CN1250287C - Application of recombinant adenovirus in treating cerebral ischemia, traumatic restoration and tissue adhesion - Google Patents
Application of recombinant adenovirus in treating cerebral ischemia, traumatic restoration and tissue adhesion Download PDFInfo
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- CN1250287C CN1250287C CN 02149418 CN02149418A CN1250287C CN 1250287 C CN1250287 C CN 1250287C CN 02149418 CN02149418 CN 02149418 CN 02149418 A CN02149418 A CN 02149418A CN 1250287 C CN1250287 C CN 1250287C
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Abstract
The present invention relates to an application of recombinant adenovirus (AdHGF) carrying human hepatocyte growth factor genes in the treatment of cerebral ischemia, wound union and tissue adhesion. More specifically, through the homologous recombination of intracellular plasmid, human hepatocyte growth factor genes are integrated into adenovirus vectors to obtain recombinant adenovirus. Parenteral solution prepared from the Ad-HGF is used for the gene therapy of cerebral ischemia, and can obviously reduce the area of cerebrum ischemic zones to improve brain functions. The parenteral solution prepared from the Ad-HGF can be used as a spraying agent, and can accelerate wound surface union for skin full thickness wounds to promote the regeneration of the hair follicle and the sebaceous gland, which indicates that the Ad-HGF can promote the growth of hair, and has important effect on the restoration of skin functions. When injected to local muscle of the spinal cord injury part, or applied to the local part of the small intestine injury part, the Ad-HGF can reduce the formation of the tissue adhesion of the injury part.
Description
Technical field the present invention relates to biomedical sector, specifically relates to the Therapeutic Method of a kind of recombinant adenovirus of carrier's liver cell growth factor gene to cerebral ischemia, wound healing and tissue adhesion.
(hepatocyte growth factor HGF) is considered to a kind of hepatocyte mitogen to the background technology hepatocyte growth factor at first, finds afterwards that it to multiple histiocytic division, diffusion and form important regulatory role had taken place.HGF has the effect that promotes that vascular endothelial proliferation and new vessels form, and can be used for the treatment of tissue ischemia disease due to a variety of causes; HGF can also regulate the synthetic and inflammatory reaction of collagen fiber, plays an important role in treatment wound healing, control tissue fibering; HGF also can promote histiocytic generation, existence and regeneration in addition, suppresses apoptosis, to the nutritious effect of nerve, can be used for prevention and treatment nerve injury.This shows that HGF is a very broad profile of cytokine of a biological activity, HGF will permeate every field in clinical treatment with its multiple pharmacological effect.
In the administration of HGF protein formulation, because of HGF in vivo the half-life shorter, thereby need heavy dose, repetitively administered just can keep local higher drug level; Yet, because of local or intravital proteolysis effect, even repeat administration also often is difficult to reach effective treatment concentration.Thereby the cost of application protein formulation is higher, and immature because of purifying process, easily produces ill effect, thereby brings side effect to clinical treatment.A kind of new treatment pattern---the gene therapy methods of rising in recent years might overcome above-mentioned shortcoming.
The gene import system that gene therapy is used is broadly divided into non-virus carrier and viral vector two classes.Viral vector commonly used has adenovirus, adeno-associated virus, retrovirus, herpes simplex virus etc., and adenovirus is wide because of its production easily, infection rate height, infection cell scope, and becomes the carrier of gene therapy widespread usage.
Raising and aged tendency of population trend along with modern society's medical condition, cerebrovascular has become one of three big killers of harm humans life, wherein see that so that ischemic diseases is the most its incidence rate and disability rate are all very high again more, bring great burden for society and family.Owing to the irreversibility of its morbidity urgency and neuronal death, clinical therapeutic efficacy is unsatisfactory at present.Neurotrophic factor is as the part of tyrosine kinase receptor, in neuronic generation.Bring into play important effect in growth and the survival, can be used to treat nervous system disease and damage.The receptor c-Met of HGF also is a kind of tyrosine kinase receptor, and is widely distributed in brain, so in close relations between HGF and the nervous system cell.HGF not only can promote angiogenesis, improves tissue ischemia, and can improve the expression of bcl-2, suppresses apoptosis, improves and therapeutical effect so HGF may have cerebral ischemia diseases.
Wound, burn comprise that the healing of operative incision remains a great problem of clinical existence, the healing of difficult healing of wound such as the healing of especially diabetes patient's wound healing, local radiotherapy wound, large-area burns or cotton-padded mattress wound, cause huge misery to the patient, also bring great burden, also do not have very effective method to promote the quickly-healing of these wounds at present to curative activity.HGF can promote effective, the high-quality healing of wound in theory from many aspects.At first, HGF can stimulate migration, the propagation of vascular endothelial cell, forms new vessels, promotes local blood circulation, thus healing acceleration; But the motion of HGF chafe horn cell, propagation are impelled the cutization again of wound.
