated with methanol or ethanol synthesis3)3Or As (OC2H5)3In 343K, 363K, 373K, 413K temperature
Drying time is respectively 43min, 19min, 14min, 3min and 116min, 55min, 17min, 4min under the conditions of degree.
As(OR)3Yield actually not with As (OR)3The change of middle alkyl and change, and perseverance is 98-99%；Work as As
(OR)3:H2O=1:During 1.6-3, reach As2O3Maximum output 99%；Work as ratio<1:When 1.6, yield drastically declines, and water increases
Add；When increasing the temperature, As2O3Yield reduces not very big；Pass through As (OR)3The As for hydrolyzing and obtaining2O3Powder, water containing 6-8%
Point and trace amounts of alcohol.In order to remove these moisture and trace amounts of alcohol, in vacuum drying chamber, to As2O3It is heat-treated.Obtained spy
Pure As2O3For the powder of high dispersive, the average diameter of particle is that 13.13 and 10.64 μ (are CH3, C corresponding to R2H5).It is obtained
Pure As2O3Roentgen's facies analysis of powder points out that they are all cubic crystal.
Another object of the present invention is to provide a kind of curative effect of medication assessed for treating angiogenesis-mediated disease
Method includes：
Arsenic trioxide is assessed to arthropathy synovial cell's TSP-1-TGF- β-CTGF-VEGF vascular proliferation function modules
Inhibitory action；
Arsenic trioxide is assessed by suppressing TSP-1-TGF- β-CTGF-VEGF vascular proliferation function modules and then to lesion
The inhibitory action of proliferation of microvascular endothelial cells in joint.
Further, the method for the curative effect of medication that the assessment is used to treat angiogenesis-mediated disease specifically includes：
Establish the thin into fiber-like synovial membrane of the fibroblast-like synoviocyte NH-FLS and RA synovial tissues of normal synovial tissue
Born of the same parents RA-FLS and microvascular dermal endothelial cell HDMECs co-culture systems；
FLS TSP-1, TGF-β 1, CTGF and VEGF in co-culture system are measured using real time fluorescence quantifying PCR method
MRNA expressions；
TSP-1, TGF-β 1, CTGF and vegf protein expression in ELISA method analysis co-culture system supernatant；So
Fresh supernatant is added into HDMECs progress Transwell experiments afterwards and is tested into pipe.
Further, the method for the curative effect of medication that the assessment is used to treat angiogenesis-mediated disease further includes：
After stimulating or disturbing respectively RA-FLS TSP-1, TGF-β 1, CTGF expression, RA-FLS and its co-culture system are detected
TSP-1, TGF-β 1, CTGF, VEGF gene and protein expression situation in culture supernatant, so as to assess vascular proliferation factor TSP-
1st, TGF-β 1, the interaction relationship between CTGF, VEGF.
Advantages of the present invention and good effect are：
The method that one aspect of the present invention provides the joint disease for treating vascular proliferation mediation, it is included to the food in one's mouth
Newborn animal applies the arsenic trioxide (As of sufficient amount in the treatment2O3), the joint disease of mammal can be made using this compound
Lesion be clinically significantly improved.In the present invention, disease symptoms be clinically significantly improved including following one or
Multinomial improvement:A) mitigation or inhibition of pain；B) mitigate or suppress swelling；C) mitigate or suppress rubescent；D) reduce or suppress by shadow
Ring the temperature of tissue；And e) reduce or suppress deformity and loss of functionality.
