CN101646425A - Method to enhance tissue regeneration - Google Patents

Abstract

The present invention provides for compositions and methods for modulating tissue growth using tissue growth modulators, which are agents that either enhance or inhibit tissue growth as desired by a particular indication by modulating the PG or Wnt signaling pathways, or employing modulators of both PG and Wnt signaling pathways for a synergistic effect or highly selective effect.

Description

Strengthen the method for tissue regeneration

Government supports

The present invention is supported by NIH NIH subsidy No.CA103846-02 at least in part.U.S. government has certain right to it.

The cross reference of related application

The application requires the U.S. Provisional Patent Application serial number No.60/853 of submission on October 20th, 2007,351, exercise question is the serial number No.60/853 that submits in the Method to Modulate Hematopoietic Stem Cell Growth method of hematopoietic stem cell growth (regulate) and on October 20th, 2007,202, exercise question is the priority of Method to Enhance Tissue Regeneration (method of enhancing tissue regeneration); And the WO/2007/112084A2, the exercise question that require on April 26th, 2007 to submit to are the priority of Method to Modulate Hematopoietic Stem Cell Growth (regulating the method for hematopoietic stem cell growth), these three applications are that Leonard I.Zon, Trista E.North and Wolfram Goessling submit to, and the full content that is incorporated herein these applications as a reference.

Technical field

That embodiment of the present invention provide is external, in the body and exsomatize and strengthen or suppress tissue development or regenerated regulator.More specifically, for example, can be used to strengthen regenerated tissue response in the organ with prostaglandin or the interactional regulator of wnt signal transduction pathway, described organ for example is liver, hematopoietic stem cell, skin, vascular and capable of regeneration other organs.

Background technology

Regenerating medicine has very big potentiality for changing the treatment development of losing the following destiny of patient of organ because of contingency, defective or disease.The signal transduction pathway of understanding development can be opened and be not only tissue regeneration and be the gate that cancer suppresses.

For example, in the liver organization growth course, undifferentiated entoderm forms liver, intestinal, pancreas and accessory organ by the effect Butut of various signal transduction pathway.Growing the plasticity of early stage entoderm CFU-GM and the mechanism of adjusting endoderm cell's destiny and organ growth is subsequently but seldom understood by the people.Therefore liver still can be repaired and be regenerated in adult, further illustrates to regulate path that liver grows and may make organizer inner equilibrium and regenerated mechanism clearer.Because the progress of the patient's condition relates to the reaction of the germinal cell program of hypertrophy and differentiation, therefore understand histoorgan better and form and to provide target for the pharmaceutical intervention that for example suppresses carcinogenesis or strengthen tissue regeneration on the contrary.

Summary of the invention

The compositions of embodiment of the present invention and method provide the tissue growth regulator, and described tissue growth regulator is the reagent that strengthens tissue development and growth or inhibition tissue development by special instructions as required.These regulators play a role to the tissue growth or the important signal transduction pathway of regenerating by stimulating or suppressing.

For example, can regulate and control the wnt signal and conduct and strengthen tissue regeneration, particularly liver regeneration, blood are replanted, vascular is grown and wound healing.Can grow and regeneration in utilize the activator of wnt signal transduction pathway to strengthen these processes, this regulator can be synthetic or solubility wnt part, the plain destructive inhibitor of β-connection or transcribe coactivator.

The conduction of prostaglandin signal interacts with the conduction of wnt signal, therefore can be used for changing the wnt activity and grow and tissue regeneration to regulate.Regulator of the present invention can be the chemical compound that changes the conduction of prostaglandin signal or its downstream effect thing, can be used for changing the conduction of wnt signal in organ growth and regenerative process.For example, can the activatory downstream effect thing of direct regulation and control prostaglandin receptor, as ring AMP, PI3 kinases and protein kinase A, so that the wnt signal transduction pathway is exerted an influence.

Also can allow " fine setting " of growth and regenerated signal thus with the regulator of prostaglandin path as regulating the active mechanism of wnt.For example, the activation of wnt signal conduction can strengthen tissue growth, in case obtain desirable result, indomethacin can be used to slow down or stop this effect.

In addition, can work in coordination with and use regulator or prostaglandin and wnt path with the total wnt activity of increase/enhancing, avoid simultaneously using high dose or repeatedly dosage give chemical compound/method and directly activate the toxicity that the wnt path brings.The present invention has confirmed these principles in fish embryo and adult and adult mammal.

The regulator of wnt or prostaglandin signal transduction pathway strengthens liver regeneration after being used in toxic damages, as behind tumor or the ill liver organization excision or the poisoning by paracetamol of the liver health used of organ donation after partly excising.These regulators can systemic fashion give or be applied directly to liver, as the input gate vein.In addition, in preparing hepatocyte transplantation, can exsomatize and external use prostaglandin dressing agent to strengthen liver stem cells and hepatocellular growth in the culture fluid, perhaps can be used for the used bioartificial livers auxiliary facilities of fulminant hepatic failure patient.

In addition, the direct adjusting of wnt signal conduction be can in its hetero-organization, utilize or organ reparation and regeneration strengthened by the adjusting of regulating and control the prostaglandin path, particularly be in hematopoietic stem cell growth and homoiostasis, at wound healing with in repairing, in vascular growth and regeneration, and in reparation and regeneration such as other organs such as heart and nervous system.

Usually, the mode systemic administration that the chemical compound of embodiment of the present invention can targeting problem organ is in the patient, or exsomatizes and be applied to cell or organ-tissue.

The regulation and control of prostaglandin path can realize by the pharmacy measure, give the activator or the inhibitor of the various ingredients of prostaglandin path as targeting.Selectively, can regulate to change the prostaglandin path by the organ that virus or other devices are paid close attention to gene target.

Embodiment provides the method that promotes histiocyte growth among the experimenter, and described method comprises and gives at least a regulator and pharmaceutically acceptable carrier.

For example, discovery can strengthen the reagent that the homeostatic regulator of tissue development and adult tissue comprises dimethyl-PGE2 (dmPGE2) and stimulates the PGE2 path.

In another embodiment, the tissue development regulator has been accelerated growth by changing the wnt path.Strengthen that regulator such as tissue growths such as liver regeneration, hematopoietic stem cell recovery, wound healing or other organ-tissue reparations for example comprises BIO or LiCl or at other chemical compounds of horizontal down-regulation joint wnt path or wnt signal transduction cascade arbitrarily by direct change wnt path.

Description of drawings

Fig. 1. the plain signal conduction of the wnt/ β-connection of raising influences the liver size.Figure 1A has compared wild type (normally) and APC ^+/-The liver size that the saltant Brachydanio rerio grows.Figure 1B has compared the APC that measures by facs analysis ^+/-The positive hepatocellular quantity of the GFP of/LFABP:GFP heterozygote.Fig. 1 C shown with the contrast fish compare APC ^+/-The increase of hepatocyte quantity in the mutant tissue slice.Fig. 1 D has reflected the plain immunohistochemical analysis of β-connection of insemination back 96 hours (hpf), has shown with wild type and has compared APC ^+/-The equal increase of intravital Cytoplasm and nucleus dyeing suddenlys change.Fig. 1 E has shown that the BrdU in the corresponding liver section is combined in APC ^+/-Significantly raised in the embryo.*=statistically-significant difference.

Fig. 2. the wnt activity of increase has been quickened liver regeneration.Fig. 2 A has shown the operation incisxal edge of the adult Brachydanio rerio of experience partial hepatectomy.Fig. 2 B has shown the result that liver inferior lobe (inferior liver lobe) epimorphosis is analyzed, and confirmed that the wnt activation provides the regeneration advantage to the wild type fish, and wnt inhibition having slowed down liver regeneration is long.

The acceleration liver regeneration of Fig. 3 .wnt mediation is conservative on evolving.In the liver regeneration process of 2/3 partially hepatectomized postoperative, APC ^Min/+The APC heterozygosis mediating growth advantage of mice.*=statistically-significant difference.

Fig. 4 .wnt signal transduction pathway figure has shown with the prostaglandin signal and has conducted interactional potential site.

Fig. 5. prostaglandin is adjusted in the Brachydanio rerio of growth the active influence of wnt.Fig. 5 A has shown the active experimental design of wnt in the mensuration TOP:dGFP fish.Fig. 5 B has confirmed to give the wnt that increases in PGE2 hindbrain activity, and the wnt activity that reduces behind the contact indomethacin.Fig. 5 C has confirmed respectively in the liver of growing and the similar influence of enteral with Fig. 5 D.

Fig. 6. prostaglandin adjusting, cAMP activation and wnt activity are to the influence of the dirty growth of entoderm regulating liver-QI.Fig. 6 A has shown wild type and APC ^+/-Indomethacin and cAMP activator Buddhist SCH (forskolin) have confirmed to compare with prostaglandin itself to the influence of entoderm progenitor cell in the Brachydanio rerio, and the downstream mediation body of prostaglandin signal conduction can have similar influence.Fig. 6 B has shown the influence to the liver form of PGE2 and indomethacin.

Fig. 7. prostaglandin signal transduction pathway regulator has changed the influence of wnt activity to expression of target gene.Fig. 7 A has shown a kind of method of using zebra fish model test prostaglandin pathway modulators.Fig. 7 B has shown the influence to wnt target and entoderm gene expression in wild type fish, wnt8 fish and the dkk fish of the prostaglandin regulator measured by quantitative PCR.

Fig. 8. prostaglandin signal conduction change liver regeneration.Fig. 8 A and Fig. 8 B have shown by measuring the liver size in the regenerative process or relating to the gene expression of this process, the method for testing prostaglandin pathway modulators in the zebra fish model by analysis.Fig. 8 C has disclosed and has suppressed the synthetic gene expression that has reduced wnt target in the liver regeneration process of prostaglandin.

Fig. 9 A and Fig. 9 B. prostaglandin are regulated and wnt activity influence liver regeneration.These figure have shown prostaglandin and wnt activation and handled with the component that influences these paths and downstream target is how to influence liver regeneration in the Brachydanio rerio.

Figure 10. prostaglandin suppresses the influence to the liver tumor growth.Figure 10 A and Figure 10 B have shown the method for test prostaglandin pathway modulators in the Brachydanio rerio carcinogenesis.Figure 10 B has shown test suppresses whether prostaglandin is synthetic can prevent that liver tumor is at APC ^+/-Form used model in the Brachydanio rerio.Figure 10 C has confirmed in this model by suppressing the synthetic tumor incidence rate that reduced of prostaglandin.