Tissue adhesion's problem that tissue injury or operation back produce is also perplexing doctor and patient, and the tissue adhesion not only influences the normal function of organ, and the adhesion that has puts to no little inconvenience for patient's life.Modern study shows, the inflammation that wound or operation back ischemic tissue cause is the major reason of adhesion, because ischemia, organizes fibrinolytic to reduce and sticks together.Therefore improve and organize the blood confession, be prevention and alleviate the important step of adhesion.HGF not only can improve the blood confession of damaged tissue as angiogenic growth factor, can also regulate and organize inflammatory reaction, reduces and organizes liquid to ooze out, and can significantly suppress the generation of TGF-β in addition.And TGF-β understands so far at most, forms the closest cytokine with collagen protein, and HGF is owing to can reduce tissue adhesion's formation to the generation of anti-TGF-beta.HGF can strengthen the activity of collagenase in addition, impels the collagen protein of unnecessary formation to degrade effectively, and these all help to prevent and alleviate tissue adhesion's generation.
Summary of the invention technical problem to be solved by this invention is the recombinant adenoviral vector (Ad-HGF) that makes up carrier's hepatocyte growth factor; Ad-HGF can alleviate nerve cell death that cerebral ischemia causes, the tissue adhesion after promoting repair in trauma and reducing damage.The technical scheme that solves the problems of the technologies described above:
1. by plasmid homologous recombination in the cell human hepatocyte growth factor gene is incorporated on the adenovirus vector, obtains recombinant adenovirus, and prepare a certain amount of virion.
2. set up cerebral ischemia animal model and suitable gene transfer method, observe the expression (Fig. 1) of adenovirus in brain, reference's hepatocyte growth factor can alleviate the nerve cell death (Fig. 2,3) that cerebral ischemia causes.
3. set up wound animal model and suitable gene transfer method, reference's hepatocyte growth factor can promote the healing of wound, promotes the regeneration (Fig. 4) of hair and sebaceous gland.
4. set up tissue adhesion's animal model and suitable gene transfer method, reference's hepatocyte growth factor can alleviate the adhesion that forms because of tissue injury.
Description of drawings
Fig. 1 is behind the big intracerebroventricular Ad-GFP in animal right side three days, makes frozen section, and (* 100) visible fluorescence albumen is at bilateral tricorn wall and inner choroid cellular expression thereof under fluorescence microscope.
Fig. 2 is that the animal ischemia was treated back three days, gets brain and on average is cut into four, carries out 1%TTC dyeing and observes, and ischemic region is not painted, and non-ischemic region is dyed brick-red.Four in left side is contrast, and four on right side is an Ad-HGF treatment group.
Fig. 3 is Ad-HGF to the influence of animal brain ischemia (experimental group with compare P＜0.05).
Fig. 4 is the tissue slice of smearing after Ad-HGF forms mouthful healing, the existence of visible hair follicle and sebaceous gland, and the division phase (* 200) of visible hair follicle and sebaceous gland.
The specific embodiment the following examples are to further describe but do not mean that any limitation of the invention of the present invention.
Embodiment 1: the structure of recombinant adenovirus and preparation
(1) structure of recombinant adenovirus
Make up the shuttle plasmid of carrier's liver cell growth factor gene, called after pXCJL1-CMV/HGF/P.With pXCJL1-CMV/HGF/P and recombiant plasmid GT4050 cotransfection 293 cells that carry adenovirus genomic dna, homologous recombination takes place in the two in cell, forms the recombinant adenovirus of carrier's liver cell growth factor gene, called after Ad-HGF, and structure is as follows:
5 ' ITR V40pA HGF hCMV promoter Ad53533-3 ' ITR
The recombinant adenovirus (Ad-GFP) that carries green fluorescence protein gene by gene therapy portion of U.S. hundred special medical supplies company (Gene Therapy Unit, Baxter Healthcare Company USA) is so kind as to give, 293 cell lines are available from ATCC.
(2) virus amplification and purification
With 293 cell inoculations in the 150mm plate, when treating that cell grows to 90% converging state, add virus, infection intensity (multiplicity of infection, MOI) be about (the plaque forming unit of 10 plaque forming units, pfu)/cell, behind the 36-48h, when complete CPE (cell pathology effect) appears in cell, collecting cell, frozen in-80 ℃, when waiting to be accumulated to 50-60 plate, unified purified virus.