The present invention can carry out following deductions but be not rely on the deduction, i.e. blood vessel forms extensive net in human body
Network, provides oxygen and nutrition, abnormal angiogenic growth and function are cancer and diseases associated with inflammation for each position of body
Mark, result in the progress of disease.In the past ten years, the understanding to angiogenesis correlation molecule mechanism is with volatile
Speed increase, part anti-angiogenic medicaments have obtained the treatment approval of cancer and eye illness.Angiogenesis are given birth to including blood vessel
The degraded of release, extracellular matrix (extracellular matrix, ECM) into the factor, endothelial cell migration, propagation, pipe
The multi-step complex process such as formation of cavity configuration.Under the pathology environment such as rheumatoid arthritis, synovial cell can secrete a large amount of thin
Intracellular cytokine, which part can influence vascular endothelial cell.TSP-1, TGF-β, CTGF, VEGF as important vascular proliferation because
Son, the high expression in RA patient synovial tissue, and there are correlation.TGF-β 1 can promote TSP-1 in RA-FLS to express, TSP-
1 can raise the vascular plasminogen activator product to play a significant role in arthritis by 1 path of TGF-β.Arthritic animals
Model gives TSP-1 derived peptide antagonism TSP-1, then can obviously relieve arthritis, and reduces synovial tissue CTGF expression.CTGF
By raising miR-210 expression, promote expression and the angiogenesis of people's synovioblast VEGF, block CTGF substantially to press down
Vegf expression processed.CTGF monoclonal antibodies block CTGF, can improve the progress of the CIA model mouse state of an illness.Using I type of TGF-β by
Body blocking agent blocks TGF-β, and good treatment prospect is also shown in CIA models.In conclusion TSP-1, TGF-β 1,
There is complicated interaction in CTGF, VEGF, formed function module, common modulating vascular propagation between multisystem.But in class
In rheumathritis, the interaction relationship between above-mentioned four kinds of protein moleculars and its influence to new vessels formation.Because
Under pathological state, above-mentioned angiogenesis factor can be expressed and secreted to articular synovial cells, and the above-mentioned factor can influence blood vessel endothelium again
Cell, has an important influence on rheumatoid arthritis vascular proliferation.Arsenic trioxide can be by suppressing TSP-1-TGF- β 1-
CTGF-VEGF function modules, and then suppress vascular proliferation, play the purpose for the treatment of vascular proliferative diseases.
The present invention relates to the new application of arsenic trioxide, particularly in blood vessels such as the rheumatoid arthritis of angiogenesis-mediated
New application in proliferative diseases treatment.The present invention has investigated arsenic trioxide on human arthropathy synovial cell TSP-1-TGF-
The inhibitory action of β-CTGF-VEGF vascular proliferation function modules, has also further investigated arsenic trioxide by suppressing above-mentioned work(
Can module and then to the suppression function of proliferation of microvascular endothelial cells in lesion joint, show that arsenic trioxide is expected to become blood vessel
Generate the active drug of the disease treatment of mediation.
The present invention provides a kind of new treatment method for the treatment of vascular proliferative diseases, and is provided for drug combination
A kind of new medicine.Its direct technology is not only in that effective reduction of patient pain, also resides in its obvious economic benefit.This experiment
Arsenic trioxide effective dose in zoopery mouse is 1mg/kg/day, equivalent to adult's human body 5mg/day, three oxidations two
Arsenic is cheap, and close to 70 yuan/5mg/ only, by taking rheumatoid arthritis as an example, generally the least expensive TNF-α antagonist is commodity price
230 yuan/day, if using arsenic trioxide in treatment, price is nearly the 30% of generally the least expensive TNF-α inhibitor, is subtracted for patient
Few financial burden nearly 70%.It is if more obvious applied to the treatment of other vascular proliferative diseases such as tumour.In addition, this
Patent drug arsenic trioxide can also heighten the effect of a treatment with TNF-α inhibitor drug combination, reduce the adverse reaction of combination medicine,
It is " cooperation ".At the same time, treatment tumour is " killing two birds with one stone " in itself to the outer lesion of arsenic trioxide in treatment tumour.In addition,
Conditions of patients improves, and study, work, viability enhancing, quality of life improve, and also brings huge economic society effect indirectly
Benefit.
Brief description of the drawings
Fig. 1 is As provided in an embodiment of the present invention2O3To RA-FLS TSP-1-TGF- β 1-CTGF-VEGF function module tables
Reach and its promote the influence figure of vascular proliferation function.
Fig. 2 is As provided in an embodiment of the present invention2O3May be by suppressing 1-CTGF-VEGF function modules of TSP-1-TGF- β
To play the vascular proliferation effect of anti-RA synovial tissues, so as to achieve the purpose that to treat RA figures.
Fig. 3 is the preparation method flow of the medicine provided in an embodiment of the present invention for being used to treat angiogenesis-mediated disease
Figure.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
The present invention includes TSP- by analyzing the fibroblast-like synoviocyte medium vessels generation factor of RA patient synovial tissue
1st, the regulated and control network of TGF-β 1, CTGF and VEGF compositions, specifies the pathogenesis of RA patient's synovial membrane medium vessels propagation.Analyze As2O3
Effect and molecular mechanism to RA patient's synovial membrane angiogenesis, explore the new method of RA treatments.
The embodiment of the present invention provides a kind of medicine for being used to treat angiogenesis-mediated disease prepared using arsenic trioxide
Thing；The medicine components for being used to treat angiogenesis-mediated disease prepared using arsenic trioxide are by mass by three oxidations
Two arsenic 5mg and sodium chloride 50mg is formed.
