CN102895652B

CN102895652B - Method for promoting osteoblast differentiation by using Runx2 and Osterix and application thereof

Info

Publication number: CN102895652B
Application number: CN201110211205.0A
Authority: CN
Inventors: 刘文广; 孟令仪; 张忠丽; 曾宪录
Original assignee: Northeast Normal University
Current assignee: Northeast Normal University
Priority date: 2011-07-27
Filing date: 2011-07-27
Publication date: 2014-07-09
Anticipated expiration: 2031-07-27
Also published as: CN102895652A

Abstract

The invention relates to a method for co-expression of Runx2 and Osterix in mesenchymal stem cells or other non-osteoblasts by a special mode that the expression level of Runx2 is less than the expression level of Osterix, thus speeding up the induction of osteoblast differentiation. The invention provides a pharmaceutical composition for prevention or treatment of osteoblast differentiation related diseases. The pharmaceutical composition contains Runx2 protein and Osterix protein, wherein the Runx2 protein content is smaller than the Osterix protein content. The invention also provides application of the Runx2 protein and the Osterix protein in preparation of the pharmaceutical composition for prevention or treatment of osteoblast differentiation related diseases, and in the pharmaceutical composition, the content of the Runx2 protein is less than that of the Osterix protein. The pharmaceutical composition and the special co-expression mode of Runx2 and Osterix provided in the invention can be used for treatment of osteoporosis, osteogenesis imperfecta, periodontal diseases, fractures and other bone diseases, and also can be used for research, development and production of tissue-engineered bones. The invention also provides a method for screening drugs preventing and treating bone diseases.

Description

Utilize Runx2 and Osterix to promote method and the application thereof of osteoblast differentiation

Technical field

The present invention relates to Runx2 and the Osterix application in the pharmaceutical composition for the preparation of prevention or the treatment disease relevant to osteoblast differentiation, in described pharmaceutical composition, the content of Runx2 albumen is less than the content of Osterix albumen, preferably, the ratio of Runx2 protein content and Osterix protein content is 1: N, wherein N is greater than 1, for example 1: 2,1: 3, to 1: 4 even lower ratio, preferred ratio is 1: 4.Particularly, the present invention is accelerated to induce the directed osteoblast differentiation of non-osteoblast or is strengthened osteoblastic function by the mode coexpression Runx2 and the Osterix that are less than expression (or active) level of Osterix with Runx2 expression (or active) level.And according to changing that Runx2 expresses the expression (or active) of (or active) level and Osterix thus the ratio of level causes the variation of osteoblast differentiation and function on purpose screens the novel drugs that osteopathia is prevented or treated.

Background technology

Osteopathia has multiple.Wherein osteoporosis is exactly a global difficult medical problem.Estimate according to World Health Organization (WHO), the whole world is annual approximately there are 2,500,000 fracture compound comminuteds that cause because of osteoporosis, and this numeral further rises the aging development of the society of various countries along with including China.Osteoporosis is not only brought misery to people, return society of various countries and bring heavy financial burden, therefore each state all the amount of having high input manpower and materials carry out basic and applied research for osteoporosis prevention and treatment.Osteoporosis occurs conventionally in situation inconspicuous, is that bone resorption causes considerably beyond bone formation because the functional equilibrium between osteoblast (being responsible for bone formation) and osteoclast (being responsible for bone resorption) is broken.Although people can develop to suppress osteoporosis therapy medicine and the method that bone resorption is object, but the active drug of promoting bone growing and method only have the exploitation of EliLilly company, parathyroid hormone (parathyroid hormone) (hereinafter to be referred as PTH) the injection Forteo being ratified by U.S. FDA 11 the end of month in 2002 so far ^tM, be used for the treatment of serious osteoporosis.People for find more and more effectively, Therapeutic Method easily, in the urgent need to the molecular mechanism of osteoblast differentiation and function thereof being had to sufficient understanding (Goltzman, 2002; Harada and Rodan, 2003; Lian and Stein, 2003).

Repair clinically bone injury, the medical treatment that promotes union of fracture and bone tissue engineer research and development and still have many Problems.Wherein one of subject matter is exactly to be limited to from other donorcells, to comprise various stem cell, obtains cell derived a large amount of, the powerful skeletonization of energy.Therefore histoorgan engineering is very paid close attention to the research contents of osteoblastic directional induction, stable, the extensive amplification in vitro of cell phenotype and tissue engineered bone scale external structure etc.But this problem does not obtain basic solution.In this area, bone morphogenetic protein(BMP) (being called for short BMPs) has been carried out clinically and has been applied and paid close attention to widely.BMP can promote non-osteoblastic ossification.But application BMP not only needs to use very large dosage; Also may have a negative impact to the cell of non-bone tissue.In a word, will have breakthrough in this field, also needing has sufficient understanding (Phillips and Garcia, 2008) to the molecular mechanism of osteoblast differentiation and function thereof.

