CN104873499B - A kind of application of compound of up-regulation Runx2 transcriptional activities in osteoporosis is prevented and treated - Google Patents
A kind of application of compound of up-regulation Runx2 transcriptional activities in osteoporosis is prevented and treated Download PDFInfo
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Abstract
The invention belongs to field of medicaments, cell model is adjusted on Runx2 transcriptional activities by building, screening obtains compound N (Cvclopropvlmethvl) 5 (base of thiophene 2) 1, the formamide of 2 azoles 3, experimental study shows that it has the expression of up-regulation Runx2 transcriptional activity, up-regulation Gegenbaur's cell alkaline phosphatase activity and skeletonization specific differentiation gene, increase the generation of calcium scoring simultaneously, with the application prospect for preparing preventing and treating medicine for treating osteoporosis.
Description
Technical field：
The invention belongs to field of medicaments, it is related to a kind of application of compound in osteoporosis is prevented and treated.Specifically, this hair
It is bright to be related to the compound and adjusted on Runx2 expression activities are prepared, adjust on intracellular alkaline phosphatase activities and suppress
Application in multinuclear mature osteoclast formation medicine.
Background technology：
Osteoporosis is that one kind is reduced with bone amount, and bone micro-structure destruction causes the increase of bone fragility, and it is spy easily to occur fracture
The general metabolism osteopathy levied.Osteoporosis can betide any crowd and any age, be more common in the elderly.Bone m etabolism is put down
Weighing apparatus is that two dynamic processes of bone information of the bon e formation and Osteoclasts mediate mediated by Gegenbaur's cell regulate and control exquisitely.Work as bone
When absorbing more than bon e formation, it may appear that bone amount is reduced, bone loss is may occur in which, occurs osteoporosis.
The medicine for the treatment of osteoporosis is generally divided into two classes：Anti- bone information medicine and promoting bone growing medicine.Anti- bone information
Medicine is by suppressing the activity of osteoclast so as to reduce bone information.It is currently available that anti-bone information medicine has diphosphonate, choosing
Selecting property estrogenic agents, calcitonin and estrogen etc..For what is seriously lost with osteoporosis or bone amount
Crowd, simple suppression bone information is obviously inadequate, promotes Osteogenesis to turn into the new Critical policies for the treatment of osteoporosis, such
Medicine can also directly facilitate the osteanagenesis after fracture complication.Current people source recombined parathyroid hormone (rhPTH), is the U.S.
The promoting bone growing medicine of the only approved listings of FDA.Its specific mechanism of action is still under study for action, it may be possible to influence many bars to lead to
Road, changes activity [the Jilka RL.Bone, 2007,40 (6) of Gegenbaur's cell, bone system cell, osteoclast and osteocyte:
1434-1446.].RhPTH is as bon e formation medicine is presently most effectively promoted, and there is also certain limitation.Therefore,
The small molecule promoting bone growing medicine of novel multi-effect is developed, is expected to that there are bright prospects in following osteoporosis therapy field.
Gegenbaur's cell is as main bon e formation cell, from the mescenchymal stem cell with Osteoblast Differentiation potential, leads to
Generation alkaline phosphatase (ALP) and bone matrix protein, including BGP (OCN) and osteopontin (OPN) are crossed, the hair of mineralising is induced
It is raw.Important function based on Gegenbaur's cell in bon e formation, it is thus regarded that skeletonization system cell propagation can be increased or promote skeletonization point
The compound of change has the effect of promoting bone growing.
In recent years, the Advances in Molecular Genetics carried out on mouse and the person, discloses participation regulation and control Gegenbaur's cell
The a variety of transcription factors occurred and its effect.These transcription factors are in processes such as the orientation of Gegenbaur's cell, differentiation and function enforcements
In all played regulating and controlling effect so that relevant cell expresses specific phenotype genes and simultaneously obtains osteoblasts in vitro.In these turns
Record the factor in, Runx2 (Runt-related transcription factor 2) be proved to be regulation Gegenbaur's cell orientation and
The central transcription factor of differentiation.Runx2 belongs to Runt domain gene family members, and table is started when interstitial cell development is bone
Reach, all exist in the whole atomization of Gegenbaur's cell afterwards.Runx2 can adjust a variety of osteoblast marker genes, including
Alp, type I collagen, Ocn, Opn expression and the generation of final mineralising.Runx2 can regulate and control embryonic stage bone development and
Bon e formation after birth.The mouse of Runx2 gene knockouts is obstructed due to osteoblast differentiation, it is impossible to pass through any intermembranous ossification or soft
The approach of bone within bone forms new bone [Ghosh S, Cell, 2002,109Suppl (0092-8674 (Print)):S81-96.].