As shown in figure 3, the preparation method of the medicine provided in an embodiment of the present invention for being used to treat angiogenesis-mediated disease,
The preparation method includes：
S101:Using As (OR)3Pure As is made in hydrolysis2O3；Wherein, R CH3,C2H5；
S102：AsCl3Generation As (OR) is reacted with alcohol3, filtered-distillation-rectifying-As (OR)3Hydrolysis-filtering-dry
To pure As2O3；
S103：Finally by pure As2O35ml is made with sodium chloride solution in powder：Arsenic trioxide 5mg and sodium chloride 50mg ratios
Example product.
As(OR)3Yield actually not with As (OR)3The change of middle alkyl and change, and perseverance is 98-99%；Work as As
(OR)3:H2O=1:During 1.6-3, reach As2O3Maximum output 99%；Work as ratio<1:When 1.6, yield drastically declines, and water increases
Add；When increasing the temperature, As2O3Yield reduces not very big；The preference temperature of hydrolysis is 293 scholar 5K.Pass through As (OR)3Hydrolysis and
The As of acquisition2O3Powder, moisture containing 6-8% and trace amounts of alcohol.It is right in vacuum drying chamber in order to remove these moisture and trace amounts of alcohol
As2O3It is heat-treated.Firing rate is 1.5 DEG C/min.Constant weight is heated under isothermal conditions.As(OCH3)3And As
(OC2H5)3Drying time needed under 343K, 363K, 373K, 413K temperature conditionss is respectively 43,19,14,3min and 116,
55、17、4min.The obtained pure As of spy2O3For the powder of high dispersive, the average diameter of particle (corresponds to R for 13.13 and 10.64 μ
For CH3, C2H5).Obtained pure As2O3Roentgen's facies analysis of powder points out that they are all cubic crystal.
The embodiment of the present invention, which provides a kind of method for the curative effect of medication assessed and be used for treating angiogenesis-mediated disease, to be included：
Arsenic trioxide is assessed to arthropathy synovial cell's TSP-1-TGF- β-CTGF-VEGF vascular proliferation function modules
Inhibitory action；
Arsenic trioxide is assessed by suppressing TSP-1-TGF- β-CTGF-VEGF vascular proliferation function modules and then to lesion
The inhibitory action of proliferation of microvascular endothelial cells in joint.
The method for the curative effect of medication that the assessment is used to treat angiogenesis-mediated disease specifically includes：
Establish the thin into fiber-like synovial membrane of the fibroblast-like synoviocyte NH-FLS and RA synovial tissues of normal synovial tissue
Born of the same parents RA-FLS and microvascular dermal endothelial cell HDMECs co-culture systems；
FLS TSP-1, TGF-β 1, CTGF and VEGF in co-culture system are measured using real time fluorescence quantifying PCR method
MRNA expressions；
TSP-1, TGF-β 1, CTGF and vegf protein expression in ELISA method analysis co-culture system supernatant；So
Fresh supernatant is added into HDMECs progress Transwell experiments afterwards and is tested into pipe.
The method for the curative effect of medication that the assessment is used to treat angiogenesis-mediated disease further includes：
After stimulating or disturbing respectively RA-FLS TSP-1, TGF-β 1, CTGF expression, RA-FLS and its co-culture system are detected
TSP-1, TGF-β 1, CTGF, VEGF gene and protein expression situation in culture supernatant, so as to assess vascular proliferation factor TSP-
1st, TGF-β 1, the interaction relationship between CTGF, VEGF.
Below in conjunction with the accompanying drawings and specific embodiment is further described the application principle of the present invention.
Method
The fibroblast-like synoviocyte (NH-FLS) of normal person synovial tissue and being slided into fiber-like for RA patient synovial tissue
Theca cell (RA-FLS) establishes co-culture system respectively with human dermis' microvascular endothelial cells (HDMECs).As2O3It is total to TNF-α
With processing co-culture system.FLS TSP-1, TGF-β 1, CTGF in co-culture system are measured using real-time quantitative fluorescence PCR method
With VEGF mRNA expressions, ELISA method analyzes TSP-1, TGF-β 1, CTGF and VEGF eggs in co-culture system supernatant
White expression, then adds to HDMECs progress Transwell experiments by fresh supernatant and is tested into pipe.The present invention also detects
After stimulating or disturbing respectively RA-FLS TSP-1, TGF-β 1, CTGF expression, TSP-1 in RA-FLS and its culture supernatant,
TGF-β 1, CTGF, VEGF gene and protein expression situation, so as to assess vascular proliferation factor TSP-1, TGF-β 1, CTGF, VEGF
Between interaction relationship.Experiment in vivo, collagen-induced property joint is established to DBA/1J mouse injection ox II collagen types
Scorching (CIA) model, gives As2O3Or methotrexate for treatment 2 weeks, joint is assessed by Joint scores and joint histopathology
The scorching order of severity, TSP-1, TGF-β 1, CTGF and VEGF in CIA model mouses synovial tissue of joint are analyzed by ImmunohistochemistryMethods Methods
Expression and angiogenesis situation.
