CN1137993C - Recombinant adenovirus of bone morphogenetic protein and its method for exciting bone generation - Google Patents
Recombinant adenovirus of bone morphogenetic protein and its method for exciting bone generation Download PDFInfo
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- CN1137993C CN1137993C CNB001271156A CN00127115A CN1137993C CN 1137993 C CN1137993 C CN 1137993C CN B001271156 A CNB001271156 A CN B001271156A CN 00127115 A CN00127115 A CN 00127115A CN 1137993 C CN1137993 C CN 1137993C
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Abstract
The present invention discloses a novel method and material for stimulating osteogenesis, particularly replication-deficient viruses and protein in the bone morphogenetic protein family coded by the replication-deficient viruses, and the replication-deficient viruses are selected from adenoviruses, adeno-associated viruses and retroviruses. The present invention also discloses mesenchymal cells transfected by the replication-deficient viruses, inocula containing the mesenchymal cells, and a method for stimulating osteogenesis in vivo and in vitro. The present invention can effectively, conveniently and/or cheaply stimulate osteogenesis.
Description
The present invention relates to biological and medical field, more specifically, the present invention relates to Delicious peptide (bonemorphogenetic protein, BMP) recombinant adenovirus, and stimulate osteogenetic method with described adenovirus.
Bone plays the support effect in Mammals.For various reasons, bone can sustain damage, for example fracture and tumor destruction etc.Therefore need exploitation to stimulate osteogenetic method and associated materials and equipment effectively.
At present clinically, still, implant the method at the position that needs reparation then adopting certain position to gather osseous tissue from patient body.This method makes patient must bear extra operation pain.And in some cases, patient itself can not provide enough osseous tissues to supply to repair the usefulness at position.
Simultaneously, before the present invention, it all is to carry out as follows that the BMP (BoneMorphogenetic Protein) that uses traditional gene engineering method to produce stimulates osteogenesis:
Utilize gene recombination technology at external generation BMP (mainly in the expressing cho cell system) → sneak in solid carrier (as collagen protein sponge) → implant patient or the animal body to stimulate living bone with protein separation technology purifying bmp protein → bmp protein.
Yet, in aforesaid method, exist following shortcoming:
(1) BMP is low in the cell inner expression amount, thereby preparation albumen is with high costs.
(2) meeting quilt enzymolysis/degraded rapidly after bmp protein is inserted in the body, thus lose activity, thereby to reach the required dosage of body endostosis high (bibliographical information is all in the milligram level).This and shortcoming (1) addition cause higher cost.
(3) owing to need solid carrier, therefore must carry out surgical operation and be implanted.In addition, implant site must have physical space to allow solid carrier to put into.This has limited the application of aforesaid method, and patient has also been caused certain pain.
Therefore, this area presses for new effective, the convenient and/or inexpensive osteogenetic method of stimulation and associated materials and equipment for a long time.
An object of the present invention is to provide new effective, the convenient and/or inexpensive osteogenetic method of stimulation.Preferably, this method pain that patient is caused is less than prior art.
Another object of the present invention provides the material that is used for described method.
A further object of the present invention provides bone or the osseous tissue precursor that forms with the inventive method.
In a first aspect of the present invention, a kind of virus of replication defect type is provided, this encoding viral is expressed the albumen in the bone morphogenetic proteins family, and described virus is selected from: adenovirus, adeno associated virus and retrovirus.Preferably, the albumen in the described bone morphogenetic proteins family is selected from: BMP-2, BMP-4, BMP-7.More preferably, the albumen in the described bone morphogenetic proteins family is BMP-2.
In a second aspect of the present invention, a kind of mesenchymal cell of conversion is provided, it expresses the albumen in the bone morphogenetic proteins family.Preferably, this mesenchymal cell be with the present invention above-mentioned virus transform.
In a third aspect of the present invention, a kind of interior inoculum with the stimulation bone growth of mammalian body that is used to implant is provided, this inoculum contains the above-mentioned mesenchymal cell of the present invention.