Necessary to this also confirms that Runx2 is Osteoblast Differentiation.The inactivation of single Runx2 allele will cause a kind of bone shape in human body
Into defective disease-clavicle cranium hypoplasia disease [Chen LF, et al EMBO J, 2002,21 (23):6539-6548.].
In addition, the rush Osteoblast Differentiation effect of a variety of micromolecular compounds is relevant with Runx2 activation.Resveratrol is that one kind exists
Naturally occurring plant polyphenol in red wine and a large amount of plants, can reverse the bone loss of Ovarian ablation rat.Experiment in vitro discovery,
The SIRT1-FOXO3A compounds of resveratrol activation can be bound to the FRE sites of Runx2 distal promoters, so as to raise
Runx2 expression, the effect that mechanisms mediate Resveratrol Inducing human mesenchymal stem cell breaks up to skeletonization direction.In addition,
Also it is reported that, the Runx2 mechanism induced osteogenesis cell differentiations that jamaicin passes through p38MAPK signal pathway activateds.In traditional Chinese medicine
In, barrenwort is usually applied to treatment osteoporosis because of effect of its strengthening the bones and muscles.What is extracted from the draft is a kind of different
Many Flavonol glycoside-the icariin of pentenyl, are its main pharmacological active substances.Research finds that icariin induced osteogenesis divides
Change also relies on Runx2.
Runx2 take part in including the paths such as BMP paths, Wnt paths, Notch paths and Hedgehog it is a plurality of into
Bone breaks up and bon e formation associated signal paths, and is probably Rendezvous Point [Lin GL, the et al J Cell of signal integration
Biochem,2011,112(12):3491-3501.].The compound of Runx2 transcriptional activities can effectively be raised by thus speculating, may
The various reactive compounds of more than targeting each path are enumerated, it has potential promotion Osteoblast Differentiation and bon e formation effect.
In summary, the important function based on Runx2 in regulation Osteoblast Differentiation, the present invention constructs Runx2 transcription work
Property on adjust cell screening model, and pass through screening, find compound N-(Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2-
Azoles -3- formamides have up-regulation Runx2 transcriptional activity, and external activity experiment shows that it has the intracellular Runx2 albumen of up-regulation
Level, up-regulation Gegenbaur's cell alkaline phosphatase activity, the expression of up-regulation skeletonization specific differentiation gene and increase calcium scoring
Generation, while being a kind of potential anti-osteoporosis candidate compound it has also been found that the compound can suppress the formation of osteoclast
Thing.Screening model of the present invention and compound N-(Cvclopropvlmethvl) -5- (thiophene -2- found based on the model
Base) -1,2- azoles -3- formamides function of resisting osteoporosis, so far there is not yet relevant report, be present invention firstly discovers that.Institute
Compound is stated compared with the medicine clinically used, structure does not have similitude, and the compound has promoting bone growing and suppression
The dual anti-osteoporosis activity of bone information, may have new anti-osteoporosis mechanism, there is wide DEVELOPMENT PROSPECT.
The content of the invention：
It is an object of the invention to provide a kind of N- (Cvclopropvlmethvl) -5- (thiophene -2- obtained by cell screening model
Base) the application of -1,2- azoles -3- benzamide compounds in osteoporosis is prevented and treated.
In order to achieve the above object, the present invention takes following technical scheme：
1, build Runx2 transcriptional activities on adjust cell screening model, and based on the model find N- (Cvclopropvlmethvl)-
5- (thiophene -2- bases) -1,2- azoles -3- formamides adjusted on Runx2 transcriptional activities are prepared in application；
2, N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides are preparing the up-regulation of Runx2 protein levels
Application in agent；
3, N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides are preparing MC3T3-E1 cell inductions
Application in being adjusted in the ALP activity of Osteoblast Differentiation；
4, N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides are preparing MC3T3-E1 cell inductions
Application in being adjusted on skeletonization specific differentiation Gene A lp, Opn, Runx2；
5, N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides are in increase C3H10T1/2 cell inductions
Application in calcium scoring number；
6, N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides are in osteoclast formation is reduced
Using.