At present, many researchs that the bone resorption participating in exploitation osteoclast suppresses all make remarkable progress, and people have developed the bone resorption inhibitor participating in the osteoclast of getting permission clinically to use up to now.Although BMPs and PTH (1-34) are considered to osteoplastic promoter, thereby and have people to screen this micromolecular of statins to induce the expression of BMP to strengthen bone formation (Mundy et al., 1999), but also do not find clinically up to now other small-molecule drug.

The cellular pathways of osteoblast differentiation is from the mescenchymal stem cell of pluripotency, via the jejune or early stage osteoblast stage, differentiation is to ripe functional osteoblast, and the bone matrix embedding of being secreted by self after osteoblast maturation is also further converted to osteocyte.Osteoblast differentiation is the result that is subject to the various effects in inside and outside, wherein, control differentiation crucial transcription factor play determine or maintain effect (Liu Wenguang, etc., 2003; Nakashima and de Crombrugghe, 2003), the molecular events (Schroeder, et al., 2005) of the cell differentiation that further regulative transcription factor causes such as various epigenetic mechanism.

Research shows, core binding factor alpha 1 (Core binding factor α 1, Cbfa1) is a kind of transcription factor of key, controls mescenchymal stem cell to osteoblast differentiation (Komori, et al., 1997).In mouse, reject Cbfa1, osteoblast differentiation can be prevented, and bone can not form.Cbfa1 is regulating and controlling much bone matrix protein gene expression.The factor (comprising hormone, somatomedin, bone morphogenetic protein(BMP) etc.) of many promotion bone developments may be by Cbfa1 and the osteoblast differentiation instructing approach thereof play a role (Yamaguchi, et al., 2000).Therefore, Cbfa1 is considered to control " major gene " (master gene) of bone formation and growth.After 2000, Cbfa1 is again by RNTO Runx2 (Runt related gene 2).Above-mentioned PTH and BMPs promote or induce the final application point of bone formation to be considered to is exactly Runx2 (Krishnan, et al., 2003; Ito and Miyazono, 2003).Runx2 can induce the directed Osteoblast Differentiation of skin flbroblast and powerful ossification (Phillips and Garcia under certain environment, 2008), and Runx2 can obviously promote the induced osteogenesis effect (Ito and Miyazono, 2003) of BMPs.

Osterix found in January, 2002, in Runx2 downstream, controlled osteoblast differentiation and osteoplastic another crucial transcription factor (Nakashima, et al., 2002).In mouse, reject Osterix, osteoblast differentiation will be prevented, and bone can not form.But the expression of Osterix be unable to do without Runx2, and bring into play pivotal role in the differentiation pathway in its downstream.The activity of Runx2 and Osterix all interacts and be affected (Koga, et al., 2005 with other factors; Schroeder, et al., 2005).

Latest Progress shows, crucial transcription factor combination plays a part very important to cell programming and the reprogrammed in stem cell field.In nearest stem-cell research field, go out the stem cell characteristic of pluripotency from somatic induction, i.e. somatic reprogrammed, research obtained many breakthrough achievements that attract people's attention.The process of the hereditary information reprogrammed of inductive pluripotent stem cells (iPS) can be by expressing four kinds of transcription factor as OCT3/4, SOX2, and Klf4, and c-Myc can reach.IPS is closely similar with embryonic stem cell, to such an extent as to can become individuality (Okita, et al., 2007 by germline transmission and embryo development procedure; Takahashi, et al., 2007).How huge the research work of this series of iPS fully given prominence to transcription factor or its and be combined in and set up and maintain or reinvent (programming of cell and reprogrammed) on cell phenotype and can bring into play power.We also notice simultaneously, induced dry-cell produce four kinds of required genes be by retroviral vector in 1: 1: 1: 1 ratio infects somatic situation.In fact, in stem-cell research field in recent years, utilize the combination of transcription factor can successfully make stem cell directional differentiation.For example there is researcher three kinds of Sox genes to be proceeded to the precursor of chondrocyte in 1: 1: 1 ratio, successfully induced cartilaginous tissue and maintained the characteristic (Ikeda, et al., 2004) of its permanent cartilage.