Regulate the influence that mouse bone marrow cells is transplanted in the time of Figure 11 .wnt and prostaglandin signal transduction pathway.The figure illustrates wnt activation after bone marrow transplantation, how enhancing formation of being undertaken by BIO of early stage spleen colony.Indomethacin has been blocked this influence.

Figure 12. prostaglandin adjusting and wnt activation are to the influence of Brachydanio rerio wound healing.Partially hepatectomized postoperative skin wound healing is faster, and uses PGE2 can reach more, at APC ^+/-In the fish too.Wound healing seriously is suppressed after giving indomethacin.

The specific embodiment

Unless this paper has definition in addition, the scientific and technical terminology relevant with the application should have the implication of those skilled in the art's common sense.In addition, unless context has requirement in addition, singular references should comprise plural form, and plural term should comprise singulative.

It should be understood that to the invention is not restricted to ad hoc approach as herein described, scheme and reagent etc., can change to some extent.Term used herein only is used to describe specific embodiment, be not used in to limit the scope of the invention, and scope of the present invention only is defined by the claims.

Except the operation embodiment in or have in addition other the explanation, all numerals of expressing component content used herein or reaction condition are construed as in all cases modifies by term " about ".When being used for percent, term " about " can refer to ± 1%.

Describe and disclosed purpose in order to reach, all patents and other publications listed all are incorporated herein by reference clearly, and for example, the method for describing in publication can be used among the present invention.These publications only are provided as the application's disclosing before submitting to day.At this on the one hand, should not be interpreted as the inventor admits owing to formerly inventing or having no right for other reasons before described disclosing.About to all explanations on date or about to the expression of these file contents all based on applicant's available information and do not constitute about any of modification and admit to date or these file contents.

Grow signal transduction pathway and hold the key that is opened into soma's regeneration and suppresses the carcinogenesis gate.The present invention understands in depth the adjusting of the hematopoietic stem cell growth of embryo and adult.The present invention also understands in depth the adjusting of the liver of embryo and adult growth, comprises the growth behind the hepatotomy.

One embodiment of the invention provide the regulation and control of genetic interaction between PGE2 and the plain signal conduction of wnt/ β-connection, thereby regulate specialization of growth and the regeneration of stem cell.In brief, prostaglandin (PG) E2 be form hematopoietic stem cell (HSC) in the body and make it have function necessary, yet its conduct mechanism in these cells is not also understood fully.People such as North, 447 (7147) Nature 1007-11 (2007).To the clinical observation of patient with APC sudden change, the center adjustment agent of wnt/ β-plain path of connection (people such as Cruz-Correa, 122 (3) Gastroenterology641-45 (2002)) and vitro data (people such as Castellone, 310 Science 1504-10 (2005)) show, prostaglandin and wnt/ β-be connected plain signal transduction pathway to interact.Steady state function people such as (, 423 Nature 409-14 (2003)) Reya of the HSC of adult is regulated in wnt signal conduction energetically, but its effect in HSC forms also is not detected.In order to confirm PGE2 and wnt signal transduction pathway direct interaction in vivo, make TOP:dGFP wnt report stable derivatives dmPGE2 (10 μ M) and the indomethacin (indomethacin) (10 μ M) (the non-selective inhibitor of cyclooxygenase (cox)) of sub-zebrafish embryo contact PGE2.The in situ hybridization of GFP has disclosed the active rapid increase of whole embryo wnt behind the contact dmPGE2 (observing 111 of 99/records), particularly in aorta-gonad-middle kidney (AGM) zone, forms permanent HSC (12 ± 3.4vs.3 ± 1.8 cell) at this.It is active and significantly reduced GFP expression (72/87) on the whole that the wnt among the AGM has been removed in indomethacin treatment.QPCR analysis by GFP has confirmed these results, discloses the active 2 times of direct influences of inducing and demonstrate PGE2 to the wnt signaling activity of wnt in the whole embryo extract behind contact dmPGE2.

Analyze the functional outcome of interaction in HSC forms of PGE2/wnt in embryo development procedure by the expression that detects HSC label runx l and c-myb.Cause 36 hours enhancing HSC of after fertilization to form (hpf at 10 body segment places to the heat shock induction of classical wnt part wnt8; 47/54).(10 μ M, in the time of 16-36hpf), the formation of HSC is reduced to or is lower than wild type level (43/46) when contact indomethacin after wnt8 induces.These results have shown that the PGE2 activity is that growth exerts an influence necessary to HSC in the wnt activation.

The negative regulator of the active inductivity of wnt and dmPGE2 handle to be combined and are used for interaction between functional PG of location and the wnt path.Dkk1 is in the startup of film in conjunction with level antagonism wnt path and wnt signal transduction cascade.Dkk in the hs:dkk1 transgenic embryo induces and has suppressed HSC growth (34/49).Contact external source dmPGE2 can succour the influence (28/51,10 μ M, 16-36hpf) of the dkk1 mediation that HSC is formed.Axin (axle Profilin) is the core component of β-plain destructive complex of connection, is the negative regulator of the indoor wnt signal transduction cascade of Cytoplasm therefore.When 10 body segments are induced, Axin severe inhibition HSC forms (47/52).In addition, can not overcome this influence by the dmPGE2 processing.Similarly, the plain leading negative form of transcribing coactivator TCF of β-connection has been removed HSC formation (60/62), and unaffected through dmPGE2 contact relief.These results show, PG and wnt path interact at β-be connected under the plain destructive complex level, thereby regulate the formation of permanent HSC in the embryo.

The wnt path can strengthen the HSC hypertrophy energetically by the signal conduction in HSC tabernacle and HSC itself.PGE2 regulates HSC and forms under vascular tabernacle level and in the HSC.People such as North, 2007.In order to identify the transducer of regulating by the wnt signal transduction pathway, analyze the gene that relates in the HSC growth by qPCR.After wnt8 induced, the expression of stem cell labeling thing runx l and cmyb was significantly strengthened, corresponding to the in situ hybridization expression data.On the contrary, induce, find that runx l and cmyb significantly reduce in response to dkkl.Common vascular label flkl and aorta specificity vascular label ephB2 increase like the wnt8 homogeneous phase, and reduce after dkkl induces.Wherein these influences to vascular tabernacle and HSC growth can change by adding the prostaglandin path dressing agent indomethacin that is fit to wnt8 and adding dmPGE2 to dkkl respectively.These data show that by regulating the growth potential level of hemopoietic tabernacle, the interaction of PGE2 and wnt influences HSC at least in part and forms.In addition, analyze wnt target gene cyclin D1 by in situ hybridization among the AGM and qPCR and show that the interaction of wnt/PGE2 also is activated in HSC self, influence their hypertrophy and self renewal probably.

Supposed wnt activation energy adjusting HSC self renewal and replanted.Yet the wnt activation also is considered to relate to carcinogenesis, makes the wnt signal conduct direct enhanced conception existing problems.Determined the active HSC homoiostasis that whether can effectively regulate adult of regulating of the wnt that is undertaken by prostaglandin by observing Brachydanio rerio by the recovery of hemopoietic after being radiated.In the past, studies show that PGE2 handled and significantly strengthened regeneration, and through after the radiation 10 days, can easily detect the influence to stem cell and CFU-GM quantity; There is not PGE2 but to suppress the hypertrophy of stem cell and CFU-GM.Report facs analysis in the sub-fish by TOP:dGFP, the wnt in the medulla renis of radiation back is active to increase by 2 times.In addition, after radiation 10 days, 24-36 hour the wnt8 in radiation back induces and makes stem cell and progenitor cell increase by 2.5 times.This influence is suppressing significantly to reduce behind the cox by indomethacin.Utilize the reservation of the activatory murine models show interaction of composition wnt in the vertebrates kind.Because APC afunction in the destructive complex, so APC ^Min/+Mice has the plain level of the β of raising-connect.Compare with compatriot's contrast, these mices show normal differential blood count at baseline.Compared with the control, behind the 5-FU chemical damage, APC ^Min/+The bone marrow of mice recovers to strengthen.Indomethacin (per 48 hours 1mg/kg) has significantly reduced APC ^Min/+Hypertrophy advantage in the mice.Thereby these data acknowledgements PG and wnt interact and regulate vertebrate hemopoietic homoiostasis.

Whether regulate wnt activity among the HSC for estimating the prostaglandin level, utilize the HSC of purification to carry out the murine transplant experiment.The isolating cKit+Scal+Lineage-of FACS-(KSL) bone marrow cell transplantation is in the receptor of the radiation that causes death.With GSK inhibitor B IO (0.05mg/kg), indomethacin (1mg/kg and 2.5mg/kg) or be used in combination and handle the receptor mice, to influence the active and PG level of wnt.Handle (p=0.03) in response to BIO, CFU-S12 demonstrates significant 2 times of increases, yet, give indomethacin simultaneously and CFU-S quantity is subtracted get back to baseline values.These results have confirmed PGE2 and wnt direct interaction in HSC.

For whether the interaction of determining the plain signal conduction of PGE2 and wnt/ β-is connected is the conservative regulator of population of stem cells and progenitor cell in its hetero-organization, detection entoderm and hepatic progenitor cells in the Brachydanio rerio growth course.The zebrafish embryo of contact indomethacin demonstrates the expression decreased (67/71) of entoderm CFU-GM label foxA3.Especially, the liver bud of growth significantly reduces, cause the liver of 72h.p.f. littler (51/56), as liver fatty acid-binding protein (1fabp) expression detected.The interpolation of dmPGE2 has caused the foxA3 group who enlarges, and the liver bastem (75/83) of increase and the liver size (88/92) that increases have disclosed the conduction of PGE2 signal at the developmental new role of entoderm.Supported this discovery by the various components that in the foxA3 positive cell, detect the conduction of PGE2 signal.