Adjustment cell screening model on Runx2 transcriptional activities of the present invention, can apply to high flux preventing and treating sclerotin and dredges
Loose disease composition screening, the compound N gone out based on the model discrimination-(Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -
3- formamides are adjusted on a kind of Runx2 transcriptional activities, raise intracellular Runx2 albumen, the work of intracellular alkaline phosphatase
Property, the number of calcium scoring, Osteoblast Differentiation specific gene mRNA level in-site and suppress osteoclast formation, be expected to be developed into preventing
Control the medicine of bone loss disorders.
Present invention also offers the composition constituted by active component of the compound with pharmaceutically acceptable carrier
Application in anti-anti-osteoporosis.
Advantages and positive effects of the present invention are, the hair of the anti-osteoporosis compound raised based on Runx2 transcriptional activities
Now there is novelty, the present invention elaborates the compound N-(Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- first
Effect of the formamide in osteoporosis preventing and treating, at home and abroad still belongs to the first time, and there is independent intellectual property right to resist China's exploitation
Medicine for treating osteoporosis has realistic meaning.
Brief description of the drawings：
Fig. 1：Runx2 expression effects in N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides up-regulation model
Should
Wherein：Ordinate-Runx2 expression effect up-regulation rates (%)；
The concentration of abscissa-N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides
Fig. 2：In N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides up-regulation MC3T3-E1 cells
Runx2 protein level
Fig. 3：N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides raise intracellular alkaline phosphatase
ALP activity
Fig. 4：N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides increase the number of calcium scoring
Fig. 5：N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides raise intracellular Alp, Opn,
Runx2 mRNA level in-site
Fig. 6：N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides suppress osteoclast formation
Fig. 7：N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides increase the number of calcium scoring
Fig. 8：N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides raise intracellular Alp, Opn,
Runx2 mRNA level in-site
Fig. 9：N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides suppress osteoclast formation
Embodiment：
Following examples can make professional and technical personnel that the present invention is more fully understood, but not limit this hair in any way
It is bright.
《Embodiment 1》Raise the structure of Runx2 Transcription Activity. Cell screening models and the discovery of upper adjustment
Compound for screening is derived from but is not limited to national new drug (microorganism) screening experiment room, is dissolved in DMSO and matches somebody with somebody
It is set to 20mM preservations.Pass through the step such as adjustment cell screening model construction, screening compound and fluoroscopic examination on Runx2 transcriptional activities
Suddenly, influence of the observation compound to Runx2 transcriptional activities in model.Concrete operations are as follows：
Model construction：
(1) structure of plasmid
According to p6OSE2 plasmids [Geoffroy V, et al J Biol Chem, 1995,270 (52):30973-9.] (such as
Information Fig. 1), devise primer with expand 6 repetitions of p6OSE2 plasmids Runx2 binding members (6OSE2) and downstream most
Small BGP promoter fragment, and add on primer cleavage sequence.Primer sequence is as follows：OSE：(5 ' contain Xho I to forward primer
Cleavage sequence)：5’-ccgctcgaggctgcaatcaccaaccac-3’(SEQ ID NO.1)；(5 ' contain Hind III to reverse primer
Cleavage sequence)：5’-cccaagcttttgtctgtctgcaccctc-3’(SEQ ID NO.2).PCR results are shown, are drawn using this
Thing can expand acquisition target fragment, and specificity is very well, and band is single.Thus directly PCR primer is entered with PCR purification kits
Row purification, is finally eluted from pillar product with 20 μ l deionized waters.By all products III pair of enzyme of Xho I and Hind
Cut through night.Carrier pGL4.17 (being purchased from promega) is equally stayed overnight with Xho I and the double digestions of Hind III.Next day, digestion products are run
Glue reclaim.By the insertion of acquisition and linear pGL4.17 carriers according to mole ratio 3:1 enters under T4DNA Ligase effects
Row connection, 16 DEG C of connections are stayed overnight.Connection product converts Escherichia coli DH-5 α competence, in containing ampicillin LB culture plates
Upper overnight incubation.Next day, monoclonal is selected, amplification cultivation simultaneously extracts plasmid, gained recombinant plasmid pGL4.17-OSE is with the Hes of Xho I
The method identification of the double digestions of Hind III.As a result such as Fig. 2,3 recombinant plasmids #4, #7, #14 are obtained.