In a fourth aspect of the present invention, the method of a kind of external generation bone or osseous tissue precursor is provided, it comprises step: under the condition that is fit to the cell growth, cultivate the mesenchymal cell of conversion of the present invention, make it at vitro differentiation osteogenic tissue precursor or osteoblast.
In a fifth aspect of the present invention, a kind of osteogenetic method that stimulates in vivo is provided, it comprises step: the inoculum that the present invention is above-mentioned is implanted the osteogenetic position of needs.
In the accompanying drawing,
Fig. 1 has shown the building process of recombinant adenovirus Adv-BMP2.
Fig. 2 has shown the expression in mesenchymal cell at external Adv-BMP2.
Fig. 3 has shown under external Adv-BMP2 influence, cell proliferation and time relation.
Fig. 4 has shown under external Adv-BMP2 influence, the dependency of cell proliferation and Adv-BMP2 dosage.
Fig. 5 has shown that the mesenchymal cell that infected by Adv-BMP2 raises at vitro culture process neutral and alkali phosphatase activity.
Fig. 6 has shown that the mesenchymal cell that is subjected to the Adv-BMP2 infection at vitro culture 15-20 days, has the sedimentary generation of calcium ion.
Fig. 7 has shown in the mouse of handling with the inventive method, the situation that new bone tissue generates in leg muscle.Wherein Fig. 7 A is no any processing; Fig. 7 B is the untreated cell of injection; Fig. 7 C is the cell that injection Adv-β gal handled; Fig. 7 D is the cell that injection Adv-BMP2 handled.
Fig. 8 is to stimulate the photo of the histological examination of new bone in the mouse muscle that generates with the inventive method.
Fig. 9 has shown the new osteogenetic situation of handling with the inventive method in rabbit backbone position.
Figure 10 is the photo that the new bone of rabbit that forms with the inventive method is carried out histological examination.
Figure 11 has shown new osteogenetic situation between the pig spinal vertebral of handling with the inventive method.Among the figure, filled arrows: cause vertebral fusion up and down with the cell after the Adv-BMP2 conversion; Hollow arrow: do not cause vertebral fusion (no change) up and down with the cell after the Adv-β gal conversion.
Figure 12 is the photo that the new bone of pig that forms with the inventive method is carried out histological examination.Wherein, Figure 12 A display organization is learned and check the visible new osseous tissue that formed between centrum; Figure 12 B has shown the soft tissue between the normal cone.
Detailed Description Of The Invention
Inventive point of the present invention is: adopt the means of transgenosis (gene therapy), between the BMP gene is changed over to In the mesenchymal cell. Utilize mesenchymal cell routinely to produce bmp protein as preparation factory. When changing BMP over to After the mesenchymal cell of gene was placed in the body, the bmp protein that mesenchymal cell produces can stimulate between transduction Mesenchymal cell autocrine stimulation (autocrine) or make that other mesenchymal cell paracrine stimulates in the recipient's body (paracrine), thus make the mesenchymal cell differentiation and development generate new bone.
As used herein, " bone morphogenetic protein (bone morphogenetic protein, BMP) " comprises bone Any member in the morphogenetic proteins family, the BMP gene family has progressively been found 14-15 similar base at present Cause, they all have the function that stimulates bone to generate. Any bmp protein all can be used for the present invention. Preferably, Bone morphogenetic protein is selected from: BMP-2, BMP-4, BMP-7. More preferably, described bone form takes place Albumen is BMP-2.
As used herein, after " replication-defective virus " refers to virus infected cell as carrier, can not the oneself Copy.
As used herein, " mesenchymal cell " refers to various have differentiation and development Gegenbaur's cell, bone precursors Cell, it comprises various stem cells, precursor and common mesenchymal cell.