As a result
RA-FLS expresses TSP-1, TGF-β 1, CTGF and VEGF mRNA level in-sites and is substantially risen compared with NH-FLS in co-culture system
Height, in RA-FLS and HDMECs co-culture system supernatants TSP-1, TGF-β 1, CTGF and vegf expression compared with NH-FLS with
The content of above-mentioned albumen substantially increases in HDMECs co-culture system supernatants, is generated which results in HDMECs migrations and tubule
Increase, illustrating the above-mentioned vascular proliferation factor of RA-FLS secretions has biological activity, can promote angiogenesis.Respectively stimulate or
Disturb RA-FLS TSP-1, TGF-β 1, CTGF expression after, TSP-1, TGF-β 1, CTGF and VEGF either in protein level also
It is that complicated change occurs in gene level, interaction intertexture forms network, and it is new that constructing function module adjusts RA synovial membrane blood vessels jointly
It is raw.TNF-α can be obviously promoted TSP-1 in RA-FLS and HDMECs co-culture systems, TGF-β 1, CTGF and vegf expression and its phase
Close angiogenesis.Regardless of whether carry out TNF-α induction, As2O3It can significantly inhibit in RA-FLS and HDMECs co-culture systems
The expression of TSP-1, TGF-β 1, CTGF and VEGF.In experiment in vivo, TSP-1 in CIA model mouses synovial tissue of joint, TGF-β 1,
The expression of CTGF and VEGF and microvessel density (MVD) are significantly raised, give As2O3After treatment, TSP-1, TGF-β 1, CTGF
Expression and MVD with VEGF are remarkably decreased, it is suppressed that the medium vessels generation of CIA model mouses synovial tissue, so as to mitigate arthritis
The order of severity.
Conclusion
TSP-1, TGF-β 1, CTGF and vegf expression are raised in systematicness in RA-FLS, and form function module, common to adjust
Vascular proliferation is controlled, is played a significant role in RA morbidities.As2O3By blocking TSP-1-TGF- β 1-CTGF-VEGF (TTCV) work(
Energy module, significantly inhibits RA-FLS and HDMECs co-culture systems and CIA synovial tissues angiogenesis, contains and treat the huge of RA
Big potentiality.
The application principle of the present invention is further described with reference to specific embodiment.
Experimental method
1) FLS and HDMECs co-culture systems and administration are established
RA-FLS and NH-FLS is separately added into 10%FBS DMEM and is made 4 × 105A cell/ml cell suspensions.Add or
Person is added without tumor necrosis factor-alpha, while adds various concentrations As2O3(0,0.5 μm of ol/L, 1.0 μm of ol/L, 2.0 μm of ol/L),
After co-culturing 48h, collect supernatant centrifugation and remove cell impurities.Next the supernatant that part is acellular is immediately available for after taking out
Transwell experiment and into pipe test, remaining supernatant frozen in -20 DEG C for follow-up ELISA test.Collect and co-culture body
It is FLS cells in bottom chamber, and extracts RNA and be used for further genetic test.
2) stimulation test
Digested when RA-FLS 85% is full using trypsase-EDTA, add 10%FBS DMEM and be made 5 × 105It is a thin
Born of the same parents/ml cell suspensions, add 6 well culture plates, make cell attachment overnight.It is 1%FBS DMEM that next day, which changes liquid, adds recombined human
CTGF, TSP-1 or TGF-β 1 make its concentration respectively reach 500ng/ml, 1000ng/ml or 5ng/ml.FLS is collected after stimulating 48h
RNA and supernatant containing albumen carry out real-time PCR (RT-PCR) and western blot detections, assessment respectively
CTGF, TSP-1,1 results of stimulation of TGF-β.
3) interference experiment
By into RA-FLS transfect TSP-1, TGF-β 1, CTGF special siRNA (siRNA) come carry out TSP-1,
TGF-β 1, CTGT interference experiments.