(2) recombinant plasmid and fluoremetry are transiently transfected
PGL4.17, p6OSE2, recombinant plasmid #4, #7 and #14 are transfected in 96 orifice plates respectively, during transfection, 10 μ are used
L/ holes opti-MEM (being purchased from Invitrogen (Shanghai) Trading Co., Ltd.) culture medium dilution 200ng DNA, 10 μ l/ hole opti-
MEM culture mediums dilute 0.5 μ l origene transfection reagents (being purchased from Origene companies of the U.S.), are incubated at room temperature after 5min and dilution
DNA merge mix, and continue be incubated 20min.The MC3T3-E1 cells in growth logarithmic phase are taken (to be purchased from China Medical Science
Institute's Institute of Basic Medical Sciences's Cell Culture Center) digested with pancreatin after, it is appropriate to add the culture medium containing 10%FBS, gently blows and beats
Single cell suspension is made, and it is 2 × 10 to adjust cell concentration5Individual/ml.Pressed in DNA- liposome complex solution after merging
Single cell suspension is added according to every μ l of hole 80, piping and druming is uniform, cell is fully mixed with DNA- liposome complexes.Will be mixed
Cell-DNA- liposome complex solution is added in 96 orifice plates, every 100 μ l.96 orifice plates are placed in CO2gas incubator and trained
Support overnight.Next day, testing compound is diluted to 2 × concentration with nutrient solution, 100 μ l are added in designation hole, continue to cultivate extremely finger
Fix time, using fluorescence detection reagent kit (Promega) fluorescence intensity.Specific method：By luciferase substrate lysate
Room temperature is put in, wait melting and equalized temperature is to after room temperature, is added in substrate and mixing of turning upside down.In 96 hole elisa Plates to be measured
Supernatant is removed, with PBS 3 times.5 × cell pyrolysis liquid (CCLR) is diluted to 1 with deionized water ×, add and treat by every μ l of hole 30
In gaging hole.37 DEG C of incubators are placed in, 30min is cracked.ELISA Plate is taken out, luciferase substrate solution 50 μ l are added, it is micro- in multiple labeling
Detected immediately on orifice detector.Carried out with positive compound genistein (being purchased from Nanjing Zelang Pharmaceutical Technology Inc.)
Preliminary identification, as a result, such as Fig. 2 D, final choice #7 plasmids, sequence results such as Fig. 2 E.
(3) stable transfected cells strain is built
By #7 restructuring reporter plasmid pGL4.17-OSE transfection MC3T3-E1 cells, stable transfection is obtained through G418 screenings thin
Born of the same parents' strain, is named as MC3T3-E1-OSE cells, so as to tentatively complete adjustment screening model construction work in Runx2 activity.With
Positive compound genistein, carries out preliminary identification, raises Runx2 activity, performance with finding positive compound energy dose-dependant
Raised for uciferase activity dose-dependant, and optimum activity (Fig. 3 A) is issued in 5 μM of concentration.In addition, also investigating
Compound processing time, cell-seeding-density, DMSO final concentrations are lived to luciferase expression in MC3T3-E1-OSE screening models
The influence of property.As a result such as Fig. 3, it is determined that the optimum condition of the cell screening model：Drug treating time is 48h；Cell is inoculated with
Density is 10000/hole；The control of DMSO final concentrations is 0.1%.
Compound processing：
MC3T3-E1-OSE cells are with 1 × 105Individual to be inoculated in 96 orifice plates per hole, every 100 μ l are placed in CO2gas incubator
Interior overnight incubation.Next day, with nutrient solution diluted compounds to 2 × concentration, 100 μ l are added in designation hole, continue to cultivate to 48
Hour.