Any virus that is used as in the art carrier all can be used for the present invention, especially is in gene therapy The virus of using in the field is such as adenovirus, adeno-associated virus and retrovirus. Preferably these viruses are multiple Defective virus processed.
The present invention is applicable to all mammals, comprising (but being not limited to): people, dog, cat, sheep, Ox, horse, pig, tiger etc. The present invention can be used for medical science, veterinary field.
In the present invention, at first make up the carrier that contains the BMP gene. This can finish with conventional method.
After having made up the carrier (as virus) that contains the BMP gene, available routine techniques will contain the virus of BMP gene and the combination of mesenchymal cell, promptly utilize virus vector that the BMP gene is imported mesenchymal cell.This is a very important step of the present invention.With the recipient cell of mesenchymal cell, not only can produce autocrine stimulation and paracrine stimulatory effect as the BMP gene.Simultaneously can produce bmp protein and effect in vivo and in vitro constantly again, thereby the excellent osteogenetic effect of stimulation is provided.
Obtained to change over to after the mesenchymal cell of BMP gene, just can further cultivate and can obtain more cell, perhaps freezing standby, perhaps directly use it for the stimulation osteogenesis.
Stimulate osteogenetic mode that two big classes are arranged.One class is to utilize the vitro tissue engineering.With the above-mentioned mesenchymal cell that changes the BMP gene over to of the present invention, can generate tissue in vitro culture technology by routine.Show, but mesenchymal cell of the present invention differentiation and development in the vitro culture process becomes into osteoblast (osteoblast).In one embodiment of the invention, shown at external bone and the osseous tissue precursor of having formed.
Another kind of is directly to use clinically.It comprises makes a kind of being used to implant in the mammalian body to stimulate the inoculum of bone growth, it has contained the above-mentioned importing of the present invention mesenchymal cell of BMP gene.A kind of example of inoculum is exactly
The suspension that contains the mesenchymal cell that has imported the BMP geneThen,, will need osteogenetic position in the described inoculum implantation mammalian body,, thereby form new bone with the stimulation osteogenesis by for example direct injection or modus operandi.The present invention's range of application clinically can contain Orthopeadic Surgery, plastic surgery and Neurological Surgery, and all use the occasion of osseous tissue, osteogenesis, bone fusion clinically.
Particularly, the present invention has following concrete application scenario:
1, common clinically have a case that can not heal after the fracture.The mesenchymal cell of Adv-BMP2 transduction can be used for being injected directly into not more position, generation autocrine stimulation and paracrine stimulatory effect promotion union of fracture of fracture.
2, often to be used for other operative site on orthopaedics and the orthopedic clinical treatment from patient's certain position intercepting bone on one's body.The inventive method can be save this intercepting bone-operating, reduces patient's misery, especially to can not bear the patient of extra operation more on physical efficiency, provides new treatment machine meeting.
3, after the present invention also was used in the bone tumor operation of orthopaedics and whole surgery, new bone was grown in the symphysis of filling out again at disappearance position.
4, the present invention can be used for the vitro tissue engineering.At the front body structure of external generation bone or osseous tissue, back skeletonization rapidly implants.
5, orthopaedics spinal fusion operation.Traditional method uses steel plate to fix or from the body bone.Present method can be save operation with injection and cause spinal fusion.
6, all use osseous tissue, and the occasion of impelling osseous tissue to generate all can be used the inventive method.
The present invention has following advantage:
(1) must be at vivoexpression and separation and purification bmp protein, thus process and the cost that batch production prepares BMP save.
(2) mesenchymal cell that shifts of receptor gene in vivo external enwergy produce bmp protein constantly and keep lasting biological action, thereby avoided immediately digesting after bmp protein implants in the past the problem of inactivation.
(3) mesenchymal cell after the transgenosis can implant with the method for direct injection or operation, thereby has eliminated the obstacle of necessary use solid carrier, thereby has reduced the restriction of clinical application.In addition, the pain that patient is caused is less than prior art.