4) RNA extractions and real-time quantitative PCR detection
Synovial cell RNA is extracted, the reverse transcription of RNA, Real-time PCR amplifications
5) ELISA is detected
CTGF, TGF-β 1, TSP-1, VEGF concentration in ELISA method detection cell supernatant
6) Transwell is tested
600 μ L/ holes of supernatant are added in 24 well culture plates, upper strata cell is put into lower floor's supernatant.Trypsase-
EDTA digests HDMECs, and HDMECs is made cell suspension with serum-free DMEM.200 μ L HDMECs cell suspensions are taken to add
In the cell of upper strata and it is incubated.Cell is taken out, film upper surface is wiped with cotton swab and does not migrate past cell, methanol is fixed, crystallization
Purple dyes, and observes and takes pictures under 100 × amplification factor of microscope, and theca cell number is worn in counting, so as to be quantified.
7) Matrigel is tested into pipe
Sample-adding pipette tips and 96 well culture plates are pre-cooled, after Matrigel glue dissolves on ice, per hole on 96 well culture plates
Adding placement 30min in 50 μ L, 5%CO2,37 DEG C of moist environments makes its solidification.96 well culture plates are taken out, before adding 50 μ L per hole
State fresh supernatant.HDMECs cells are made with serum-free DMEM.100 μ L HDMECs cells are added to hang per hole on 96 well culture plates
Liquid.It is incubated after 6h and is observed using phase contrast microscope Human Umbilical Vein Endothelial Cells into pipe.
8) zoopery
The present invention tests the validity of the anti-angiogenic propagation of arsenic trioxide using animal model.
Collagen-induced Arthritis is the experimental model of rheumatoid arthritis, which is widely used for many kinds
The preclinical test of rheumatoid medicine.The characteristics of model is for multi-joint inflammation that is strong, easily surveying, with synovial membrane
Vascular proliferation and obvious osteoclasia.This analysis is used for determining that the effect of intraperitoneal administration (IP) arsenic trioxide and dosage are anti-
Should, apply daily with for suppress to produce in mouse collagen Induced Arthritis evolution inflammation (foot is swollen), cartilage damage
And bone resorption.
9) CIA mouse models modeling process is briefly described below:
Ox II collagen types (CII) are dissolved in 0.05M acetic acid to 2mg/ml, addition and contain the heat-killed knots of 4mg/mL in equal volume
Complete Freund's adjuvant (CFA) mixing of core mycobacteria H37Ra, is fully ground to mixture and emulsified completely, instilled with emulsion
It is not loosely degree (being operated in ice bath) in water, contains the 100 μ L of 100 μ g CII in every DBA/1J mouse tails root intracutaneous injection
Emulsion carries out first immunisation.The 21st day mouse carries out booster immunization after first immunisation, i.e. injection and isometric incomplete Freund
CII100 μ g after adjuvant (IFA) emulsification.There are 6 mouse as the non-induced arthritis of Normal group.
21 days after first immunisation, mouse is divided into following 6 groups (every group 6)：I group：Normal group；II group：CIA models
Control group；III group：As2O31.0mg/kg/day treatment group；IV group：As2O32.0mg/kg/day treatment group；V group：As2O3
5.0mg/kg/day treatment group；VI group：MTX1.5mg/kg/week treatment groups.The 26th day to 39 days CIA are small after first immunisation
Mouse intraperitoneal administration As2O3And MTX.Normal group and model group mouse give isometric phosphate buffered saline (PBS) (PBS).Treatment phase
Between daily monitoring mouse hair color, weight, meal situation.40th day, slaughter all mouse and carry out subsequent experimental.
Osteoarthritis clinical symptom is assessed daily the 19th day after first immunisation, joint was commented in every 3 days
Divide until experiment terminates.In order to quantify arthritis severity, the present invention refer to the points-scoring system having been reported.According to lesion
The order of severity is evaluated by visual observation limbs, completes scoring by two independent observers, each limbs are according to inflammation journey
Degree divides 0-4 points, and standards of grading are as follows：
0 point=without erythema and swelling；
1 point=erythema and mild swelling, are confined to shank or ankle-joint, and ankle-joint has certain rubescent and swelling, or
There is rubescent and swelling in indivedual toes；
2 points=erythema and mild swelling, extend to shank, moderate erythema and swelling occurs in ankle-joint from ankle-joint；
3 points=erythema and moderate swelling, plantar joint is extended to from ankle-joint；
4 points=erythema and serious swelling, enclose ankle-joint, foot and toes, or arthrocleisis.
Four limbs of every mouse to be evaluated respectively, the sum of scoring is Joint scores, and every animal highest is divided into 16 points,
Scoring diagnosable more than 4 points is arthritis.After mouse is slaughtered, HE dyeing carries out Histological assessment, and immunohistochemical staining assessment is micro-
The important indicators such as vessel density.