Fluoroscopic examination：
Fluorescence is detected using fluorescence detection reagent kit (Promega).Method is as described above.
As a result as shown in figure 4, finding that N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides (are purchased from hundred
Ling Wei Science and Technology Ltd.s) the Runx2 transcriptional activity in dose-dependant ground mode transfer type in the range of 0.001-25 μM.
《Embodiment 2》N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides raise mouse bone precursor
Runx2 protein level in cell MC3T3-E1
300,000 MC3T3-E1 cells are inoculated with six orifice plates after after cell attachment, N- (the cyclopropyl first of various concentrations is added
Base) after -5- (thiophene -2- bases) -1,2- azoles -3- formamides processing 48h, cell is collected, and clean with precooling PBS cell 2 times,
Collect cell precipitation.According to 5 times of volume addition cell pyrolysis liquids of cell precipitation (adding PMSF in advance makes its final concentration of 1mM),
On ice after cracking 30min, with 12000rpm rotating speeds, 4 DEG C of centrifugation 15min collect supernatant, determine protein concentration, managed according to 40 μ g/
Packing, and 5 × albumen sample-loading buffer is added, loading volume is supplied with autoclaved ultra-pure water, 100 DEG C are boiled 7min, centrifuged
Get rid of lower wall built-up liquid.Using LaemmLi systems, lamination gum concentration is 5%, and resolving gel concentration selects 10% electrophoresis, when before dyestuff
Along entering after separation gel, voltage is brought up to 110V and continues electrophoresis, after the completion of albumen Marker position judgments race glue, turned
Film.Transferring film buffer solution (25mM Tris, 192mM glycine, 20% (v/v) methanol) is prepared during electrophoresis, 4 DEG C are placed in advance
It is cold.Pvdf membrane activates 1min with methanol in advance, is then dipped in transferring film buffer solution, puts and vibrate to remove membrane removal surface gas on shaking table
Bubble.Filter paper 15min before transferring film immerses precooling in transferring film liquid.After electrophoresis terminates, gel is carefully removed, transferring film buffer solution is transferred to
Middle balance 5min, carries out half-dried transferring film afterwards.3 filter paper are spread successively from bottom to top in the anode of half-dried transferring film instrument, it is pvdf membrane, solidifying
Glue, 3 filter paper, stack neatly, notice that driving away for bubble is removed.Make sandwich to finish, dried with blotting paper outside sandwich structure
Excessive transferring film liquid, to prevent current leakage.Finally the negative electrode of transferring film instrument is covered.Voltage sets 10~15V, two pieces when turning one piece of glue
20~25V of glue.After transferring film terminates, film is removed, rocked at room temperature closing 1h, confining liquid is 5% skim milk (PBS-T dilutions).
4 DEG C of overnight incubations of Runx2 (Santa Cruz Biotechnology) primary antibody.Next day, primary antibody solution is removed, PBS-T is washed 3 times,
Each 7min.(diluted again with the two corresponding anti-solution of horseradish peroxidase-labeled with the PBS-T containing 5% skim milk, mouse is anti-with 1：
5000 dilutions) 1h is incubated, PBS-T is washed 3 times, each 7min.Finally it is incubated with ECL western blot reagents, passes through Chemi
The gel imager capture images of Imager 5500.As a result such as Fig. 5, N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -
3- formamides can raise the expression of Runx2 albumen in MC3T3-E1 cells.