For the progress of the above-mentioned combination about transcription factor, we further guess that the different ratios between transcription factor also may play a significant role in the process of stem cell directional differentiation, we find subsequently, the also degree of the directed osteoblast differentiation of profound influence mescenchymal stem cell of the ratio of the expression between the crucial transcription factor of these two control osteoblast differentiation of Runx2 and Osterix.We further infer, in bone development process, osteoblastic formation dissimilar or different developmental phases is also probably to realize by set up the intergenic expression of different Runx2 and Osterix or active ratio in osteoblast.

Applicant publish thesis October calendar year 2001 under the little Shou Wen of the keeping doctor of Japan instructs (Liu, etal., J.Cell Biol.155:157-166,2001).We study discovery by transgenic mouse, and Runx2 is this cell maturation of overexpression meeting severe inhibition (Liu, et al., 2001) in osteoblast.Its main manifestations is that the osteoblast of comparative maturity and the osteocyte that is further transformed by osteoblast sharply reduce in transgenic bone, and more jejune osteoblast obviously increases, so that osteodysplasty.Therefore we think, Runx2 promotes early stage osteoblast differentiation, suppresses the conversion to osteocyte, and in the osteoblast of functional, comparative maturity, its expression or its transcriptional activity must maintain relatively low level.Runx2 transgenic mouse, though fail promoting bone growing, promotes early stage osteoblast differentiation and quantity, and therefore, we are proposing to utilize the hypothesis (Liu, et al., 2001) of Runx2 promoting bone growing.This hypothesis is exactly suggestion improves Runx2 in early days expression or activity in osteoblast differentiation, reduces expression or the activity of Runx2 in the ripe osteoblast differentiation stage.And much external research shows, Runx2 can induced osteogenesis cell differentiation and bone formation effect under certain environment.In this body, show with external contradiction, people can't effectively utilize Runx2 to promote osteoblast differentiation and bone formation.

The inventor proves that different Runx2 and Osterix relative expression/active ratio have Different Effects for osteoblast differentiation by experiment, the protein expression ratio of a kind of special Runx2 and Osterix of having found can accelerate to promote osteoblastic differentiation, thereby promoting bone growing, thereby complete the present invention.

Summary of the invention

The present invention relates to Runx2 and the Osterix application in the pharmaceutical composition for the preparation of prevention or the treatment disease relevant to osteoblast differentiation, in described pharmaceutical composition, the content of Runx2 albumen is less than the content of Osterix albumen, preferably, the ratio of Runx2 protein content and Osterix protein content is 1: N, wherein N is greater than 1, for example from 1: 2,1: 3, to 1: 4 even lower ratio, preferred ratio is 1: 4.Particularly, the present invention is accelerated to induce the directed osteoblast differentiation of non-osteoblast or is strengthened osteoblastic function by the mode coexpression Runx2 and the Osterix that are less than expression (or active) level of Osterix with a kind of Runx2 expression (or active) level.And according to changing that Runx2 expresses the expression (or active) of (or active) level and Osterix thus the ratio of level causes the variation of osteoblast differentiation and function on purpose screens the novel drugs that osteopathia is prevented or treated.

Technical problem:

Object of the present invention is by accelerating to promote osteoblast differentiation according to the Runx2 of special ratios and Osterix coexpression in mescenchymal stem cell or other non-osteoblast.The method can promoting bone growing and is contributed to treat various osteonosus, for example osteoporosis, fracture, osteogenesis imperfecta etc.

Another object of the present invention is to provide a kind of for preventing or the pharmaceutical composition of the disease that treatment is relevant to osteoblast differentiation, it comprises Runx2 albumen and Osterix albumen, and wherein the content of Runx2 albumen is less than the content of Osterix albumen, preferably the ratio of Runx2 protein content and Osterix protein content can be any ratio in following proportion: 1: N, wherein N is greater than 1, for example, 1: 2,1: 3, to 1: 4 even lower ratio.Preferably, described pharmaceutical composition also comprises pharmaceutical carrier.

Another object of the present invention is to provide the application in the pharmaceutical composition for the preparation of prevention or the treatment disease relevant to osteoblast differentiation of Runx2 albumen and Osterix albumen, in described pharmaceutical composition, the content of Runx2 albumen is less than the content of Osterix albumen.Preferably, in described pharmaceutical composition, the ratio of Runx2 protein content and Osterix protein content can be any ratio in following proportion: 1: N, wherein N is greater than 1, for example, 1: 2,1: 3, to 1: 4 even lower ratio.