In multigelation between-80 ℃ and 37 ℃ of water-baths three times, the centrifugal 10min of 2500rpm is to remove cell debris with 293 cells that CPE occurs; Preparation 1.5g/ml CsCl solution (is got 30g CsCl, add PBS to final volume 42.5ml), 1.35g/ml CsCl solution (gets the CsCl liquid of 15ml 1.5g/ml, add PBS to final volume 21ml) and 1.25g/ml CsCl solution (get the CsCl liquid of 11ml1.5g/ml, add PBS to final volume 20ml); Preparation CsCl density gradient: 0.5ml 1.5g/ml CsCl solution, 2.5ml 1.35g/ml CsCl solution and 2.5ml 1.25g/ml CsCl solution are added in the 12ml ultracentrifugation pipe successively; To be purified viral liquid after the freeze thawing is added on the CsCl gradient liquid of each pipe; 150,000g, the vaporific virus band of white appears in 10 ℃ of centrifugal 1h between 1.35g/mlCsCl solution and 1.25g/ml CsCl solution; Collect the virus band, and mixed with 1.35g/ml CsCl solution; 150,000g, 10 ℃ of centrifugal 18h are further purified virus; Sucking-off virus band with the Hanks liquid virus dilution of 1-2 times of volume, is that dialysis solution is viral in 4 ℃ of dialysis with Hanks liquid, and every 2h changes a dialysis solution, changes liquid altogether 5 times; Take out the viral liquid of purification, adding aseptic glycerol to final concentration is 10%, frozen in-80 ℃ after the packing.
Embodiment 2: the mediated by recombinant adenovirus human hepatocyte growth factor is to the experimentation of brain focal ischemia gene therapy
(1) animal model and gene transfer
Wistar male rat body weight 200-250g, lumbar injection amobarbital sodium (100mg/kg) anesthesia, aseptic separation right side neck is total, outer and the internal carotid artery of neck, ligation external carotid artery branch's section start and common carotid artery proximal part, bulldog clamp is clamped internal carotid artery, cut an osculum at common carotid artery, the nylon wire level that is ball-type 4-0 to end is inserted about about 20mm to the internal carotid artery end, causes rat right side brain focal ischemia, sew up wound to middle cerebral artery.Following assisting of stereotaxic instrument then, in rat brain anterior fontanelle tail side 1.5mm, right side 2mm, degree of depth 3.5mm injects 10 with microsyringe to tricorn
7Pfu/10 μ l adenovirus vector.The HGF group gives Ad-HGF, and matched group gives normal saline.
(2) carry expression and the distribution of recombinant adenovirus (Ad-GFP) in rat brain of green fluorescent protein
Anesthesia in three days behind the rat tricorn injection 107pfu/10 μ l Ad-GFP, after heart carries out the normal saline perfusion, get brain and make full brain frozen section, visible fluorescence albumen mainly is expressed in rat bilateral tricorn wall and ICV choroid cell (Fig. 1) under the fluorescence microscope.
(3) effect of brain focal ischemia gene therapy
Animal is in treatment anesthesia after three days, after heart carried out normal saline perfusion, get brain and on average be cut into four, dyeed 30 minutes at 37 ℃ with the phosphate buffer that contains 1%TTC (tetrazole is red), observe with the fixing back of 4% formaldehyde PBS solution then, ischemic region is not painted, and non-ischemic region is dyed brick-red (Fig. 2).Brain sections after the dyeing is in high-resolution scanner scans into computer, with the ImageTool computed in software go out brain not colour attaching area account for the percentage ratio of full brain volume, with the significance of TTEST statistical experiment group (10) and matched group (8) the animal difference of Excel, the result shows that HGF gene therapy treated animal cerebrum ischemia volume is significantly less than matched group (Fig. 3).
The effect that embodiment 3. mediated by recombinant adenovirus human hepatocyte growth factors (Ad-HGF) are repaired skin trauma
(1) animal model and gene transfer
Select 10 of female new zealand white rabbits healthy about 2.5kg for use, Nembutal vein anesthetic (30mg/kg) excises circular holostrome skin until cartilage at the ears veutro with the aseptic operation method, and otch diameter 0.7cm whenever picks up the ears 5.Be divided into 5 groups at random, two every group: Ad-HGF1 group, Ad-HGF2 group, Ad-HGF3 group, Ad-GFP group and group of solvents.Operation is at once with 6 * 10
7Pfu Ad-HGF, 6 * 10
8Pfu Ad-HGF, 6 * 10
9Pfu Ad-HGF, 6 * 10
9Pfu Ad-GFP or be applied in the above-mentioned wound surface of respectively organizing successively with the solvent once of volume.