Wnt signal conduction has demonstrated that to be that entoderm and liver form necessary.Be used to characterize the influence of wnt/PG interaction in foxA3+ entoderm CFU-GM of 48hpf as the APC saltant Brachydanio rerio of wnt signal transduction pathway composition activation model.The compatriot compares with wild type, APC ^+/-The embryo has strengthened the foxA3 expression and has increased liver bud (88/93) and increased liver size (68/75).Compare with untreated wild type contrast, indomethacin makes APC ^+/-Positive CFU-GM (33/39) of intraembryonic foxA3 and liver size (61/67) reduce, and dmPGE2 has increased entoderm CFU-GM (47/54) and liver size (75/81) greatly.The use of heat shock induction transgenic lines has confirmed that when in HSC the interaction of PG and wnt path takes place with destructive complex level in the entoderm growth course.QPCR has disclosed entoderm label (foxA3) and hepatic progenitor cells label (hhex) all is conditioned, and shows that the interaction of PG and wnt relates to different progenitor cell in the entoderm growth course.The expression of insulin is not subjected to the influence that the PG path is regulated, and shows that it is not the general regulator that different entoderms are that PG/wnt regulates.Cyclin D1 and cmyc are all regulated by wnt and prostaglandin in growth course altogether, and this can show that PGE2 exerted an influence to stem cell by enhanced cell cycle and hypertrophy.

Utilize the hepatotomy model of Brachydanio rerio can clearly confirm the lasting importance of PG/wnt path in the adult hepatic homoiostasis.1/3 partially hepatectomized postoperative, Brachydanio rerio liver were regenerated in 5 days to 7 days.This process is at APC ^+/-Be accelerated in the fish.The regenerative index of handling significantly to have reduced wild type and APC saltant fish with indomethacin in 6 to 18 hours after excision.The plain immunohistochemistry of β-connection has disclosed APC ^+/-Fish is the enhanced cell nuclear staining after excision.Yet indomethacin descends the plain level of whole β-connection, and wild type and APC ^+/-There is not nuclear beta-connection plain in the fish.

By to wild type and APC ^Min/+Mice carries out partial hepatectomy can further illustrate PGE2 and influence the mechanism that β-the plain level of connection is utilized, and confirms the conservative role of this interaction in mammalian liver regeneration.Here, APC sudden change makes total β-plain level of connections and nuclear beta-be connected plain level increase, particularly in the portal vein peripheral region.Contact indomethacin (2.5mg/kg bid sq) significantly reduces the plain level of two kinds of genotypic β-connect.Cell culture studies shows, PGE2 makes the GSK3b inactivation can strengthen the plain level of β-be connected by phosphorylation and activation by adenyl cyclase and protein kinase A (PKA); At wild type and APC ^Min/+Behind the contact indomethacin, the IHC of P-GSK3b (at serine 9) discloses its amount to be reduced in the mice.These discoveries have been confirmed by immunoblotting.β-connection element may increase hyperplasia by its target cell cyclin D1.In conjunction with measurement, cyclin D1 level and the hyperplasia that obtains are at APC by BrdU ^Min/+Increase in the mice and behind the contact indomethacin, reduce significantly.

By utilizing the Buddhist SCH to increase the generation of cyclic adenosine monophosphate and utilizing H89 to suppress the functional interaction that PKA explores the signal conductive process in PG downstream in the Brachydanio rerio.In HSC and entoderm CFU-GM, the contact of Buddhist SCH has the potentiation similar to dmPGE2.The Buddhist SCH can be succoured the inhibition effect of indomethacin in wild type and wnt8 genetically engineered fish.By H89 suppress PKA reduced increase that dmPGE2 brings out HSC form.In addition, dmPGE2 is eliminated by H89 the relief of dkk influence.These data show, PGE2 strengthens the plain level of β in various population of stem cells and the progenitor cell-be connected by the activation of cyclic adenosine monophosphate and PKA and the inactivation of GSK3b thereafter.

Another embodiment of the invention provides the effect of the plain signal conduction of wnt/ β-connection in liver growth and growth course.In brief, adenomatous polyposis coli (adenomatouspolyposis coli) gene (APC ^+/-) that liver is grown is bigger for the hybridization embryo of (wnt signal conduction important regulator).On the contrary, APC ^-/-The embryo can not specialization formation liver.β in the wnt signal conduction of raising and the cell that increases-be connected element to mediate two kinds of APC liver phenotypes.Utilize to express the transgenic zebrafish of plain signal conduction inductivity activator of wnt/ β-connections and inhibitor, demonstrate in embryo's forming process in two stages: need to suppress the wnt signal after hepatocyte destiny forms special catabolic gastrula and conduct the demand of wnt; On the contrary, normal liver growth needs activation wnt signal conduction.Brachydanio rerio and mice are carried out hepatotomy to estimate the functional demand of wnt signal conduction in liver regeneration.Interesting is that the APC heterozygote demonstrates and can quicken liver regeneration, and suppresses wnt signal conduction seriously having reduced regrowth.The present invention has disclosed the conservative effect of the wnt/ β-connection evolution of plain signal conduction in specialization of hypoblastic organs, hepatic cell growth and liver regeneration, and this has enlightenment to regenerating medicine.

Another embodiment of the invention provides the prostaglandin signal transduction pathway as the grow effect of effective dressing agent of liver.Compared with the control, insemination is hatched zebrafish embryo with cox1-, cox2-or bispecific inhibitor (for example, indomethacin) after 72 hours the liver size is significantly reduced.On the contrary, contact dimethyl-PGE2 (dmPGE2) has strengthened the liver growth.The morpholine of cox1 or cox2 strikes to subtract and has similarly suppressed growth, but by contact external source dmPGE2, this growth is succoured fully.Compared with the control, experience partial hepatectomy and the adult Brachydanio rerio that contacts indomethacin demonstrate that liver regeneration is long significantly to be reduced.The inhibition of cox also stops wound healing.On the contrary, compare with untreated fish, contact dmPGE2 in excision back can cause long enhancing of liver regeneration and liver vessel to distribute obviously to be increased.Also observe the faster healing of wound in the fish that dmPGE2-handles.Brachydanio rerio similar studies confirm that the medulla renis that dmPGE2 can strengthen after the damage replants.Therefore, the adjusting of prostaglandin path can influence such as the repair/regeneration of all kinds tissues such as heart tissue, osseous tissue and wounded tissue long.

Undifferentiated entoderm forms liver, intestinal, pancreas and accessory organ by signal transduction pathway effect Butut.People such as Cui, 180 Dev.Biol.22-34 (1996); Zaret, 3 Nat.Rev.Genet.499-512 (2002).The conduction of wnt signal organizes Butut, cell fate decision and hypertrophy to play an important role for control by its main transcriptive intermediate body β-connection is plain in many embryo's environment, comprises allelotaxis and differentiation.Clevers，127?Cell?365-69(2006)。Do not having wnt signal when conduction, β-connections is plain by Axin, APC and the destructive complex effect of glycogen synthase kinase (GSK) 3 β and phosphorylation, and targeting is degraded.The wnt part is attached to surface receptor and allows the plain accumulation and transfer to nucleus, regulator gene expression there in Cytoplasm of β-connections.

In gastrointestinal neoplasia process, frequently detect the gene mutation in several components of wnt/ β-plain signal transduction pathway of connection.The most significant is that the patient who carries the apc gene sudden change just produces colon cancer at the very little age.People such as Kinzler, 251 Sci.1366-70 (1991).The child who has the APC sudden change has 1000 times probability to produce the hepatoblastoma of embryo's form of hepatocarcinoma.People such as Hirschman, 147 J.Pediatr.263-66 (2005).In hepatocarcinoma (HCC), find β-sudden change of connections element and the sudden change of Axin-1 and Axin-2 people such as (, 21 Oncogene 4863-71 (2002)) Taniguchi.Based on the component ubiquity defective of the wnt path of finding in the hepatoma of original and differentiation, so several aspects that liver is grown are probably regulated in the plain signal conduction of β-connection.

In embryo's forming process, liver comes from preceding entoderm CFU-GM.After the entoderm CFU-GM convergeed to center line, the hypertrophy and specialization of entoderm bar were activated.In zebrafish embryo, the following entoderm CFU-GM that can form liver can be confirmed as thickening in the pro-entoderm at after fertilization 22-24 hour (hpf).People such as Field, 253 Dev.Bio.279-90 (2003).Along with entoderm further grows, original liver looks like an outstanding bud, is extended left by the yolk sac center line.Between 28hpf and 30hpf, the liver specificity genetic transcription starts in the cell of specificity formation liver.Liver is grown fully through 48hpf and is expressed ripe liver specificity gene such as liver fatty acid-binding protein (LFABP).People such as Her, 538 FEBS Lett 125-33 (2003).Along with the Brachydanio rerio liver is expanded forward and left, the liver growth continues to carry out.Specialization of the intraembryonic liver of vertebrates, the mechanism that startup and control utilized of sprouting and growing seem vertebrates kind high conservative.

Demand to the conduction of wnt signal in entoderm grows is documented in the nematicide (C.elegans) at first, and is to evolve to go up to guard.People such as Lin, 83 Cell 599-609 (1995).It is deadly and slow down that the branch factorial of the plain signal of wnt/ β-connection conduction effect in the vertebrates entoderm grows is had the body early embryo of the plain mice of homozygosity disappearance β-connections.People such as Haegel, 121 Devel.3529-37 (1995).APC ^MinHomozygosity mutant mice also is an embryonic death, although heterozygote be live and when adult, develop into tumor.People such as Su, (1992).In the XenoPus animal, need wnt in the forming process in the gastrula of entoderm Butut.People such as Heasman, (2000).By the plain inductivity inactivation of β-connection, it is that intestinal is grown and the intestinal structure forms needed that the plain signal conduction of wnt/ β-connection is shown.People such as Ireland, (2004).In addition, the wnt-dependent form of intestinal crypt dissection is regulated and still is retained in the adult.People such as Pinto, (2003).The recent research of wnt signal conduction in the liver growth has been caused seeming the discovery of contradiction.The data that the XenoPus animal occurs show, need to suppress wnt to allow the generation of specialization of liver in the entoderm CFU-GM in early days.On the contrary, Brachydanio rerio wnt2b mutant prometheus (Prometheus) has disclosed in adjusting liver necessary for growth and has derived from mesodermal wnt signal.People such as Ober, (2006).Homozygosity prometheus mutant be live and finally develop into liver.This shows that when not having wnt2b, hepatic progenitor cell can still not had the primary wave of mesoderm wnt2 signal conduction can weakening liver growth by correct specialization.Do not know whether other wnt factors can compensate the shortage of wnt2b in specialization of liver and the growth, or the liver cell proliferation later stage does not need wnt.