Histologic analysis
HE is dyed
After abundant decalcification, wax stone is made in dehydration, paraffin embedding knee joint, and slice thickness is 5 μm, carries out hematoxylin-eosin
(hematoxylin-eosin staining, HE) is dyed.
HE dyeing staining procedures are as follows：
1) dimethylbenzene dewaxes, graded ethanol aquation.
2) it is put into hematoxylin aqueous solution and dyes.
3) color separation in sour water and ammonium hydroxide, each several seconds.
4) flowing water enters distilled water a moment after rinsing 1h.
5) enter in 70% and 90% alcohol and be dehydrated each 10min.
6) alcohol eosin stains liquid dyeing 2-3min is entered.
7) it is dehydrated transparent：Section after dyeing is dehydrated through absolute alcohol, then makes section transparent through dimethylbenzene.
8) upper natural gum is dripped into section, covered, marks, and preserves.
Arthritic tissues are assessed
The arthritic order of severity of qualitative assessment, is broken by two observers in a manner of blind comment according to cellular infiltration, cartilage
Bad and bone erosion parameter, scores with reference to existing standard[147]。
Standards of grading are as follows：
0=natural joint structures；
1=mild changes, slight synovitis, pannus and the cartilage stove erosion being dispersed in；
2=moderates change, cartilage large defect, and pannus corrodes, synovial hyperplasia and inflammatory cell infiltration；
3=severe synovitis, cartilage and bone erosion, joint structure destroy.
Immunohistochemical analysis
Take CIA mouse knee joints wax stone to carry out immunohistochemical staining, comprise the following steps that：
1) dimethylbenzene dewaxes knee joint section.
2) graded ethanol aquation.
3) under room temperature, 3%H2O2Block the activity of endogenous peroxydase.
4) being incubated in citrate buffer (pH 6.0) exposes antigen.
5) when 10% lowlenthal serum, 37 DEG C of closings 1 are small.
6) 120min is incubated at room temperature with anti-TSP-1, TGF-β 1, CTGF, VEGF, vWF antibody respectively.
7) the goat anti-rabbit immunoglobulin polymer incubation at room temperature 60min of horseradish enzyme mark.
8) DAB substrate solutions 1ml and DAB concentrates 1 drip (about 50 μ L) and are sufficiently mixed, and add on slide sample and develop the color.
9) it was observed that after brown color dyeing (7-10min), stopping colour developing, develop a film, haematoxylin is redyed, the differentiation of salt water-alcohol,
Ammonium hydroxide returns indigo plant, graded ethanol aquation.
10) mounting.
Brown color dyeing is positive staining position.Rabbit-anti mouse vWF is used to identify blood vessel endothelium.After immunostaining, aobvious
Observation assessment is carried out to each section upper joint synovial tissue under micro mirror, is observed under 200 × amplification factor of microscope and takes 3 at random
The visual field, counts positive cell numbers and total cell number by two different observers, obtains respective average, draw positive cell number with
The ratio of total cell number.The Microvessel Count of vWF stained positives is carried out in above-mentioned same area.
With reference to experimental result, the invention will be further described.
First, TSP-1, TGF-β 1, CTGF, VEGF are overexpressed and have rush vascular proliferation activity in RA-FLS.Experiment in vitro,
Normal human articular's synovioblast (NH-FLS) and rheumatoid arthritis patients synovium of joint fibroblast (RA-FLS)
Co-cultured respectively with human microvascular endothelial cell (mvec) (HDMECs), for A. compared with NH-FLS co-culture systems, RA-FLS co-cultures body
It is significantly raised to fasten TSP-1-TGF- β 1-CTGF-VEGF function modules member protein expression in clear liquid；B, the NH- with co-cultivation
FLS is compared, and TSP-1-TGF- β 1-CTGF-VEGF function module members mRNA expression is significantly raised in the RA-FLS of co-cultivation；C.
Compared with NH-FLS co-culture systems, RA-FLS co-culture systems supernatant is because of TSP-1-TGF- β 1-CTGF-VEGF function modules
It is overexpressed and causes endothelial cell angiogenesis ability to be remarkably reinforced.
2nd, As2O3TSP-1-TGF- β 1-CTGF-VEGF function modules in RA-FLS are can inhibit to be overexpressed so as to suppress blood vessel
Propagation.