《Embodiment 3》PNPP methods determine alkaline phosphatase activities (ALP)
MC3T3-E1 cells are inoculated in 6 orifice plates, 3 × 105/ hole, after cell growth is converged, is changed to the μ of osteogenic induction liquid 25
G/ml ascorbic acid, 5mM β-phosphoglycerol., and various concentrations decoction is added, change liquid once within every three days, collected in specified day
Sample.When receiving sample, cell ice-cold PBS 2 times, then cleaned with 1ml Milli-Q H2O, exhaust liquid afterwards, tries one's best
There should not be residual.1ml is added per hole through autoclaved Milli-Q H2O, cell is scraped with cell scraper, is transferred to precooling
In 2ml EP pipes.Ultrasound 30s, totally 2 times on ice.Gained cell pyrolysis liquid is in 4 DEG C, 12000rpm centrifugations 15min；Shift supernatant
To new precooling EP pipes.Take 100 μ l albumen supernatants, plus 100 μ l Fresh pNPP solution (1.0mg/mL PNPP,
1M diethanolamine buffers, 0.5mM MgCl2)；100 μ L albumen supernatants, plus 100 μ l BCA reaction solutions are taken in addition, in 37 DEG C
30min is incubated, ALP determines the NaOH terminating reactions that hole adds 50 μ l concentration to be 3M, ALP reaction products are determined at 405nm to nitre
Albumen and the reacted absorbance of BCA liquid are determined at the absorbance of base phenol, 570nm.The absorbance of measure is substituted into pair respectively
Linear equation is answered, is quantified.OD405 and OD570 in calculation formula have subtracted background values respectively.With respect to alkaline phosphatase
Active (%)=dosing group (ALP amount/Tot Prot)/control group (ALP amount/Tot Prot) × 100%..The institute of result figure 6
Show, N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -
1,2- azoles -3- formamides raise the activity of intracellular alkaline phosphatase.
《Embodiment 4》The measure of mineralising
C3H10T1/2 cells osteogenic induction liquid (complete culture solution contain 25 μ g/ml ascorbic acid, 5mM β-phosphoglycerol,
10nM dexamethasone) middle culture 21d measure calcium tubercle formational situations.Cell PBS 2 times, exhaust PBS.With pre- under normal temperature
70% cold ethanol fixes cell 1h, and fixer is removed afterwards, is washed 2 times with Milli-Q.Add the alizarin red dye of Fresh
Liquid (pH4.2,40mM), dyes after 10min, is gently cleaned with Milli-Q water 3 times at normal temperatures, removes unspecific staining.
Scanner scanning image is used, setting is output as tif. forms.As a result such as Fig. 7, N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,
2- azoles -3- formamides increase the number of calcium scoring.
《Embodiment 5》Osteogenesis gene RT-PCR is detected
MC3T3-E1 cells are inoculated in 6 orifice plates, 3 × 105/ hole, after cell growth is converged, is changed to the μ of osteogenic induction liquid 25
G/ml ascorbic acid, 5mM β-phosphoglycerol.Trizol cell lysis, extracts intracellular mRNA.Determine after RNA concentration and purity,
Carry out reverse transcription and prepare cDNA.1 μ g total serum IgEs and 4 μ l 5 × PimeScript RT Master Mix are taken to be added in PCR pipe,
And supplied with the water without RNase to 20 μ l systems.Reverse transcription condition：37 DEG C of 30min, 85 DEG C of 5s, 4 DEG C of coolings.PCR uses 20 μ l
Reaction system：2 × Easy Taq, 10 μM of PCR primers and 50ng cDNA templates.Primer sequence is as follows：Alp：Forward primer：
5’-tgaccttctctcctccatcc-3’(SEQ ID NO.1)；Reverse primer：5’-cttcctgggagtctcatcct-3’
(SEQ ID NO.2)；Runx2：Forward primer：5’-agagtcagattacagatcccagg-3’(SEQ ID NO.3)；Reversely
Primer：5’-tggctcttcttactgagagagg-3’(SEQ ID NO.4)；Opn：Forward primer：5’-
tccaaagccagcctggaac-3’(SEQ ID NO.5)；Reverse primer：5’-tgacctcagaagatgaactc-3’(SEQ
ID NO.6)；GAPDH：Forward primer：5’- catggccttccgtgttccta-3’(SEQ ID NO.7)；Reverse primer：5’-
cctgcttcaccaccttcttgat-3’(SEQ ID NO.8)；Reaction condition：94℃5min；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C
1min, 30 circulations；72℃10min.As a result such as Fig. 8：N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formyls
Amine raises intracellular Alp, Opn, Runx2 mRNA level in-site.