The wherein said disease relevant to osteoblast differentiation is selected from osteoporosis, fracture, osteogenesis imperfecta or periodontal disease etc.

Another object of the present invention is to provide Runx2 gene and the application of Osterix gene in the test kit for the preparation of promotion osteoblast differentiation, the recombinant precursor of the recombinant precursor that described test kit comprises Runx2 gene and Osterix gene.In the time using described test kit, with the recombinant precursor of described Runx2 gene and the recombinant precursor of the Osterix gene suitable retrovirus production cell of transfection respectively, collect respectively retroviral supernatant, by the retroviral supernatant that contains Runx2 gene and the retroviral supernatant that contains Osterix gene with 1: (wherein M is greater than 1 to the ratio of M, for example, 1: 2, 1: 3, the even lower ratio to 1: 4) mix, jointly infect mescenchymal stem cell or other non-osteoblast, thereby the ratio that makes Runx2 protein content and Osterix protein content reaches any ratio in following proportion: 1: N, wherein N is greater than 1, for example, 1: 2, 1: 3, the even lower ratio to 1: 4.Wherein, M and N are greater than any number of 1, and those skilled in the art can determine suitable numerical value.Described test kit can for the production of tissue engineered bone and with the gene therapy of osteoblast differentiation relevant disease.

A further object of the invention is to provide a kind of method of the medicine that screens promoting bone growing, and it comprises the steps:

(1) build respectively the recombinant precursor of Runx2 gene and the recombinant precursor of Osterix gene;

(2) utilize suitable retrovirus, with the recombinant precursor of the Runx2 gene in step (1) and the recombinant precursor infected cell of Osterix gene, screen following three kinds of monoclonal reconstitution cells: (i) wherein Runx2 protein expression level is less than Osterix protein expression level, (ii) wherein Runx2 protein expression level equals Osterix protein expression level, and (iii) wherein Runx2 protein expression level be greater than Osterix protein expression level; With

(3) step (2) is screened to the independent effect of accepting or not accepting compound to be selected of the every kind of cell obtaining, observe the variation of Runx2 protein expression level and Osterix protein expression level ratio, and in conjunction with the variation observation analysis osteoblast differentiation of described ratio or the enhancing of function or weaken, thereby complete the screening of the medicine of promoting bone growing;

Wherein in step (2), cell used is selected from mescenchymal stem cell or other non-osteoblast, for example, and bone marrow stem cell, Skin Cell, adipose cell etc.

Runx2 gene of the present invention and Osterix gene can derive from any mammalian species, preferably derive from mice and people, more preferably derive from people.

The present invention is also provided for the screening system of the medicine that screens promoting bone growing, described system comprises following three kinds of monoclonal reconstitution cells: (i) wherein Runx2 protein expression level is less than the monoclonal reconstitution cell of Osterix protein expression level (R < O), (ii) wherein Runx2 protein expression level equals the monoclonal reconstitution cell of Osterix protein expression level (R=O), (iii) wherein Runx2 protein expression level is greater than the monoclonal reconstitution cell of Osterix protein expression level (R > O).

In described screening system, cell used can be selected from mescenchymal stem cell or other non-osteoblast, for example, and bone marrow stem cell, Skin Cell, adipose cell etc.

Technical solution:

The invention provides by Runx2 and express the mode coexpression Runx2 of expression (or active) level that (or active) level is less than Osterix and Osterix in mescenchymal stem cell or other non-osteoblast, thereby accelerate the method for induced osteogenesis cell differentiation.