(4) adenovirus of taking on transgenosis can cause the intravital immune response of receptor because of the antigenicity of himself, is finally removed.Therefore, avoided the mesenchymal cell after the transgenosis unrestrictedly to stimulate the consequence of giving birth to bone in vivo.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment
(1) makes up the recombinant adenovirus that carries the BMP-2 gene
People BMP-2 gene is from ATCC (American type culture collection).With the molecular cloning means people BMP-2 gene is inserted formation plasmid PAC-BMP2 among the expression plasmid PAC.In this plasmid, the BMP-2 gene is subjected to the control of cytomegalovirus early promoter/enhanser (cytomegalovirus, early gene promoter/enhancer).With XhoI PAC-BMP2 is cut into wire.
With adenovirus/5 types (adenovirus type 5) protease K digesting (56 ℃ are spent the night) of purifying preparation, discharge the genomic dna of virus with the degraded coat protein.With the ClaI restriction endonuclease genomic dna of adenovirus is digested (37 ℃, 60 minutes), thus produce one big, little two dna fragmentations.Separating these two fragments with the agar electrophoresis method gathers in the crops big dna fragmentation then and is called the big fragment of ClaI.
With lipofectamine PAC-BMP2 and the big fragment cotransfection of adenovirus ClaI (co-transfection) after the XhoI cutting are gone in 293 cells.Point is got the virus plaque of formation after 10 days.After further plaque purifying and amplification, this recombinant adenovirus is called Adv-BMP2 (building process is seen Fig. 1).
This recombinant adenovirus 5 type Adv-BMP2 are preserved in Chinese typical culture collection center (CCTCC, China, Wuhan City) on July 11st, 2000, and the CCTCC preserving number is V200004.
Embodiment 2
Change the BMP gene over to mesenchymal cell
The adenovirus of reorganization has lost the propagation in ordinary cells and the function of cell killing.Only cell is gone in the BMP-2 transgenosis that it can be carried as carrier.Infect various mesenchymal cells (the medulla mesenchyma cell that comprises mouse C3H10T1/2 cell and rabbit and pig) with Adv-BMP2, then vitro culture 5 days.
The collecting cell nutrient solution carries out immunoprecipitation (immunoprecipitation) back with the antibody of specific anti-human BMP2 and does immunity printing and dyeing (Western blot) test.Visible special BMP-2 band in the cell culture fluid that Adv-BMP2 infects.In other control group, then do not have.This BMP-2 transgenosis that shows the Adv-BMP2 mediation can produce people BMP-2 albumen (Fig. 2) in mesenchymal cell.
Mesenchymal cell after the Adv-BMP2 transfection is at external differentiation and development
After infecting various mesenchymal cells (as mouse, rabbit and pig) with Adv-BMP2, in the vitro culture process, following variation appears in the mesenchymal cell that is contaminted:
A, mesenchymal cell number.7th day comparable contrast mesenchymal cell (untreated mesenchymal cell and non-activity recombinant adenovirus infect mesenchymal cell) the propagation 3 multiple orders (Fig. 3) of mesenchymal cell after propagation is contaminted after cultivation.And the effect of mesenchymal cell propagation be directly proportional with the consumption of Adv-BMP2 (Fig. 4).The Adv-BMP2 consumption is big, and mesenchymal cell propagation is many, otherwise then few.
B, mesenchymal cell differentiation.The mesenchymal cell that infected by Adv-BMP2 can be to becoming osteoblast to break up in the vitro culture process.It is masked as alkaline phosphatase activities and raises.The contrast mesenchymal cell is then kept alkaline phosphatase activity constant (Fig. 5).
C, calcium ion precipitation.The mesenchymal cell that is subjected to the Adv-BMP2 infection can present Van Cossa stained positive at vitro culture 15-20 days, and this indicates the sedimentary generation of calcium ion.This phenomenon is typical external one-tenth osteoblast/osteoblastic another sign.And the contrast mesenchymal cell does not have this reaction (Fig. 6).