Identified the Brachydanio rerio that the APC sudden change is arranged by TILLING (the local sudden change of directional induction genome) in the past.People such as Hurlstone, 425 Nature 633-37 (2003).Although APC ^+/-Mutant 96hpf death, but APC ^+/-Fish but lives and the susceptibility that spontaneous gastrointestinal neoplasia develops is increased.People such as Haramis, (2006).APC ^+/-The liver tumor that the saltant Brachydanio rerio produces is similar to hepatoblastoma, this means that the APC sudden change has caused the defective of the hepatic progenitor cells differentiation of wnt adjusting.

The APC sudden change and the transgenic type Brachydanio rerio of the inductivity activator and the inhibitor of the plain signal conduction of wnt/ β-connection expressed in utilization, the invention provides the sign that the embryo development procedure neutralization is adjusted to the temporary transient demand of in soma's homoiostasis process the plain signal of wnt/ β-connection being conducted.The APC disappearance is grown liver the differentiation influence, regulates by the temporary transient demand that changes the conduction of wnt signal in the liver generative process.The wnt activation has influenced the destiny decision of entoderm CFU-GM, and this has caused, and pancreatic tissue forms cost increase liver and intestinal is grown to sacrifice.Be used for the zebra fish model of partial hepatectomy by generation, the present invention has confirmed to need wnt in the liver regeneration process in the body.In addition, the present invention has shown that the potentiation that the wnt signal of raising conducts regenerative process is retained in Brachydanio rerio and mice.These data acknowledgements the conduction of the plain signal of wnt/ β-connection be essential and in liver is grown the process of several respects by altitude mixture control, and in the organ homoiostasis, keep central role.Therefore, the invention provides the method for the wnt signal being conducted instantaneous rise, can be used as enhancing mammal and the regenerated attractive mechanism of human liver.

By making APC ^+/-The differentiation influence that the APC disappearance forms liver organ is illustrated in Brachydanio rerio and the hybridization of LFABP:GFP fluorescence report daughter cell system, and grows by the fluorescence microscope evaluating liver.72hpf, the compatriot compares with wild type, APC ^+/-The embryo demonstrates the liver size sharply increases (265/297).On the contrary, all do not detect LFABP at homozygosity APC in any stage of growing ^+/-Expression among the saltant embryo (134/134).For guarantee that the developmental phenotypic alternation of observed liver is because the in situ hybridization and the transfer of sterol carrier protein are carried out in the change that LFABP expresses, and obtains analog result remarkablely.

APC ^+/-: the flow cytometry analysis of the GFP+ cell among the LFABP:GFP heterozygote offspring has disclosed APC ^+/-Hepatocyte quantity among the embryo has increased by 3 times, has confirmed APC ^+/-There is not the GFP+ hepatocyte in the mutant.Hepatocyte cell check figure in the corresponding tissue slice has been proved conclusively the differentiation influence that the APC disappearance is grown liver; Fabric analysis has shown with wild type compares APC ^-/-The embryo does not have hepatocyte fully, and APC ^+/-The embryo then demonstrates remarkable increase.At wild type and APC ^+/-Do not observe the change of whole cell form between the sample.

Because APC regulates the plain availability in nucleus of β-connection altogether, so detect wild type and APC by immunohistochemical method (IHC) at 72hpf ^+/-Plain cell content of β-connection and location in embryo's the hepatocyte.The wild type liver has shown that the bonded β of cell membrane-connection is plain basically.At APC ^+/-In the fetal liver, β-connection uniformly dyeing color significantly increases, and Cytoplasm dyeing has strengthened 4 times, and nucleus dyeing has increased by 5 times.The influence to various in-house cell cycles, hyperplasia and apoptosis is regulated in the plain signal conduction of known wnt/ β-connection.Alonso ﹠amp; Fuchs, 17 Genes Devel.1189-1200 (2003); People such as Pinto, 17 Genes Devel.1709-13 (2003); People such as Reya, 243 Nature 409-14 (2003).In order to measure APC ^+/-Whether the increase of total hepatocyte quantity is caused by the proliferative activity enhancing in the embryo, detects the BrdU combination at 72hpf.Compare with wild type, see at APC ^+/-The percent of the BrdU positive cell of each liver significantly increases in the embryo.See analog result with PCNA dyeing.

Although not existing the liver growth to get rid of estimates in the intracellular distribution of embryonic liver β-connection is plain, it is plain very strong along the whole length dyeing of gastrointestinal that but contiguous entoderm tissue demonstrates β-connection, and almost all seeing the BrdU combination in each cell.Because the unusual wnt signal that the disappearance of APC causes in the brain of enteral and growth has caused differentiation to be obstructed and final apoptosis.Chenn ﹠amp; Walsh, 297 Sci.365-69 (2002); People such as Sansom, 18 Genes Devel.1385-90 (2004).Although have a large amount of wnt signal conduction why but APC for further analyzing ^+/-The reason that the embryo but can not make hepatocyte grow is at APC ^+/-Embryo tissues section inner evaluation TUNEL dyeing.Find that along endoblastic whole length high-caliber TUNEL-positive apoptotic cells is arranged, comprise the zone that the liver differentiation can not take place.APC ^+/-Intraembryonic apoptosis marker Capsase enzymatic activity is 2 times of wild type.These data show, APC ^+/-Not having liver in the mutant is reason owing to the death of entoderm CFU-GM.

The increase of β-plain level of connection has caused the liver phenotype of differentiation in the APC mutant.The disappearance gradually that interesting discovery is APC does not cause linear effect to the liver size.For confirming that β-connection element makes APC ^-/-The interior liver of embryo increases and can not make APC ^+/-Specialization of liver of mutant reduces the plain level of β-connect by morpholine antisense oligonucleotide (MO) strategy.To stop the plain MO that begins to occur of Brachydanio rerio β-connections people such as (, 286 Devel.Bio.427-39 (2005)) Lyman Gingerich to inject the APC that enters the unicellular stage ^+/-The heterozygote offspring.By using low concentration MO (40 μ M), the embryo through injecting can successfully continue to grow by forming gastrula, compares with the contrast embryo of MO-injection, and noticing does not influence whole general morphology.

The plain targeting of β-connection strikes and subtracts the drastic shift that has caused the liver phenotype distribution.Most of embryos (74%) have shown normal liver, comprise wild type and APC and gene type has thereafter disclosed this cell mass ^+/-The embryo.Some APC ^+/-Embryo's (15%) has still shown the liver that increases, and may reflect that plain insufficient functional the striking of β-connection that the MO low dosage causes subtracts.Injecting the APC that MO is still survived until 72hpf ^+/-Among the embryo, 43% evidence that has LFABP to express; Yet these embryos still demonstrate serious developmental defect, and surpass 120hpf death.These data show that the plain level of β-connection just is enough to separately produce two kinds of liver phenotypes, and demonstrating wnt/ β-plain path of connection is the mediation body of these influences.The striking of classical wnt2b, wnt3 and wnt8 subtracts the fact that causes the liver size to reduce and supported this conclusion.

The plain signal conduction of wnt/ β-connection influences the entoderm progenitor cell.The inductivity transgenic zebrafish of expressing the activator of wnt signal conduction or inhibitor is used to measure the conduction of wnt signal influence entoderm in which of fetal development in stage and breaks up and the liver size.The hs:wnt-GFP fish is expressed wnt part wnt8 under the condition of control heat shock-inducible promoter, and hs:dkk-GFP and hs:dnTCF-GFP allow respectively curl acceptor levels nucleus is transcribed or the level of nucleus transcription complex under carry out wnt/ β-the is connected inhibition that plain signal conducts.Caused stopping or death at the tail bud that grows (10hpf) all induces wnt8 to cause forming gastrula before the stage heavy damage and whole embryo's Butut in the 24hpf growth.At 1 body segment and 5 body segments between the stage, the wnt activation has caused in the embryo that 72hpf is also survived that significant cardiac edema, body are long to shorten and do not form liver, APC ^-/-The memory of mutant.Compare with the wild type contrast of heat shock, the heat shock induction of the wnt8 of 10～18 body segments has caused enlarging markedly of liver.Except the integral body increase of organ size, 50% liver of suffering a shock at 10 body segment intermittent fever can not break away from fully from the entoderm bar, and this causes the liver specificity gene to increase in the expression of center line, and liver cell extends back; This phenotype is confirmed by confocal microscope and tissue slice analysis.The instantaneous wnt activation that time point between 24hpf and 36hpf carries out produces moderate influence at 72hpf to the liver size.Similarly, it is the most remarkable when 10～18 body segments that the wnt that induces generation of dkk or dnTCF suppresses influence that liver is grown, more moderate in embryo's maturation of back.Whole inhibition of 5 body segment plain signal conduction of wnt/ β-connection before the stage have caused body early embryo to cause death.

The about 44hpf that is expressed in that transcribes such as liver specificities such as LFABP begins, and the zone that entodermal canal is isolated future and can be become liver is considered to finish at 22hpf.The result that heat shock test obtains shows, the influence that liver is grown of wnt mediation is before ripe organ forms, produce in determined stage of the destiny of entoderm CFU-GM or forward a little stage.For the influence of research wnt activation, in the expression of heat shock induction wnt8 post analysis pan-entoderm label foxA3 to the entoderm progenitor cell.48hpf observes liver bud size after 10 body segments carry out thermal activation significantly increases; See that also pancreas bastem size reduces, and the influence that later wnt8 brings out (behind the 24hpf) is more not obvious.Wnt8 activation also has dosage-dependent form influence to the entoderm CFU-GM: compared with the control, caused liver to increase gradually in 5 minutes, 20 minutes and 60 minutes 18 body segment stage heat shocks, but be cost to sacrifice pancreatic tissue.

β-connection is plain to have caused entoderm destiny to be changed in the APC intravital activation that suddenlys change.The heat shock experiment shows that the hepatocyte quantity enhancing of wnt mediation takes place in entoderm CFU-GM level.The APC disappearance detects APC to the influence of entoderm CFU-GM at 48hpf in order to estimate gradually ^+/-Expression in the heterozygote offspring.Compare APC with wild type ^+/-The embryo demonstrates the liver increase and the pancreas bud reduces.By carry out the confocal microscope analysis of APC at 48hpf; The gut:GFP heterozygote, and confirm this phenotype in the facs analysis body.APC ^+/-The embryo organizes Butut and reduces by facs analysis gut:GFP+ CFU-GM quantity the entoderm that 48hpf can not clear display, this means all not generations of expansion of definite organ and entoderm CFU-GM.