Runx2 and Osterix are the crucial transcription factor that determines osteoblast differentiation, in the formation of bone and the process of growth, leave any a kind of factor wherein and all can cause bone formation not occur.The two all regulates and controls the expression of the special gene of bone, and much research shows, in cultured cells in vitro, abduction delivering the two one of, can make these cells all give expression to the special gene of multiple bone, thereby cause osteoblastic differentiating characteristic.But, the inventor finds, with identical protein level, single expression Runx2 or Osterix do not cause similar Osteoblast Differentiation level in C3H10T1/2 mesenchyme versatile stem cell respectively, no matter Osterix is in alkaline phosphatase activities level, calcification level and induce the ability of the expression of the special gene of multiple bone to be all significantly smaller than Runx2.But when by the two protein expression level co expression in varing proportions during in the versatile stem cell of same, the inventor finds, in the time that Runx2 is lower than the protein expression level of Osterix, co expression Runx2 and Osterix be the important symbol-calcification of induced osteogenesis cell differentiation extremely significantly.In the time that the protein expression level of Runx2 and Osterix is close, when lower than the protein level of Osterix with Runx2, to compare, calcification effect significantly reduces, but is still significantly improved compared with when higher than the protein expression level of Osterix with Runx2.When Runx2 is higher than the protein level of Osterix when only expressing Runx2 compared with, calcification effect does not change, although the protein level of Runx2 does not have notable difference under both of these case.No matter all further on the basis of single expression Runx2, do not induce the activity of the important symbol-alkali phosphatase of early stage osteoblast differentiation with which kind of ratio protein level co expression Runx2 and Osterix.Analyze and show by RT-PCR, in the ability of expression of inducing the special gene of multiple bone, for example type i collagen albumen, osteocalcin (Osteocalcin), these ripe osteoblastic marker gene of bone sialoprotein (Bonesialoprotein), compared with when higher than the protein level of Osterix with Runx2, can induce extremely significantly the expression of these genes than the low mode co expression Runx2 of the protein level of Osterix and Osterix with Runx2.Moreover, the inventor also finds, when Runx2 is lower than the protein level of Osterix, co expression Runx2 and Osterix can also earlier induce calcification, and at synchronization, in the time that the protein level of Runx2 and Osterix is close, Runx2 is when higher than the protein level of Osterix or while only expressing Runx2, calcification effect does not all occur.

For obtain Runx2 than the protein level of Osterix low, coexpression the two in homocellular effect, can be by the retroviral supernatant that contains Runx2 gene and the retroviral granule supernatant that contains Osterix gene be mixed and jointly infect C3H10T1/2 cell with the ratio of 1: 4.

Many micromolecular compounds may promote the differentiation of induced osteogenesis cell or the formation of bone, for example TSA (, hachimycin).But the inventor finds, TSA processes not to be had further promote protein expression level co expression Runx2 and Osterix in varing proportions or only express the calcification level of the C3H10T1/2 mesenchyme versatile stem cell of Runx2, has but reduced on the contrary the calcification level of these kind of cell.Consider inevitable Runx2 and the Osterix of simultaneously containing of osteoblast, perhaps due to the environment inside and outside different cells, different osteoblastic Runx2 and mRNA, albumen or the activity level of Osterix are also different, the inventor thinks, TSA processes has negative effect for osteoblastic impact.This prompting the inventor consider to utilize the method for the different cells of foundation provided by the present invention to evaluate the promotion of various micromolecular compounds and affect osteoplastic ability and effect, also can directly be used as the method for the drug screening of bone disease treatment.

Due to the high conservative of Runx2 and Osterix, the Runx2 that the present invention is used and Osterix can derive from any mammalian species, preferably derive from mice or people, most preferably derive from people.In one embodiment of the invention, Runx2 and Osterix derive from mice.Mice Runx2 compares with people Runx2, is 92% at the homology of aminoacid sequence; Be 92%-96% at the homology of nucleotide sequence.Mice Osterix compares with people Osterix, is 95% at the homology of aminoacid sequence; Be 89% at the homology of nucleotide sequence.

In one aspect, can directly take the medicine of the Runx2 of comprising albumen of the present invention and Osterix albumen to the experimenter who suffers from the osteopathia relevant to osteoblast differentiation, in described medicine, Runx2 protein content is lower than Osterix protein content.For example, can and use etc. at treatment place local injection by oral, intramuscular injection or intravenous injection.It should be appreciated by those skilled in the art that and can have a variety of conventional methods to reach the object of using.

Except protein level, exist in certain proportion Runx2 and Osterix gene and expression vector thereof also to can be used as a kind of active constituents of medicine, its corresponding seemingly protein-based.The known variety carrier that exists of those skilled in the art can be transported this two kinds of genes, or has its expression vector of multiple expression.Similarly, also can, by injection or local application, be less than the ratio of Osterix protein expression level according to Runx2 protein expression level, Runx2 gene recombinaton construct and Osterix gene recombinaton construct are administered in body or relevant intralesional.