Embodiment 4
Mesenchymal cell after the Adv-BMP2 transduction generates new bone in vivo
To can generate new bone in the mesenchymal cell implantation animal body after the Adv-BMP2 transduction, but the method direct injection or the operation of implantation are put into
A, in the mouse body, generate new bone
Three weeks behind the muscle of thigh of the C3H10T1/2 injection cell after the Adv-BMP2 transduction being gone into mouse.X-ray examination can be found new bone (Fig. 7) at the intramuscular of injection site.Further confirm to have complete various osseous tissue forms with histological examination, as osteocyte, chondrocyte, medullary space fine jade (Fig. 8) at the new bone that intramuscular generates.The control group no change.
B, in the rabbit body, generate new bone
When the spinal bone position of self the rabbit bone marrow mesenchymal cell after the Adv-BMP2 transfection being implanted rabbit back with operation method.The visible new osteogenesis (Fig. 9) of the X-ray examination of 4-5 after week.Further complete osseous tissue form (Figure 10) is confirmed in histological examination.The control group no change.
C, in the pig body, generate new bone
When self the Medulla Sus domestica mesenchymal cell after the Adv-BMP2 transduction is implanted space between spinal vertebral with operation method.The back X-ray examination of 6 weeks can be found the new bone (Figure 11) that forms in interbody space.Newly-generated osseous tissue can fuse and causes fusion going up hypocentrum.The histologic characteristics (Figure 11, Figure 12 A and 12B) of new bone is confirmed in histological examination.The interbody space of control group is kept original soft tissue structure.X-ray and histological examination no change.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. the virus of a replication defect type is characterized in that, the albumen in this encoding viral bone morphogenetic proteins family, and described virus is adenovirus, the albumen in the described bone morphogenetic proteins family is BMP-2.
2. virus as claimed in claim 1 is characterized in that, described virus is adenovirus 5 types.
3. virus as claimed in claim 1 is characterized in that, described virus is recombinant adenovirus 5 type Adv-BMP2, and the CCTCC preserving number is V200004.
4. the mesenchymal cell of a conversion is characterized in that, it expresses the protein BMP-2 in the bone morphogenetic proteins family.
5. mesenchymal cell as claimed in claim 4 is characterized in that, described mesenchymal cell is the medulla mesenchyma cell.
6. mesenchymal cell as claimed in claim 5 is characterized in that, it is transformed by the described virus of claim 1.
7. one kind is used to implant the interior inoculum with the stimulation bone growth of mammalian body, it is characterized in that it contains the described mesenchymal cell of claim 4.
8. inoculum as claimed in claim 7 is characterized in that, described mesenchymal cell is the medulla mesenchyma cell.
9. the method for external generation bone or osseous tissue precursor is characterized in that it comprises step: under the condition that is fit to the cell growth, cultivate the described mesenchymal cell of claim 4, make it at vitro differentiation osteogenic tissue precursor or osteoblast.
10. method as claimed in claim 9 is characterized in that, described mesenchymal cell is the medulla mesenchyma cell.
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| KR20050111593A (en) * | 2003-02-19 | 2005-11-25 | 가부시키가이샤 디나벡크 겐큐쇼 | Method of treating ischemic disease |
| US20040223953A1 (en) * | 2003-03-17 | 2004-11-11 | Hsiang-Fu Kung | Combined adeno-associated virus and adenovirus cocktail gene delivery system for high efficiency gene expression without eliciting immune response in immuno-competent subjects |
| CN100418582C (en) * | 2005-11-25 | 2008-09-17 | 上海第二医科大学附属第九人民医院 | Microcapsule for promoting bone regeneration and its use |
| CN101564555B (en) * | 2009-05-27 | 2013-01-23 | 深圳市第二人民医院 | Tissue engineering bone implant and method for constructing the same |
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