Influence because all groups of entoderm CFU-GM seem to be subjected to the APC disappearance or wnt is activatory, therefore ripe hypoblastic organs is grown the detection of the consequence of early stage change.APC at 72hpf ^+/-In the embryo, insulin and tryptic expression (being respectively the indication of endocrine and exocrine pancreas differentiation) all reduce.At APC ^-/-In the mutant, tryptic expression is undetectable basically; The expression of insulin is although reduce but still can observe.APC disappearance is to similar with liver by the influence of the intestinal of the differentiation of the presentation markup of tripe tallow fat acid binding protein (IFABP): compare APC with wild type ^+/-Embryo's IFABP dyeing increases, and APC ^-/-The embryo can not express IFABP.Induce wnt8 similar at 10 body segments, but cause more disorderly Butut, particularly pancreas each hypoblastic organs's influence.These data acknowledgements, the plain signal conduction of initial wnt/ β-connections was regulated entoderm and is grown before specialization of organ, and this influence is that cost is regulated the entoderm CFU-GM and broken up and turn to liver with the sacrifice pancreatic tissue.In addition, can cause specialization of entoderm and outgrowth failure at tail bud and early stage over-drastic wnt/ β of body segment stage-is connected plain activation, the result is that dead the raising with not growing of endoderm cell is sophisticated hypoblastic organs.

The plain signal conduction enhancing of wnt/ β-connection hepatic cell growth.For measuring the whether also influence of the hepatic cell growth of mediation differentiation of the plain signal conduction of wnt/ β-connection, the expression of inducing wnt8 at 48hpf.By at 72hpf the LFABP:GFP fish being carried out the confocal microscope analysis and the GFP+ cell being carried out FACS find that this has caused the liver size to increase by 2 times.Along with specialization hepatic progenitor cells begins hypertrophy, the liver size is sharply expanded: at 72～120hpf, the intraembryonic liver cell quantity of wild type increases by 2～3 times.By facs analysis, the embryo that wnt8 brings out still has the GFP+ hepatocyte of accelerating at 120hpf, although this difference no longer easily displays by the whole detection that LFABP expresses.The wnt signal conduction of bringing out in the 48hpf heat shock suppresses the liver growth needs wnt that confirmation is optimized; By at the 72hpf in situ hybridization, compared with the control, dkk and dnTCF embryo's liver size reduces.The conduction of these data acknowledgements wnt signal is still very important in the hepatocellular hypertrophy of differentiation.

The plain signal conduction of wnt/ β-connection is activated in the liver regeneration process and is required.The vertebrates liver is dynamic organ, can repair limited damage in whole life process.In order to estimate the effect of the plain signal conduction of wnt/ β-connection in keeping the adult hepatic homoiostasis, researched and developed Brachydanio rerio liver regeneration model.The adult Brachydanio rerio has the SANYE liver; Carry out 1/3 partially hepatectomized postoperative by removing inferior lobe, the wild type Brachydanio rerio greater than 95% recovers immediately and their liver was all regenerated in 7 days.In the plain sub-fish of report of wnt/ β-connection (TOP:dGFP), back 24 hours (hpr) can observe GFP fluorescence in the hepatotomy edge in excision, shows the activation of wnt signal transduction pathway in the liver regeneration early process.This is associated with and sham-operation contrast regeneration liver inner cell nuclear β-the is connected increase of element of comparing.

Whether provide the regeneration advantage for measuring excessive wnt activation, in the expression of 6～18hpr by heat shock induction wnt8.Compare with the wild type contrast, this processing produces significant liver growth at 3hpr and quickens.Similarly, APC ^+/-Mutant demonstrates enhanced regeneration capacity compared with the control.The genetically modified use of dnTCF has disclosed liver regeneration and wound healing and has been seriously undermined and confirmed that the Brachydanio rerio liver regeneration needs the plain signal conduction of wnt/ β-be connected.Fabric analysis has all confirmed these discoveries in regenerated all stages.Plain level of nucleus and cytoplasmic β in the Brachydanio rerio-be connected and PCNA dyeing increase and have strengthened regeneration with the raising of wnt signal conduction.The plain signal conduction of wnt/ β-connection lasting importance to liver homoiostasis and growth in the organic life-span has been given prominence in these experiments.

Importantly, the plain signal conduction of the β-connection of raising can strengthen mammiferous liver regeneration.For testing this prediction, that is, the plain signal conduction of the wnt/ of increase level β-connection can give mammal partially hepatectomized postoperative conservative regeneration advantage, to APC ^Min/+Carry out hepatotomy with wild-type mice.Carrying out 2/3 partially hepatectomized postoperative of standard, to liver weight: the analysis of the ratio of body weight has disclosed APC ^Min/+Mice increases than regeneration capacity compared with the control, and this is long the most remarkable in early days in liver regeneration.At APC ^Min/+In the mice, the plain level of β-connection increases near the door pipe basically at baseline, and in the early stage significantly increase of liver regeneration.APC ^Min/+The data acknowledgement wnt activation of mice has strengthened liver regeneration kinetics, shows that in addition pharmacology's regulation and control are carried out in the plain signal conduction of wnt/ β-connection can quicken to damage the back liver regeneration.

The molecular mechanism indication that the control liver is grown has been emphasized the Basic of Biology that liver tumor forms and the target of treatment regulation and control is provided.Because wnt/ is β-defective of the plain signal conduction of connection is prevalent in the hepatoma of original and differentiation, the invention provides the effect of wnt signal conduction in regulating specialization of liver and growth.The activator by analyze expressing the conduction of wnt signal and the transgenic zebrafish of inhibitor and have misadjustment β-is connected plain active saltant Brachydanio rerio the invention provides the wnt/ β that liver is grown and the homeostatic several aspects of adult tissue are required-be connected element adjusting.

The disappearance gradually of APC does not have linear effect to liver size in the embryo development procedure.APC disappearance causes Cytoplasm and nuclear beta-the is connected increase of gathering of element.Although at APC ^+/-This causes hepatocyte quantity to improve in the embryo, but APC ^-/-There is not plain adjusting of β-connection but to cause apoptosis to increase and the hypoblastic organs that can not grow in the mutant fully for differentiation.The use of the heat shock induction genetically engineered fish of this paper report has confirmed that the functional demand of the wnt signal conduction in the entoderm CFU-GM changes in embryo development procedure.Amazing is that although the plain signal conduction of over-drastic wnt/ β-connection (about 1～5 body segment) can suppress suitable liver growth during body constitution formed in early days, the wnt8 activation has caused the liver increase when 10 body segments.Enhanced CFU-GM hypertrophy by the plain signal conduction of wnt/ β-connections that improves (10 body segments are to 24hpf) mediation can be fast with enlarge CFU-GM exponentially and compile, cause at wnt8 and APC ^+/-Observe the liver size in the embryo and the cell quantity difference is very big.After the liver of differentiation formed, the wnt signal of raising conduction (48hpf) is enhance hepatocyte hypertrophy and whole organ growth once more.In a word, these data show be used for several stages-dependent form demand that the wnt signal conduction of suitable specialization of liver and growth is regulated.

The two stage wave patterns of the plain signal conduction of wnt/ β-connection to entoderm CFU-GM and the influence of specialization hepatocyte have been supported in observation.In addition, this makes the plain signal conduction of wnt/ β-connection grow entoderm, and particularly the difference that exists in the impact statement in the liver becomes consistent.In the early stage body constitution forming process, high-caliber wnt signal conduction is to specialization of liver and grow harmful.APC ^-/-Saltant embryo has shown that suitable entoderm CFU-GM needs some inhibition of the plain signal conduction of β-connection.After having specified endoblastic destiny, confirm as this paper, need wnt to start CFU-GM expansion and organ growth.At this stage A PC ^+/-Growth vigor has been given in the plain signal conduction of the β-connection that improves in the embryo, and this is reflected by the increase of outgrowth liver blast cell quantity and the enhancing of liver cell quantity thereafter.

The challenge that this paper proposed finds it is the growth destiny that the plain signal conduction of wnt/ β-connection can change non-specialization entoderm CFU-GM.In heat shock induction wnt8 embryo, wnt/ β-connection element is induced the influence of hypoblastic organs's differentiation thereafter very remarkable.The plain signal of wnt/ β-connection conducts and has changed specialization of liver longitudinal axis district and the CFU-GM distribution is turned to specialization of liver cell fate.The most significant is that it is unfavorable especially that over-drastic wnt signal conduction seems pancreas is grown.The endoderm cell district that can respond the liver conditioning signal can enlarge, and can clip the zone that is applicable to that normally pancreas is grown like this.Selectively, if there is the liver-pancreatic progenitor cell of economic benefits and social benefits, the pressure of then being partial to be divided into liver cell destiny will effectively reduce the CFU-GM quantity that is used to generate pancreas.In embryo's outer planting was analyzed, gut entoderm demonstrated not such as liver inducement signals such as fibroblast somatomedin the time and activates the pancreas gene program before the abdomen, shows to have the economic benefits and social benefits cell.People such as Deutsch, 128 Devel.871-81 (2001).Can regulate and control to therapeutic the character of these multiple-effect progenitor cell.Although can be divided into the hepatic progenitor cells of hepatocyte and bile duct cell people such as (, 101P.N.A.S.USA 8360-65 (2004)) Strick-Marchand on the books, the CFU-GM plastic detection relevant with the pancreas differentiation but also do not finished.

The present invention introduces the research and development of partial hepatectomy and allows to carry out in the Brachydanio rerio of liver regeneration research as new technique.The Brachydanio rerio liver was regenerated as its original dimension in 7 days, be equivalent to the long kinetics of murine liver regeneration.Brachydanio rerio size and the complexity relevant with the excision that does not encapsulate organ have been got rid of based on the long accurate quantification analysis of the liver regeneration of the ratio of liver/body weight, but the progress of the monoblock dissection and analysis that this paper describes in detail, and regenerative process several stages tissue characterization completely, thereby allow accurately and detail analysis.For example, use wnt-to report sub-fish, studies confirm that in the body that the conduction of the wnt of margins of excision place signal is activated in 24 hours.

In addition, the invention provides first example that the plain signal conduction of activation wnt/ β-connection can strengthen the liver regeneration rate.Wnt8 transgenic and APC ^+/-Nuclear beta-connection element that the analysis of fish has disclosed the increase level causes hyperplasia enhancing and liver regrowth to be quickened.In addition, APC ^Min/+Regeneration advantage after the Mouse Liver excision is to evolve to go up to guard.(according to Green ﹠amp; Puder, the murine hepatotomy is carried out in the record of 16 J.Investigational Surgery 99-102 (2003)).Therefore, the invention provides behind hepatotomy or from the acute hepatic failure recovery process of bringing out, by the method for the regulation and control wnt/ β-plain path enhancing of connections patient liver regeneration by poisonous substance (as acetaminophen).