After smearing recombinant adenovirus, observe the wound healing situation of respectively organizing every day, and the wound area that do not heal is measured.Animal all lives and kills during to the 23rd day, and with wound surface and close on no site of injury skin holostrome and downcut in the lump, 10% formalin fixed is done the routine paraffin wax section, carries out HE and dyes.
(2) effect assessment
Behind the rabbit ear wound surface partial smearing recombinant adenovirus the 6th day, general appearance promptly can be observed Ad-HGF, and to form mouthful healing rate fast than Ad-GFP and group of solvents, during to the 8th day, two form the mouth area percentage significance statistically that do not heal: Ad-HGF1, Ad-HGF2 and Ad-HGF3 are respectively 9.36%, 4.46% and 4.59%, Ad-GFP and group of solvents are respectively 26.88% and 32.16% (p＜0.05), and there was no significant difference (p＞0.05) between Ad-GFP and the group of solvents.During to the 23rd day, smear Ad HGF and form all healings (20/20) of mouth, and Ad-GFP and group of solvents respectively there are the not healing fully significantly of 2/20 and 6/20 wound.And Ad-HGF to form mouthful healing comparatively smooth, have in addition with contiguous normal skin is identical on every side, and Ad-GFP and group of solvents all obviously thicken.Optical microscope is observed down and is shown, smear Ad-HGF and form and mouthful have only 2/20 not cutization fully more as yet, and Ad GFP and group of solvents respectively has 10/20 not cutization fully again.Smear Ad-HGF and form fiber thin and marshalling in mouthful corium, and the fiber of matched group is thick and row disorderly.
In addition, form mouthful the existence of visible hair follicle and sebaceous gland in the tissue smearing Ad-HGF, and the division phase of visible hair follicle and sebaceous gland, especially with heavy dose group (10
9Pfu and 10
8Pfu organizes) comparatively obviously (Fig. 4), show that HGF can promote the growth of hair, significant to the recovery of skin function.
Embodiment 4: mediated by recombinant adenovirus human hepatocyte growth factor (Ad-HGF) is to tissue adhesion's effect
(1) Ad-HGF treatment spinal cord half is from disconnected effect assessment
Wistar female rats body weight 200-250g, amobarbital sodium lumbar injection (100mg/kg) anesthesia, back preserved skin, skin, subcutaneous tissue and paraspinal muscle are cut in layering, sting and remove the prominent and vertebral plate of ridge, expose breast 10 sections spinal cords and do left side half, the local intramuscular injection 10 of experimental group from disconnected
9PfuAd-HGF, matched group is with 100 μ l normal saline, layer-by-layer suture otch.In six weeks of postoperative, the PBS solution of cardiac perfusion normal saline and 4% paraformaldehyde is animal fixedly, gets spinal cord and observes, and the local spinal cord of visible medication treated animal obviously alleviates than matched group with muscle adhesion on every side, and cicatrix also obviously reduces.
(2) Ad-HGF treatment intestinal adhesion effect assessment
Wistar male rat body weight 200-250g cuts off the abdominal cavity after the anesthesia, expose the small intestinal ileocecus, is played clamp ileum three places, 1.5cm at interval by ileocecus with mosquito forceps.Folder metagaster local cyanosis has a small amount of oozing of blood, smears 10 at the clamp position
9PfuAd-HGF or normal saline feelings 20 μ l close the abdominal cavity.Put to death after 10 days, observations of cutting open the belly, the less and adhesive band of the adhesion of medication treated animal formation is shorter than matched group.
Claims (5)
1. recombinant adenovirus is used for the treatment of application in cerebral ischemia, repair in trauma and tissue adhesion's the medicine, wherein said recombinant adenovirus carrier liver cell growth factor gene in preparation.
2. application according to claim 1, wherein this medicine is used for treating cerebral ischemia.
3. application according to claim 1, wherein this medicine is used for treating repair in trauma.
4. application according to claim 1, wherein this medicine is used for the treated tissue adhesion.
5. according to each described application of claim 1-4, wherein said adenovirus is made into injection, spray liniment or lyophilized preparation.
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| CN109771642B (en) | 2017-11-13 | 2022-09-20 | 同济大学苏州研究院 | c-MET agonistic antibodies and uses thereof |
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Address after: 100850 Taiping Road, Beijing, Haidian District, No. 27 Patentee after: INSTITUTE OF RADIATION MEDICINE, ACADEMY OF MILITARY MEDICAL SCIENCES, PLA Patentee after: Beijing Haitai Joint Medical Technology Development Co.,Ltd. Address before: 100850 Taiping Road, Beijing, Haidian District, No. 27 Patentee before: Institute of Radiation Medicine, Chinese Academy of Military Medical Sciences Patentee before: Beijing Lu Yin Li Hua Pharmaceutical Science Technology Development Co.,Ltd. |
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