Cell and functional experiment
In order to imitate the microenvironment of RA synovial tissues, the present invention by Transwell devices establish RA-FLS with
HDMECs co-culture systems, while TNF-α is added to imitate RA synovial membrane inflammation environment.Drug-treated：To RA-FLS and HDMECs
Tumor necrosis factor-alpha (TNF-α is added or is added without in co-culture system；100ng/ml), while various concentrations As is added2O3
(0,0.5μmol/L,1.0μmol/L,2.0μmol/L).ELISA method detection co-cultures TSP-1, TGF-β 1, CTGF in supernatant
With vegf protein expression.TSP-1, TGF-β 1, CTGF and VEGF mRNA expression in the RA-FLS that RT-PCR detections co-culture
Situation.Collect after above-mentioned dosing it is fresh co-culture supernatant carry out respectively Transwell experiments up to 6h and Matrigel into
Pipe is tested, and further detection co-cultures influence of the supernatant to HDMECs angiogenesis abilities.
As a result such as Fig. 1 is shown, TNF-α can induce TSP-1 in supernatant, TGF-β 1, CTGF and vegf protein expression, and
As2O3Point of TSP-1, TGF-β 1, CTGF and VEGF can be significantly inhibited in 1.0 μm of ol/L or the concentration more than 1.0 μm of ol/L
Secrete, and be in concentration dependent, and with whether to give TNF-α unrelated.TSP-1, TGF-β in the RA-FLS that RT-PCR detections co-culture
1st, CTGF, VEGF mRNA expressions are consistent with protein expression variation tendency in supernatant.Because co-culture TSP-1- in supernatant
1-CTGF-VEGF function modules of TGF-β are suppressed, and the present invention carries out Transwell experiments and tests further detection into pipe
Under the influence of supernatant is co-cultured, As2O3Effect to HDMECs angiogenesis abilities.
3rd, As2O3Suppress the progress of Collagen-induced Arthritis.
The present invention has carried out As in 25 days to 39 days after first immunisation2O3Treatment, and established pass was applied since 19 days
Section points-scoring system assesses arthritis severity.Mouse As2O3Dosage is 1.0,2.0,5.0mg/kg/d.This
In analysis, the present invention is not observed using As2O3Any obvious toxicity or lethal effect afterwards.After CII is immunized, the pass of mouse
The scorching progress of section is rapid, and shows obvious clinical symptoms.MTX 1.5mg/kg/week groups are as treatment positive control, performance
Go out the effect of stronger suppression arthritis progress.Although As2O31.0mg/kg/day treatments are very weak to CIA mouse inhibitory action,
2.0mg/kg/day and 5.0mg/kg/day As2O3Arthritis index, model comparison can be persistently substantially reduced from 25 days to 39 days
Group and As2O3The the 1st, 7, the 14 day typical mouse claw photo of 5.0mg/kg/day treatment groups upon administration.As2O3Treatment group
Joint symptoms and scoring are significantly improved compared with CIA model control groups, especially As2O32.0mg/kg/day treatment groups and As2O35.0mg/
Kg/day treatment groups.
4th, As2O3Improve the histological change of CIA mouse synovium of joint.
In order to confirm As2O3Protective effect, the present invention further pass through knee joint Histological section HE dyeing assessment CIA
Mouse As2O3The order of severity of posterior joint inflammation is administered.The results show that, As consistent with the trend for improving joint symptoms2O3And MTX
Inflammation, cartilage and the osteoclasia for the treatment of group significantly reduce.As2O3Treatment group, mouse destruction of joint degree subtract in dose dependent
It is weak, and especially suitable for As2O32.0mg/kg/day and 5.0mg/kg/day treatment groups.In fact, As2O35.0mg/kg/day
Change of the treatment group to MTX 1.5mg/kg/week treatment groups on synovial tissue is learned is similar.
5th, As2O3Suppress the TSP-1-TGF- β 1-CTGF-VEGF (TTCV) of model mouse synovial tissue of joint systematicness up-regulation
Function module and vascular proliferation.
As is analyzed by immunohistochemical staining2O3TSP-1, TGF-β 1, CTGF, VEGF are in synovial tissue of joint after administration
In expression, and Angiogenesis situation in synovial tissue.The results show that TSP- in CIA mouse synovial tissue of joint
1st, TGF-β 1, the staining power of CTGF, VEGF are in As2O3Treatment group significantly reduces.Moreover, in As2O35mg/kg/d treatment groups with
And MTX 1.5mg/kg/week treatment groups are almost not detected by TSP-1, TGF-β 1, CTGF and VEGF dyeing.
Finally, microvessel density (MVD) is measured by immunohistochemical markers vWF, so as to assess As2O3CIA is small after administration
Mouse synovial tissue angiogenesis situation, As2O3Relevant blood vessel formation against function is also confirmed using the method.The results show that
MVD is in As2O3Treatment group significantly reduces, and is in dose dependent, especially sees As2O32mg/kg/day and 5mg/kg/day are controlled
Treatment group.These results explanation, As2O3Anti- RA may be played by suppressing 1-CTGF-VEGF function modules of TSP-1-TGF- β to slide
Membrane tissue vascular proliferation acts on, so as to achieve the purpose that to treat RA.