As shown here, the plain signal conduction of wnt/ β-connection influences several entoderms and liver cell mass in the liver growth course, and this program is activated again to regulate liver regeneration.Having a lot of documents to put down in writing the conduction of the plain signal of wnt/ β-connection relates to various forms of hepatomas and form: hepatoblastoma shows that APC frequently suddenlys change, and cancer of biliary duct and HCC confirm β-the is connected variation of element, Axin and GSK-3 β.This shows that as the plain signal of wnt/ β-connection conducts the hepatocyte destiny that can regulate in the liver growth course several stages the plain signal conduction of wnt/ β-connection can be made contributions for the carcinogenesis in several cell masses of hepatocyte type.Understanding the plain signal conduction of wnt/ β-connection can open the mechanism of carcinogenesis and it is suppressed in each cell type the development impact of these cell masses.Zebra fish model provides unique chance for determining the new treatment of regulating the conduction of wnt signal.The chemical genescreen of the dressing agent of the adjusting that the present wnt that is carrying out cell growth in embryo's forming process mediates; Further estimate this method compounds identified possibly in adult Brachydanio rerio liver regeneration process and at APC ^+/-Conservative function in fish and the chemically induced trouble hepatocarcinoma zebra fish model in the carcinogenesis adjustment process.As extensive chemistry screening, the function of the plain signal conduction of exhaustive analysis wnt/ β-connection in the growth of Brachydanio rerio liver, liver cell proliferation and carcinogenesis strengthened more efficient diagnosis and the ability for the treatment of cancer.

Another embodiment of the invention provides by the prostaglandin signal transduction pathway and has regulated vertebrates tissue growth or regenerated compositions and method.For example, PGE2 has strengthened the vertebrates tissue regeneration.The effective dressing agent of prostaglandin signal transduction pathway as liver growth in the embryo development procedure determined in the chemistry screening of Brachydanio rerio.Compare with the wild type contrast, hatch the embryo with cox1-, cox2-or bispecific inhibitor the liver size is significantly reduced at 72hpf, and contact dimethyl PGE2 (dmPGE2) has strengthened the liver growth.The morpholine of cox1 or cox2 strikes to subtract and has similarly suppressed the liver growth, and is succoured fully by contact external source dmPGE2.Because the organizer inner equilibrium in the molecular pathway of the many control fetal development mediation adult, so detect the influence of prostaglandin signal conduction in the liver regeneration process.Designed the new method of a kind of lasting excision Brachydanio rerio 1/3 liver alive; Give after the tricaine anesthetics, carry out little cutting, and use micro dissector to remove the inferior lobe of SANYE liver at the abdominal part place at heart rear portion.Make fish bring back to life and in fish and water, heal.

Be the demand of functional prostaglandin signal conduction in the test regenerative process, partially hepatectomized postoperative 6～18 hours makes fish contact bispecific cox inhibitor indomethacin.Compared with the control, significantly reduced liver regeneration at the 1st and the 3rd day indomethacin, can not holomorphosis after 5 days in excision.In addition, the indomethacin contact has caused liver structure regional at incisxal edge just and the whole not damaged portion of organ to change.Cox suppresses also to have stoped the wound healing of cut place.

Compare with untreated fish, excise back contact dmPGE2 and caused the long enhancing of liver regeneration, being accompanied by the liver vessel distribution significantly increases.Just can see this enhancing in back 1 day, and cause the faster holomorphosis of organ as far back as excision.In addition, it is very fast to observe the wound healing of joining tissue.The adjusting of PGE2 level can play a role to the repair/regeneration such as various types of organizations such as heart, bone and wound repair.

In addition, prostaglandin path and the conduction of wnt signal interact: prostaglandin path activator dmPGE2 is found in brain, liver and the intestinal growth and strengthens the conduction of wnt signal, and indomethacin causes in fact not having the conduction of wnt signal.In addition, cox suppresses to alleviate the influence that the wnt activation is grown liver and the regenerated promotion of liver is grown.These results show that the prostaglandin path directly influences the center mediation body β-plain transcriptional activity of connection of wnt path.Similarly, as putting down in writing among the WO 07/112084, the conduction of wnt signal can also be regulated the formation and the recovery of hematopoietic stem cell.As liver, can stop the HSC quantity enhancing of wnt mediation by the inhibition of prostaglandin signal conduction.This interaction that shows wnt and prostaglandin path is guarded in the growth of many tissues with in repairing.The wnt signal transduction pathway is the target that potential captivation is arranged of treatment regulation and control: the activation of path has strengthened the growth and the regeneration of damage back tissue, and on the contrary, inhibition may be very important to treatment of cancer.Yet the wnt inhibitor of Fa Xianing does not also develop into fully and can clinically use up to now, perhaps is the reason owing to toxicity or side effect.Using prostaglandin or prostaglandin inhibitor to regulate the conduction of wnt signal provides the selectable method of taking into account: wnt activation to offer help for damaging the acute repairing phase in back, and suppresses undesirable influence that prostaglandin can be used for preventing the conduction of wnt signal.

The tissue growth regulator of the present invention that influences the prostaglandin path and tissue growth is suppressed comprises indomethacin, NS398, SC560, celecoxib (Celecoxib), sulindac (Sulindac), fenbufen (Fenbufen), aspirin, naproxen (Naproxen), ibuprofen (Ibuprofen), AH6809 (EP 1/2 antagonist) and AH23848 (EP4 antagonist).

The tissue growth regulator that influences the prostaglandin path and tissue growth is enhanced comprises dmPGE2, PGE2, PGI2, linoleic acid, 13 (s)-HODE, LY171883, ONO-259, Cay10397, eicosatrienoic acid, epoxy eicosatrienoic acid and arachidonic acid.

The tissue growth regulator that influences the wnt path and growth is suppressed comprises Kenpaullone (the HDAC effect is not GSK3b), valproic acid (the HDAC effect is not GSK3b) and solubility dkk.

On the contrary, influence the wnt path and make the tissue growth regulator that is enhanced of growing comprise BIO, LiCl and solubility wnt part.

In addition, influence the initial prostaglandin signal conduction downstream of cAMP/PI3K/AKT second message,second messenger dressing agent effect and tissue growth regulator that tissue growth is suppressed comprises H89, PD98059, KT5720, U0126, LY294002 and wortmannin (Wortmannin).

The tissue growth regulator that influences the initial prostaglandin signal conduction downstream of cAMP/PI3K/AKT second message,second messenger dressing agent effect and tissue growth is enhanced comprises Buddhist SCH, 8-bromo-cAMP and Sp-5,6, and-DCI-cBiMPS.

Other tissue growth regulators that can act on initial prostaglandin signal conduction downstream comprise Ca ⁺Second message,second messenger's dressing agent.Those of inhibition tissue growth comprise BayK 8644 and thioridazine.Those of reinforcing agent of being considered to grow comprise Bapta-AM, fendiline, nicardipine, nifedipine, pimozide, apocymarin and lanatoside.

According to the present invention, NO/ angiotensin signal conduction dressing agent path can interact with prostaglandin and the conduction of wnt signal.Those of inhibition tissue growth comprise L-NAME, enalapril, captopril, AcSDKP, Losartan, telmisartan, histamine, ambroxol, chrysin, cycloheximide, methylene blue, epinephrine, dexamethasone, proadifen, benzyl isothiocyanate and ephedrine.

Can interact with prostaglandin and the conduction of wnt signal and comprise L-arginine, sodium nitroprusside, vanadic acid sodium and Kallidin I with the NO/ angiotensin dressing agent that strengthens tissue growth.

Early stage experimental evidence shows that the liver growth can be suppressed such as tissue growth regulators such as norethindrone, 3-estradiol, beta-carotene and BMS189453.On the contrary, the liver growth can be strengthened by flurandrenolide, retinoid acid, vitamin D and retinol.

The product PGE2 (PGE2) of cyclooxygenase (COX) is by being attached to 4 kinds of protein bound receptors of G (EP1-EP4) performance function.Therefore, tissue growth regulator of the present invention comprises PGE2 receptor stimulating agent and PGE2 receptor antagonist.

The EP4-selective agonist comprises ONO-AE1-734 (methyl-7-[(1R, 2R, 3R)-the 3-hydroxyl-2-[(E)-(3S)-3-hydroxyl-4-(-the methoxy phenyl)-the 1-butylene base]-the 5-oxocyclopentyl]-5-thia heptanoate), ONO-AE1-437, ONO-AE1-329, ONO-4819 is (all from OnoPharma.Co., Osaka, Japan), APS-999 NA (Toray Indus., Inc., Tokyo, Japan), analog AGN205203 (Allergan, Inc., the Irvine of the 8-azepine piperidones series of EP4 agonist, CA), L-902,688 (Merck Frosst Canada, Ltd.), 1,6-disubstituted piperidine-2-ketone, 3,4-dibasic 1,3-oxazine alkane-2-ketone, 3,4-is dibasic 1,3-thiazan-2-ketone and 4, the dibasic morpholine of 5--3-ketone derivatives, referring to U.S. Patent No. 7,053,085 (Merck ﹠amp; Co.Inc, Rahway, NJ).

On the contrary; the EP4-selective antagonist comprises ONO-AE3-208 (4-{4-cyano group-2-[2-(4-fluoronaphthalene-1-yl) propionamido] phenyl } butanoic acid) (Ono Pharma.Co.; Osaka; Japan); CJ-023; 423 (N-[({2-[4-(2-ethyls-4; 6-dimethyl-1H-imidazo [4; 5-c] pyridine-1-yl) phenyl] ethyl } amino) carbonyl]-the 4-methyl benzenesulfonamide) (Pfizer); BGC20-1531 (BTC Int ' l; Ltd.); AH23848 ((4Z)-7-[(rel-1S; 2S; 5R)-5-((1; 1 '-biphenyl-4-yl) methoxyl group)-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid half calcium salt hydrate); AH22921 ([1 α (Z); 2 β a; 5 α]-(±)-7-[5-[[(1; 1 '-biphenyl-4-yl) methoxyl group]-2-(4-morpholinyl)-3-oxocyclopentyl]-the 5-heptenoic acid) (GlaxoSmithKline); L-161; 982 (N-[[4 '-[[3-butyl-1; 5-dihydro-5-oxo-1-[2-(trifluoromethyl) phenyl]-4H-1; 2; 4-triazole-4-yl] methyl] [1; 1 '-biphenyl-2-yl] sulfonyl]-3-methyl-2-thenoyl amine) (Merck Frosst Ltd., Canada).