Also can prepare express the mescenchymal stem cell that Runx2 protein level is lower than Osterix protein level (as, bone marrow stem cell) or other non-osteoblast (as, autologous skin or adipose cell etc.), such reconstitution cell can be prepared by following step: build respectively the recombinant precursor of Runx2 gene and the recombinant precursor of Osterix gene, produce cell with the suitable retrovirus of these two kinds of recombinant precursor transfections respectively, collect respectively retroviral supernatant, by the retroviral supernatant that contains Runx2 gene and the retroviral supernatant that contains Osterix gene with 1: the ratio of M is mixed, jointly infect mescenchymal stem cell or other non-osteoblast, wherein M is greater than 1, for example, 1: 2, 1: 3, the even lower ratio to 1: 4, thereby the ratio that makes Runx2 protein content and Osterix protein content reaches any ratio in following proportion: 1: N, wherein N is greater than 1, for example, 1: 2, 1: 3, the even lower ratio to 1: 4.It should be appreciated by those skilled in the art that the method that obtains in the art such reconstitution cell has a lot, for example, by adenovirus or retroviral mode, also can, by non-viral mode, for example, utilize the transfection reagent of various applicable treatments to carry out transfection.

Can be by injection or the mode of local application by the expression Runx2 protein level of above-mentioned preparation than the low mescenchymal stem cell of Osterix protein level or other non-osteoblast is administered in subject or intralesional is treated osteopathia.Preferably, described experimenter is mammal, and described Runx2 gene and Osterix gene source are in described experimenter to be treated.Preferably, described experimenter is mice or people, more preferably people.Therefore,, when for people experimenter, described Runx2 gene and Osterix gene preferably derive from people.

The expression Runx2 protein level that the present invention obtains also goes for the production of tissue engineered bone than the low mescenchymal stem cell of Osterix protein level or other non-osteoblastic method.Utilize method of the present invention can accelerate to promote tissue engineered bone to produce.

Brief description of the drawings

In detailed description below in conjunction with accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:

Fig. 1 is photo, shows the effect to alkaline phosphatase activities level and calcification level in C3H10T1/2 cell with identical protein level single expression Runx2 or Osterix.Figure 1A shows protein expression situation, and Figure 1B shows the situation of alkaline phosphatase activities, and Fig. 1 C shows the situation of calcification level.

Fig. 2 is photo, shows the effect to alkaline phosphatase activities level and calcification level in C3H10T1/2 cell by Runx2 and Osterix protein expression level co expression in varing proportions.Fig. 2 A shows protein expression situation, and Fig. 2 B shows the situation of alkaline phosphatase activities, and Fig. 2 C shows the situation of calcification level.

Fig. 3 is photo, and on the retroviral supernatant that demonstration contains Runx2 gene and the retroviral that contains Osteix gene, feelings liquid mixes and jointly infects after C3H10T1/2 cell with the ratio of 1: 4, and Runx2 is than the ratio of the protein level of Osterix.

Fig. 4 is photo, shows that TSA processes the impact of the calcification level of the C3H10T1/2 cell of Runx2 on expressing different proportion protein level and Osterix.

Sequence table explanation:

Serial number	Title
		SEQ.ID.No.1	The aminoacid sequence of mice Runx2 albumen
SEQ.ID.No.2	The DNA sequence of mice Runx2
		SEQ.ID.No.3	The aminoacid sequence of mice Osterix albumen
SEQ.ID.No.4	The DNA sequence of mice Osterix

Detailed description of the invention

Reality of the present invention and the currently preferred embodiments property illustrated, as shown in the following examples.But, will understand those skilled in the art in the time considering present disclosure, can in concept of the present invention and scope, make and modify and improve.

Embodiment 1: plasmid construction and cell culture

<1-1> cell culture

Plat-E cell (Cell Biolabs Inc.) is incubated at and contains 10% hyclone (Hyclone), 1 μ g/ml puromycin (puromycin, Sigma), 10 μ g/ml blasticidin (brasticidine, Sigma), 100U/ml penicillin (Sigma), also adherent in being coated with 6 orifice plates of rat tail type i collagen albumen in the DMDM culture medium (Gibco) of 100 μ g/ml streptomycins (Sigma).Other cell strain obtaining by C3H10T1/2 cell strain (ATCC, CCL-226) and from C3H10T1/2 cell is incubated at and contains 10% hyclone, 100U/ml penicillin, and in the DMDM culture medium of 100 μ g/ml streptomycins.Above cell is all incubated at 37 DEG C, 5%CO ₂incubator in.

<1-2> plasmid construction

By the total length Runx2 (SEQ.ID.No.2 of FLAG labelling, derive from mice) and the total length Osterix (SEQ.ID.No.4 of FLAG labelling, derive from mice) cDNA be inserted into respectively in pMXs/neo carrier (Cell Biolabs Inc), or the total length Osterix of FLAG labelling (SEQ.ID.No.4) is inserted in pMXs/puro carrier (Cell Biolabs Inc).By the recombinant precursor of above-mentioned acquisition called after Runx2-pMXs/neo respectively, Osterix-pMXs/neo, Osterix-pMXs/puro.