This conclusion can be briefly summarized such as Fig. 2.Experiment made on the living, A-D are prompted respectively, with normal mouse synovial tissue of joint phase
Than four members of TSP-1-TGF- β 1-CTGF-VEGF function modules in rheumatoid arthritis CIA model mouses synovial tissue of joint
It is overexpressed, after giving arsenic trioxide in treatment, TSP-1-TGF- β 1-CTGF-VEGF function modules are substantially lowered, and its curative effect
It is close with MTX.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should all be included in the protection scope of the present invention.
Claims (6)
1. a kind of medicine for being used to treat angiogenesis-mediated disease prepared using arsenic trioxide, it is characterised in that described
The medicine components for being used to treat angiogenesis-mediated disease prepared using arsenic trioxide are by mass by arsenic trioxide 5mg
Formed with sodium chloride 50mg.
2. a kind of medicine for being used to treat angiogenesis-mediated disease for utilizing arsenic trioxide to prepare as claimed in claim 1
Preparation method, it is characterised in that the preparation method includes：
Using As (OR)3Pure As is made in hydrolysis2O3；Wherein, R CH3,C2H5；
AsCl3With methanol or ethanol synthesis generation As (OR)3, filtered-distillation-rectifying-As (OR)3Hydrolysis-filtering-drying, obtains
To pure As2O3；
By As2O35ml is made with sodium chloride solution in powder, wherein, arsenic trioxide 5mg, sodium chloride 50mg.
3. preparation method as claimed in claim 2, it is characterised in that As (OR)3In hydrolysis, As in mass ratio (OR)3:H2O=
1:1.6-3；The temperature of hydrolysis is 293 scholar 5K；
In drying steps, including：In vacuum drying chamber, to the As after filtering2O3It is heat-treated；Firing rate for 1.5 DEG C/
min；Constant weight is heated under isothermal conditions；
AsCl3As (the OCH generated with methanol or ethanol synthesis3)3Or As (OC2H5)3In 343K, 363K, 373K, 413K temperature strip
Part lower drying time is respectively 43min, 19min, 14min, 3min and 116min, 55min, 17min, 4min.
4. a kind of assess the medicine for being used to treat angiogenesis-mediated disease for utilizing arsenic trioxide to prepare described in claim 1
The method of curative effect, it is characterised in that the method for the curative effect of medication that the assessment is used to treat angiogenesis-mediated disease includes：
Assess suppression of the arsenic trioxide to arthropathy synovial cell's TSP-1-TGF- β-CTGF-VEGF vascular proliferation function modules
Make and use；
Arsenic trioxide is assessed by suppressing TSP-1-TGF- β-CTGF-VEGF vascular proliferations function modules and then to lesion joint
The inhibitory action of middle proliferation of microvascular endothelial cells.
5. the method for the curative effect of medication for treating angiogenesis-mediated disease is estimated as claimed in claim 4, it is characterised in that institute
The method that the curative effect of medication for treating angiogenesis-mediated disease is estimated in commentary specifically includes：
Establish the fibroblast-like synoviocyte RA- of the fibroblast-like synoviocyte NH-FLS and RA synovial tissues of normal synovial tissue
FLS and human dermis' microvascular endothelial cells HDMECs co-culture systems；
FLS TSP-1, TGF-β 1, CTGF and VEGF mRNA tables in co-culture system are measured using real time fluorescence quantifying PCR method
Up to level；
TSP-1, TGF-β 1, CTGF and vegf protein expression in ELISA method analysis co-culture system supernatant；Then will
Fresh supernatant adds to HDMECs and carries out Transwell experiments and tested into pipe.
6. the method for the curative effect of medication for treating angiogenesis-mediated disease is estimated as claimed in claim 5, it is characterised in that institute
The method that the curative effect of medication for treating angiogenesis-mediated disease is estimated in commentary further includes：
After stimulating or disturbing respectively RA-FLS TSP-1, TGF-β 1, CTGF expression, RA-FLS and its co-culture system culture are detected
TSP-1, TGF-β 1, CTGF, VEGF gene and protein expression situation in supernatant, thus assess vascular proliferation factor TSP-1,
TGF-β 1, the interaction relationship between CTGF, VEGF.
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| CN111228301A (en) * | 2020-01-14 | 2020-06-05 | 哈尔滨医科大学 | Compound preparation for treating angiogenesis-mediated diseases and preparation method and application thereof |
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