《Embodiment 6》TRAP dyeing detection osteoclast formations
By RAW264.7 cells with 2.5 × 103It is individual to be inoculated in 96 orifice plates per hole, culture medium is discarded after cell is covered with, is trained
Support based component：The high sugared complete mediums of DMEM, then by following packet Fiber differentiation：A groups：control；B groups：50ng/ml
RANKL；C groups：50ng/ml RANKL+1 μM N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides；D groups：
50ng/ml RANKL+5 μM N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides；E groups：50ng/ml
RANKL+10 μM of N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamide；F groups：50ng/ml RANKL+20μ
M N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides.After 6 days, TRAP staining kits (sigma,
386A) dye.As a result such as Fig. 9, N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides suppress osteoclast
Formed.
Claims (4)
1. a kind of application of compound of up-regulation Runx2 transcriptional activities in preventing and treating medicine for treating osteoporosis is prepared；
The compound is N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- formamides.
2. the application described in claim 1, it is characterized in that, the compound is based on adjusting cell sieve on Runx2 transcriptional activities
What modeling type MC3T3-E1-OSE was obtained.
3. the application described in claim 1 or 2, it is characterized in that, the cell screening model M C3T3-E1-OSE is to utilize
P6OSE2 information, design primer with expand on p6OSE2 plasmids 6 repetitions Runx2 binding members (6OSE2) and downstream most
Small BGP promoter fragment and pGL4.17 plasmids, obtain recombinant plasmid pGL4.17-OSE, and transfection MC3T3-E1 cells are obtained
's.
4. by active component of N- (Cvclopropvlmethvl) -5- (thiophene -2- bases) -1,2- azoles -3- benzamide compounds and pharmaceutically
Application of the composition of acceptable carrier composition in preventing and treating medicine for treating osteoporosis is prepared.
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| CN201510319767.5A CN104873499B (en) | 2015-06-11 | 2015-06-11 | A kind of application of compound of up-regulation Runx2 transcriptional activities in osteoporosis is prevented and treated |
| PCT/CN2015/000908 WO2016197284A1 (en) | 2015-06-11 | 2015-12-16 | Use of compound for up-regulating transcriptional activity of runx2 for preventing and treating osteoporosis |
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| CN201510319767.5A CN104873499B (en) | 2015-06-11 | 2015-06-11 | A kind of application of compound of up-regulation Runx2 transcriptional activities in osteoporosis is prevented and treated |
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| CN104873499B (en) * | 2015-06-11 | 2017-10-17 | 中国医学科学院医药生物技术研究所 | A kind of application of compound of up-regulation Runx2 transcriptional activities in osteoporosis is prevented and treated |
| CN109722413B (en) * | 2019-03-19 | 2022-08-16 | 上海揽微赛尔生物科技有限公司 | Osteogenesis inducer and application thereof in osteogenic induced differentiation of adipose mesenchymal stem cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101528706A (en) * | 2006-10-02 | 2009-09-09 | 财团法人国家卫生研究院 | Pyrazole compounds |
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| KR100848073B1 (en) * | 2004-12-14 | 2008-07-24 | 주식회사바이오러넥스 | 2 2 Method to enhance the bone formation activity of BMP by Runx2 acetylation |
| CN100396667C (en) * | 2006-02-20 | 2008-06-25 | 中国医学科学院医药生物技术研究所 | Unsaturated five heterocycle compound for up-regulating bone formation protein BMP-2 expression activity |
| BRPI0920764A2 (en) * | 2008-10-31 | 2015-12-22 | Snu R&Db Foundation | spiro chiral carbon structure compound, method of preparation thereof, and pharmaceutical composition containing the same |
| CN104873499B (en) * | 2015-06-11 | 2017-10-17 | 中国医学科学院医药生物技术研究所 | A kind of application of compound of up-regulation Runx2 transcriptional activities in osteoporosis is prevented and treated |
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| CN101528706A (en) * | 2006-10-02 | 2009-09-09 | 财团法人国家卫生研究院 | Pyrazole compounds |
Non-Patent Citations (2)
| Title |
|---|
| Gene promoter polymorphism of RUNX2 and risk of osteoporosis in postmenopausal Indonesian women;Elza I Auerkari等;《SAGE Open Medicine》;20140114;1-6 * |
| RUNX2与骨代谢的调控;李彬等;《中国骨质疏松杂志》;20090131;第15卷(第1期);63-67 * |
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| CN104873499A (en) | 2015-09-02 |
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