The antibody that the present invention uses is the antibody (Sigma) for FLAG label.

Embodiment 2: stably express Runx2 or Osterix and Simultaneous Stabilization are expressed the foundation of the C3H10T1/2 cell strain of Runx2 and Osterix respectively

The present embodiment infects by cell transfecting, retrovirus and the immune marking completes.

Particularly, using pMXs/neo (as negative control), and the Runx2-pMXs/neo building in embodiment 1, Osterix-pMXs/neo plasmid proceeds to respectively in Plat-E cell with Fugene 6 transfection reagents (Promega).After 72 hours, the supernatant of culture medium (containing the DMEM culture medium of 10% hyclone) has contained retroviral.Filter through 0.45 μ m filter membrane (Milipore), after filtrate is mixed according to 1000: 1 with 5mg/ml polybrene (polybrene, Sigma), be added to and carry in adherent good C3H10T1/2 cell the previous day.After 6 hours, change fresh culture and continue and cultivate 24 hours.Cell obtains with G418 screening the cell being infected by retroviral, and the cell not infecting is killed.Cellular expression Runx2 and Osterix protein level can detect by the immune marking, and use the antibody of anti-FLAG to do first antibody detection.Reconstitution cell called after Ctrl, Runx2 and the Osterix respectively of pMXs/neo, Runx2-pMXs/neo, Osterix-pMXs/neo will successfully have been transformed respectively.The cell infecting from the above-mentioned retroviral that is contained Runx2 or Osterix, choose respectively a representational monoclonal by selecting monoclonal method, no matter these monoclonals are respectively on Runx2 or Osterix protein level, or all representative in the activity level of the alkali phosphatase causing at Runx2 or Osterix and calcification level.By the cell of selecting above called after Runx2-10 and Osterix-2 respectively.

In order to obtain the cell strain of expressing Runx2 and Osterix by different proportion simultaneously, the Osterix-pMXs/puro plasmid building in embodiment 1 is proceeded in Plat-E cell with Fugene 6 transfection reagents, and infect the representational Runx2-10 monoclonal cell of acquisition before this according to aforementioned process, jointly screen and again select by monoclonal by 400 μ g/ml G418 and 20 μ g/ml puromycins, obtaining and contain the Runx2 of different proportion and the cell strain of Osterix protein level simultaneously.By the cell of screening above called after R > O respectively, R=O and R < O (represent that respectively the protein expression level of Runx2 is higher than Osterix protein expression level, the protein expression level of Runx2 equals Osterix protein expression level, and the protein expression level of Runx2 is less than Osterix protein expression level).Runx2 and Osterix protein level and can detect by the immune marking in same intracellular ratio, and use the antibody of anti-FLAG to make first antibody to detect.

In addition, for Runx2 in the same cell of convenient acquisition is than the low result of the protein level of Osterix, can respectively Runx2-pMXs/neo and Osterix-pMXs/neo plasmid be proceeded in Plat-E cell with Fugene6 transfection reagent, after 72 hours, the retroviral supernatant that contains Runx2 gene and the retroviral supernatant that contains Osteix gene be mixed and jointly infect C3H10T1/2 cell with the ratio of 1: 4.Runx2 and Osterix protein level ratio can detect by the immune marking, and use the antibody of anti-FLAG to do first antibody detection.The results are shown in Figure 3.

A little less than the energy force rate Runx2 of embodiment 3:Osterix induced osteogenesis cell differentiation

By the Ctrl obtaining in embodiment 2, Runx2 and Osterix cell (add 100 μ g/ml ascorbic acid (Sigma) and 5mM β-phosphoglycerol (β-glycerophosphate, Sigma)) in osteoblast differentiation culture medium in normal culture medium, and alkaline phosphatase activities dyeing and von Kossa dyeing are carried out in differentiation after 6 days.Found that, Osterix is in induction alkaline phosphatase activities level or the ability of calcification level is all significantly smaller than Runx2.Result is referring to Figure 1B and Fig. 1 C.

By the Ctrl obtaining in embodiment 2, Runx2 and Osterix cell carry out alkaline phosphatase activities dyeing after being also cultured in normal culture medium and covering with.Result still shows that Osterix is weaker than Runx2 in the ability of induction alkaline phosphatase activities level.Result is referring to Figure 1B.