The EP2-selective agonist comprises ONO-AE1-259, ONO-8815Ly, ONO-8815, (L-lysine (Z)-7-[(1R, 2R, 3R, 5R)-5-chloro-3-hydroxyl-2[(E)-(S)-4-(1-ethyl cyclobutyl)-4-hydroxyl-1-butylene base] cyclopenta]-the 5-heptenoic acid esters) (Ono Pharma.Co., Osaka, Japan), AH13205 (trans-2-[4-(1-hydroxyl hexyl) phenyl]-5-oxo-cyclopentane-enanthic acid) (GlaxoSmithKline).

Several derivatives of prostaglandins have shown the relative effectivenes of the increase liver growth shown in the following table:

Derivatives of prostaglandins

(

Expression increases the relative effectivenes of liver growth):

PGE2

PGI2

16-phenyl tetranor PGE2

16,16-dimethyl PGE2

19 (R)-hydroxyl PGE2

16,16-dimethyl PGE2p-(p-acetylamino benzamido) phenylester

9-deoxidation-9-methylene-16,16-dimethyl PGE2

The PGE2 methyl ester

BAY-q 4218

15 (S)-15-methyl PGE2

15 (R)-15-methyl PGE2

20-hydroxyl PGE2

11-deoxidation-16,16-dimethyl PGE2

9-deoxidation-9-methylene PGE2

9-ketone fluprostenol

PGE2 serinol amide

Sulprostone

17-phenyl trinor PGE2

8-is different-15-ketone PGE2

The different PGE2 isopropyl esters of 8-

The trans PGE2 of poisonous 5-

The example that relates to the wnt path and can be used as other molecules of the regulator that is included in the scope of the present invention is in the news.Referring to, for example, Barker ﹠amp; Clever, 5 Nature Rev.DrugDiscovery 997-1014 (2007); People such as Janssens, 24 Investigational New Drugs263-80 (2006).Yet, should be noted that, reported that the wnt signal conduction of attempting directly to suppress the cancer patient has significant adverse side effect.Barker?&?Clever，2007。Therefore, as indicated in the present invention, can in the treatment research and development, be proved to be more effective through prostaglandin path indirect regulation wnt signal transduction pathway.

Can determine the tissue growth regulator in the scope of the invention in every way, as the Brachydanio rerio genetic system.Brachydanio rerio (Danio rerio) is the fabulous genetic system of growth of research vertebrates and disease.For example referring to, Hsia ﹠amp; Zon, 33 (9) Exp.Hematol.1007-14 (2005); De Jong ﹠amp; Zon; 39 Ann.Rev.Genet.481-501 (2005); Paffett-Lugassy ﹠amp; Zon, 105 Meth.Mol.Med.171-98 (2005); Haffner ﹠amp; Nusslein-Volhard, 40 Int ' l J.Devel.Biol.221-27 (1996).Outside fetal development is transparent and can easily sees each organ.Brachydanio rerio is shared many identical gene program with mammal in growth.When the Brachydanio rerio copulation, they produce a large amount of (100-200 weekly) transparent embryos.Many embryos can be placed on less relatively space, and arranged very short generation time (about 3 months).Extensive screening produced have in fact influence the embryo form each side specific defects surpass 2000 gene mutation bodies.People such as Driever, 123 Devel.37-46 (1996); Eisen, 87 Cell 969-77 (1996).Many blood mutants in the critical event of describing hemopoiesis of great use.Dooley?&Zon，10?Curr.Op.Genet.Devel.252-56(2000)。Brachydanio rerio has been used to carry out based on whole organic micromolecule screening, because a large amount of embryo can be aligned in the microtitration plate that contains from the chemical compound of chemical libraries.For example, Peterson and colleague have tested 1,100 kind of chemical compound and have been used for developmental defect research.People such as Peterson, 97 P.N.A.S.USA 12965-69 (2000).In this screening, about 2% chemical compound is a lethal, and 1% causes the specificity phenotype.For example, a kind of chemical compound inhibition is known as the formation of the interior ear structure of otolith, but does not but cause other defect.

Can also screen the chemical inhibitor of mutation type surface.People such as Peterson, 22 Nat.Biotech.595-99 (2004); People such as Stern, 1 Nat.Chem.Biol.366-70 (2005).In the screening of this class, find (gridlock) mutant that the relief of chemicals mass-energy is stopped up, promptly a kind of congenital aortic contracting model.People such as Peterson, 2004.This relief mechanism relates to induces VEGF, forms defective to correct vascular.These data acknowledgements use Brachydanio rerio can determine highly effective and specific chemical compound.

Be to have made up the dense genetic map of the sequence label (EST) that comprises microsatellite marker thing, gene and expression further about Brachydanio rerio.People such as Knapuk, 18 Nat.Genet.338-43 (1998); People such as Shimoda, 58 Genomic 219-32 (1999); People such as Kelly, 10 GenomeRes.558-67 (2000); People such as Woods, 20 Genome Res.1903-14 (2000).Also carry out full-length cDNA and be intended to be the expansion that Brachydanio rerio EST plans.Make up intensive RH figure at the Sanger center and integrated the genome project data.The important Internet resources that NIH supports are the Brachydanio rerio information networks (ZFIN) as the public attention focus.The reserve center and the support laboratory that are known as Brachydanio rerio international resource center (ZIRC) have also been supported this area energetically.The Sanger center is to the Brachydanio rerio gene order-checking.

Use the techniques described herein, can make wild type contact a large amount of chemical compounds with transgenic zebrafish, with the influence of assessing compound as prostaglandin and/or wnt/ β-plain signal transduction pathway regulator of connection.For example, test compounds can be had the genetically engineered fish that contains the external source construct of reporting sub-protein expression sequence.By to the contact test compounds and not the fish of engaged test chemical compound report the comparison of sub-protein expression, can measure the influence of chemical compound to the adjusting of prostaglandin signal transduction pathway.Similarly, the comparison of reporting sub-protein expression to the fish and the negative contrast of contact test compounds, can assessing compound to the influence of the plain signal transduction pathway adjusting of wnt/ β-connections.Test compounds can be used as the report subbase because of inhibitor or activator.Importantly, the regulator of individual channel can make up and contact with the sub-fish of report, and will report that sub proteic expression and the positive and negative contrast that is fit to compare.By this way, determine that and as described herein, wnt/ β-plain signal transduction pathway of connection may be subjected to the influence of prostaglandin pathway modulators as treatment and wnt/ β-be connected the regulator of the medicine of plain signal transduction pathway associated conditions.

Regulator of the present invention comprises the regulator of direct adjusting wnt signal transduction pathway; Regulate the regulator of prostaglandin path to realize that the wnt signal transduction pathway is regulated; Or the downstream influences of adjusting prostaglandin is to regulate the regulator of wnt.In addition, these regulators can be combined with " fine setting " signal.For example, can utilize wnt signal conduction activator, limit the influence of wnt activator afterwards with prostaglandin inhibitor until noticing desirable enhancing.Perhaps, for example, low dosage wnt activator and low dosage prostaglandin activator can be made up to avoid toxicity.Therefore, the interaction of the regulator of wnt and prostaglandin signal transduction pathway be can use,, toxicity or undue growth limited simultaneously to obtain desirable response with any direction or combination in any.Can simultaneously or use regulator in turn.

The patient can several modes benefit from the present invention: for example, the patient of experience hepatotomy operation can faster recovery liver function, reduces complication and hospitalization.Compellent is that the patient who accepts liver transplantation can have higher organ survival rate.When being used for other aspects of organ and tissue regeneration, for example, can the positive impact myocardial infarction after and the enhancing in the wound healing process of fracture back recover.In addition, for example, regulator of the present invention can be added in experimenters' such as suffering traumatic damage, drug intoxication, poisoning (for example, amanita picked-up), industrial poison, operation, liver donation, cancer, skin transplantation, burn the therapeutic scheme.Growth regulator of the present invention can be used for regenerating, any tissue of reparation or regrowth, comprises hematopoietic stem cell, liver, skin or vascular.

The directly stripped regulator that gives can make in-vivo tissue growth or regeneration significantly, and is therefore promptly enough than being organized in the transplanting of a small amount of even.The source of this tissue is unrestricted.Selectively, can gather, in the presence of such as regulators such as PGE2, store immediately then, before introducing the experimenter, in the presence of regulator, begin to hatch (before transplanting) such as tissue-derived samples such as skins.

In addition, can use one or more regulators to strengthen tissue-derived function.For example, regulate the ability that the wnt/PGE2 path can accelerate to transplant its physiological role of performance, and therefore shorten the time that the experimenter has insufficient tissue (for example liver organization), thereby reduced the danger that leads to complications.In addition, can after removing, tissue give LD with healing acceleration with regulator.

The using-system growth regulator is to strengthen tissue growth and stripped using-system growth regulator to increase tissue growth in can body.This can be by giving the experimenter with one or more chemical compounds or cut tissue is realized.For example, in the uterus again in the structure process, under the environment of biocompatible scaffold, can handle the tissue of excision (referring to for example with the tissue growth regulator, people's such as Atala U.S. Patent No. 7,049,057, " Tissue engineered uterus "), to transplant prerequisite for the growth of enhanced autologous tissue.

This area known be used to collect, all ingredients box and the gathering-device of processing and storage source cell.In collection, processing and/or storage process, regulator of the present invention can be introduced cell.Therefore, be not subject to any specific collection, processing or save scheme, embodiment of the present invention provide and will be added in the tissue samples such as regulators such as wnt activator, PGE2 or dmPGE2 or their analog, cAMP activator.This can carry out when collecting or when preparing to store or before when thawing and the transplanting.

Therefore, the inventive method provides following advantage: (1) allows the patient who is not taken as candidate owing to have graft failure or elementary portability function to fail this unacceptable excessive risk is transplanted; (2) reduced to produce the minimum required donor tissue size of accepting to gather; (3) tissue samples that is suitable for transplanting by increase has reduced the incidence rate of primary and secondary graft failure; (4) reduced the required time of elementary implantation by the growth that strengthens the tissue of transplanting.