Embodiment 4: the protein level co expression Runx2 and the Osterix that are less than Osterix with Runx2 protein level in same C3H10T1/2 cell can accelerate the differentiation of induced osteogenesis cell

By the Runx2-10 obtaining in embodiment 2, R < O, R=O and R > O monoclonal cell (add 100 μ g/ml ascorbic acid (Sigma) and 5mM β-phosphoglycerol (Sigma)) respectively in osteoblast differentiation culture medium in normal culture medium to be broken up after 4 or 6 days and carries out von Kossa dyeing.Found that, R < O cell is significantly induced calcification level.Result is referring to Fig. 2 C.

By the Runx2-10 obtaining in embodiment 2, R < O, R=O and R > O monoclonal cell carry out alkaline phosphatase activities dyeing after being also cultured in normal culture medium and covering with.Result shows that these cells do not affect alkaline phosphatase activities.Result is referring to Fig. 2 B.

Embodiment 5: the effect to osteoblast differentiation or function effect that can verify TSA with the C3H10T1/2 cell of different protein level co expression Runx2 and Osterix.

By the R < O obtaining in embodiment 2, R=O and R > O monoclonal cell (add 100 μ g/ml ascorbic acid (Sigma) and 5mM β-phosphoglycerol (Sigma)) respectively in osteoblast differentiation culture medium in normal culture medium, add or do not add the TSA (hachimycin of 50nM, Sigma), differentiation was carried out von Kossa dyeing after 6 days.Found that, TSA significantly suppresses R < O, R=O and R > O cell calcification level.Consider that in body, osteoblast is co expression Runx2 and Osterix, this presentation of results TSA is negative to the effect of osteoblast differentiation and function effect.Result is referring to Fig. 4.

Industrial applicibility

As described above, by being less than the mode coexpression Runx2 of expression (or active) level of Osterix and Osterix with a kind of Runx2 expression (or active) in mescenchymal stem cell or other non-osteoblast, can accelerate the differentiation of induced osteogenesis cell; Due to co expression Runx2 and Osterix in osteoblast, therefore, in osteoblast, can strengthen osteoblastic function by the expression (or active) that reduces Runx2 and/or increase Osterix again.The present invention can be used for treating the osteopathia such as osteoporosis, osteogenesis imperfecta, periodontal disease, fracture, also can be used for research and development and the production of tissue engineered bone.The present invention is also applicable to the method for the screening of the medicine of osteopathia prevention and treatment.

It will be appreciated by one of skill in the art that disclosed concept and specific embodiment in description above can be easily with making an amendment or being designed for the basis of other embodiments of implementing identical object of the present invention.Those skilled in the art also will understand that this type of is equal to embodiment and does not deviate from the spirit and scope of the present invention that provide in appended claims.

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Claims

The gene of 1.Runx2, the gene of cDNA or RNA and Osterix, cDNA or the RNA application in pharmaceutical composition or test kit for the preparation of promotion osteoblast differentiation, the gene that described pharmaceutical composition or test kit comprise Runx2, the recombinant precursor of cDNA or RNA and the gene of Osterix, the recombinant precursor of cDNA or RNA, the gene of wherein said Runx2, the recombinant precursor of cDNA or RNA and the gene of Osterix, the content of the recombinant precursor of cDNA or RNA in described compositions or test kit makes Runx2 and Osterix co expression in asking mesenchymal stem cells or other non-osteoblast, and make the Runx2 protein content of expression and the ratio of Osterix protein content reach any ratio in following proportion: mol ratio is 1:N, wherein N is greater than 1.
2. application claimed in claim 1, wherein makes the Runx2 protein content of expression and the molar ratio of Osterix protein content reach 1:2.
3. application claimed in claim 1, wherein makes the molar ratio of Runx2 protein content and Osterix protein content reach 1:3.
4. application claimed in claim 1, wherein makes the molar ratio of Runx2 protein content and Osterix protein content reach 1:4 or lower.
5. application claimed in claim 1, gene, cDNA or the RNA of wherein said Runx2 and gene, cDNA or the RNA of Osterix derive from mammal.
6. application claimed in claim 5, gene, cDNA or the RNA of wherein said Runx2 and gene, cDNA or the RNA of Osterix derive from mice or people.
7. application claimed in claim 1, its for the production of tissue engineered bone and with the gene therapy of osteoblast differentiation relevant disease.
8. application claimed in claim 7, the wherein said disease relevant to osteoblast differentiation selected white osteoporosis, fracture, osteogenesis imperfecta or periodontal disease.