Regulator of the present invention for example suppresses the regulator of tissue growth, also can be used for treating the experimenter who suffers from the high proliferation disease, comprises those diseases of hemopoietic system or other cancers.Especially, described regulator can be used in the treatment of hepatic disease.

Regulator of the present invention also comprises the derivant of this class regulator.Derivant used herein comprises the chemical compound of chemical modification; wherein said modification is the route of chemist's approval of common skill, as extra chemical part (for example, Suan ester or amide; blocking group is as for the benzyl of alcohol or mercaptan with for the tertbutyloxycarbonyl of amine).Derivant also comprises the conjugate (for example, biotin or avidin have such as enzymes such as horseradish peroxidases, have luminescent biological agent, chemiluminescence agent or fluorescent agent) of radiolabeled regulator, regulator.In addition, described part can be added in the part of regulator or regulator to increase the half-life in the body.Derivant used herein also comprises analog, as comprises the specific compound of chemical modification form or the chemical compound of its compounds, keeps the chemical compound of described chemical compound or distinctive pharmacy of its compounds and/or pharmacological activity to be also included among the present invention.Derivant used herein also comprises the prodrug of regulator, and known its can strengthen the many desirable character of medicine (for example, dissolubility, bioavailability, preparation etc.).

The compounds of this invention or reagent can be included in the pharmaceutically acceptable preparation.This pharmaceutically acceptable preparation can comprise pharmaceutically acceptable carrier and/or excipient." pharmaceutically acceptable carrier " used herein comprises any and all solvent, disperse medium, coating agent, antibiotic and antifungal, isotonic agent and absorption delay agent etc. of physical compatibility.For example, carrier can be suitable for injection and enters cerebrospinal fluid.Excipient comprises pharmaceutically acceptable stabilizing agent.The present invention is suitable for any pharmaceutically acceptable preparation, synthetic or natural polymer, Nano capsule, microsphere or the pearl that comprise the macromolecular complex form, with the preparation that is suitable for based on lipid, comprise the erythrocyte of emulsion oil-in-water, micelle, mixed micelle, synthetic membrane vesicle and heavy involution.

When reagent or chemical compound are flowed to the patient, can give by any suitable approach, comprise that for example oral (for example, with capsule, suspending agent or tablet) or parenteral give.Parenteral for example give to comprise intramuscular give, comprise be administered to directly that pylic intravenous gives, give in the articular cavity, intra-arterial gives, give in the capsule, subcutaneous give or intraperitoneal gives.Reagent can also orally give, transdermal administration, topical administration, suction give (for example, suck in the bronchus, intranasal sucks, per os sucks or intranasal splashes into) or rectum gives.Can give according to indication part or whole body.Can also use viral vector delivery of therapeutic agents well known to those skilled in the art.

The present invention includes topical administration and whole body gives.The desirable characteristics of topical administration comprise the effective reactive compound local concentration of acquisition, have avoided whole body to give the adverse side effect of reactive compound simultaneously.In a preferred embodiment, antagonist is by topical administration.Local conveying technology is documented in for example 51 J.Biomed.Mat.Res.96-106 (2000); 100 (2) J.Control Release211-19 (2004); 103 (3) J.Control Release 541-63 (2005); 15 (3) Vet.Clin.North Am.Equine Pract.603-22 (1999); Among 1 (1) Semin.Interv.Cardiol.17-23 (1996).

Pharmaceutically acceptable preparation can be suspended in the aqueous carrier and by conventional hypodermic needle or use delivery pump and introduce.

Be administered to individual amount of reagent and depend on personal feature, as general health, age, sex, body weight and medicine allowance and repulsion degree, severity and type.Depend on these and other factors, those skilled in the art can determine the dosage that is fit to.

Further describe several embodiments below by non-limiting example.

Embodiment

The technology that embodiment 1. is relevant with zebra fish model.

The management of Brachydanio rerio: preserve Brachydanio rerio according to the IACUC scheme.Use LFABP:GFP (G.M.Her ﹠amp; J.L.Wu, Nat ' l Cheng Kung Univ., Taiwan gives), gut:GFP, hs:wnt8-GFP, hs:dnTCF-GFP and hs:dkk-GFP transgenic lines.People such as Dorsky, people such as 2002:Her, 538 FEBS Lett 125-133 (2003); People such as Lewis, 131 Devel.1299-1308 (2004); People such as Ober, 120 Mech.Devel.5-18 (2003); People such as Stoick-Cooper, 134Devel.479-89 (2007); People such as Weidingder, 15 Curr.489-500 (2005).By the gene type of being put down in writing that carries out the APC mutant.People such as Hurlstone, 2003.

Heat shock activation/the inhibition of wnt signal conduction: except as otherwise noted, embryo's heat shock of carrying out the persistent period be under 38 ℃ 20 minutes is tested.After the thermal induction 3 hours, in the presence of GFP fluorescence, measure genotype, non-fluorescence (wild type) compatriot is in contrast.

Morpholine strikes and subtracts: with concentration be 40 μ M at Brachydanio rerio β-connection element, wnt2b, wnt3, wnt5, wnt8 and wnt11 (people such as Buckles, 121 Mech.Devel.437-47 (2004); People such as Lekven, 1 Cell Devel.103-14 (2001); People such as Lele, 30 Genesis 190-94 (2001); People such as Lyman Gingerich, 2004; People such as Ober, 442 Nature 688-91 (2006)) MO (Gene Tools, LLC, Philomath, OR) or the contrast of mispairing injection enter the zebrafish embryo in unicellular stage.

In situ hybridization: use at for example ZFIN: the standard Brachydanio rerio scheme of finding on zebra fish model organism data base's (Univ.Oregon, Eugene, the main frame of OR) the Internet is handled the fixed embryo of paraformaldehyde (PFA), carries out in situ hybridization.Following rna probe is used to detect entoderm and the developmental variation of liver: GFP, LFABP, sterol carrier protein, transferrins, foxA3, insulin, trypsin and IFABP.The expression variation that each genotype is compared with the wild type contrast is registered as # change/# record; Each analyzes 3 independent experiments of minimum work.

Immunohistochemical analysis: with fixedly embryo, adult and the section of whole abdominal part of paraffin-embedded PFA, and be cut into the continuous step section of 40 μ m, be used for fabric analysis.Use standard technique that alternate slices is carried out hematoxylin/eosin dyeing.(BD 610154, BD Transduction Laboratories to make β-connection plain (1: 100) by DAB ^TM, San Jose, CA), TUNEL (Chemicon/Millipore, Billerica, MA), BrdU (1: 2000) (Clone BU-33, B2531, Sigma-Aldrich, St.Louis, MO) and PCNA (1: 80) (Clone PC10, NA03, Calbiochem/EMD Chemicals, Inc., San Diego, CA) antibody is visual, and with hematoxylin or methylene is green redyes.

Caspase test: with the lysis buffer single embryo and centrifugal that manually dissociates.(Promega Corp., Madison WI) are used for Caspase-Glo with supernatant (100ml) according to manufacturers protocol

3/7 test system.Use is confirmed the apc gene type from cell ball separated DNA.

Confocal microscope is analyzed: culture dish contained in the 1% low melting point agar of 0.4mg/ml tricaine-S (Tricaine-S) at the bottom of GFP transgenic zebrafish embryo was embedded in glass, by Zeiss LSMMeta confocal microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY) visual.

Flow cytometry analysis: each embryo that in 0.9%PBS, manually dissociates, and carry out GFP fluorescence and forward scattering detection.By unnecessary cell PCR being carried out the gene type of APC, carry out facs analysis then.

Hepatotomy: after giving the tricaine anesthetics, under the bright visual field of anatomic microscope, carry out 1/3 partial hepatectomy of adult Brachydanio rerio liver.Abdomen left part with micro dissector cutting heart rear portion.Excise the inferior lobe of whole length then with tweezers.

Embodiment 2. Brachydanio rerio tumor models.

Although Brachydanio rerio is research carcinogenesis of great value vertebrates model, and is opaque because of the adult fish, so a challenge is still had in the noninvasive imaging.Yet, utilize the ultrasonic in-vivo tumour that can easily carry out of high resolution microscope to detect.This technology has promoted perfused tissue calculating, Cellular respiration, tumour progression analysis and treatment response.The ultrasound biological microscopy allows zebra fish model is carried out the longitudinal study of tumor growth and the Real-Time Evaluation of therapeutic effect.Visualization technique used herein is documented in people such as Goessling, among 4 (7) the Nature Methods 551-53 (2007).

As shown in figure 10, make Brachydanio rerio contact DMBA during ages with 4 weeks 3.Monitoring tumor takes place about more than 5 months.Differentiate tumor by ultrasound biomicroscopy UBM.Suffered from the cancer fish 12 hours with the indomethacin treatment of per 3 days of every month spending the night then, and by the observation tumor of record.People such as Goessling, 2007.In representative fish, treat 3 times after tumor size increase and structural change.After treating 6 times, the entity part size decreases of tumor liquefaction zone occurs in the abdomen back, shows neoplasm necrosis.After treating 10 times, tumor is atrophy greatly, and comparing during with initial ultrasound seems that size reduces.After the Drug therapy 1 month, it is very healthy that this fish seems, confirmed to exist cancer but kill by the histology.These observations have confirmed that prostaglandin suppresses the influence of liver tumor growth and the successful chemotherapy of fish.

In another experiment, see Figure 10 B and Figure 10 C for details, for prophylaxis of tumours is treated fish weekly.Here, cause the wild type fish to produce liver tumor, APC with the carcinogen processing ^+/-The saltant fish produces the tumor of twice quantity.With the indomethacin treatment tumor of two kinds of wild type fishes is formed weekly and reduce, particularly APC ^+/-Fish.

Claims (3)

1. one kind is used to promote tissue growth or regenerated method, and described method comprises makes described tissue contact with at least a tissue growth/regeneration regulator that raises prostaglandin signal transduction pathway or its downstream mediation body, strengthens the wnt signaling activity thus.

2. one kind is used to promote growth of experimenter inner tissue or regenerated method, and described method comprises at least a tissue growth/regeneration regulator that raises prostaglandin signal transduction pathway or its downstream mediation body, strengthens the wnt signaling activity thus.

3. pharmaceutical composition, it comprises at least a tissue growth/regeneration regulator that raises prostaglandin signal transduction pathway or its downstream mediation body, strengthens the wnt signaling activity thus.

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