CN1284563A - Recombined carrier carrying human liver cell growth factor gene and its application in treating ischemic diseases - Google Patents
Recombined carrier carrying human liver cell growth factor gene and its application in treating ischemic diseases Download PDFInfo
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Abstract
The present invention relates to the field of biomedicine. Human liver cell growth factor gene whole-length code region cDNA is inserted into plasmid vector and the recombined plasmid can express human liver cell growth factor gene effectively in extracorporeally cultured cell and can be used to promote angiogenesis increase blood flow rate obviously. Therefore, the recombined vectors may have vast application prospect in the gene treatment of ischemic diseases.
Description
The present invention relates to biomedical sector, specifically relate to the recombinant vectors of carrier's liver cell growth factor gene and the application in ischemic disease thereof.
Vascular occlusive disease is one to have a strong impact on the disease of human life's health.Along with the broad application such as coronary angioplasty of surgical operation and percutaneous puncture, prevention and treatment vascular occlusive disease have been obtained very big progress, and the blood supply and the clinical symptom of ischemia organ all were greatly improved after patient performed the operation.But in the clinical application of operative treatment vascular occlusion vascular disease, exist the operation cost high, the conditional request strictness, dangerous big drawback focuses mostly at present and carries out in the hospital preferably in condition.The development of modern molecular biology technique becomes a reality gene therapy application in a lot of fields, the experimental study of gene therapy ischemic disease is also flourish, shift the generation of the gene promotion neovascularity of angiogenic growth factor by the suitable carriers mediation, can be in local collateral circulation, foundation " molecule bridging " mechanism of forming of ischemic.The gene therapy of ischemic disease has shown tempting prospect, attracts tremendous attention.
Known many gene products all have the effect of regulating revascularization, as vascular endothelial growth factor (VEGF), fibroblast growth factor and pHGF (HGF) etc.PHGF thinks at first that by the mesenchymal cell synthesis secretion its target cell is a liver cell, can cell cultured supernatant carry out mitotic division.Found afterwards that it also was that the mitogenesis of strong vascular endothelial cell is former.
In sum, there is limitation in the operative treatment vascular occlusive disease, and this cytokine that has than the effect of strong stimulation blood vessel hyperplasia of pHGF is not applied to clinical.We have made up the expression plasmid pcDNA3-HGF of human hepatocyte growth factor for this reason, behind the exposed recombinant DNA of local intramuscular injection, can obviously promote the formation of limbs acute ischemia position neovascularity.Therefore the prospect that has potential clinical application treatment ischemic disease.
First purpose of the present invention is to provide a kind of recombinant plasmid, this recombinant plasmid carrier liver cell growth factor gene; Second purpose of the present invention is to use the recombinant plasmid treatment ischemic disease of this carrier's pHGF.
Specific embodiments of the present invention is as follows:
1. human hepatocyte growth factor coding region cDNA is inserted into the multiple clone site of pcDNA3 carrier, obtains the recombinant vectors pcDNA3-HGF of carrier's liver cell growth factor gene, human hepatocyte growth factor gene is subjected to the CMV promoter regulation.
2. observe the propagation of pHGF expression product energy significant stimulation vascular endothelial cell.
3. behind the recombinant plasmid of the local intramuscular injection purifying in rat limbs acute ischemia position, histological chemistry's detection method detects the expression product of pHGF, can be observed the formation of a large amount of neovascularity on the tissue slice.
The invention provides a kind of recombinant vectors of carrier's liver cell growth factor gene, can be preserved by freeze-drying after this recombinant vectors is purified, can be made into injection liquid etc. and be used for the treatment of the ischemic disease patient.Ischemic disease can be a peripheral vascular disease, also can be the ischemic of myocardial ischemia and other organs.Embodiment will be by describing carrier's pHGF recombinant vectors the gene therapy of peripheral vascular disease is used for further setting forth the present invention.
Use recombinant vectors provided by the invention treatment ischemic disease and be from the purpose that promotes vasculogenesis, sets up collateral circulation and improve local blood circulation and start with, in patient's body, import the gene of short angiogenesis factor HGF.Therefore it is more simple and convenient than therapies such as operative treatments to use recombinant vectors provided by the invention, and cost is also cheaper.If the present invention is implemented, will provide a kind of alternative new treatment means for the clinical treatment of present ischemic disease.
Further specify the present invention in conjunction with the accompanying drawings:
Fig. 1.Sequence chart 2 such as the poly a-signal of structural representation 1.CMV enhancers/promoters 2. liver cell growth factor genes 3. bovine growth hormone genes of pcDNA3-HGF and transcription termination sequence 4.SV40 replication initiation fragment 5.E.coli replication origin 6. ampicillin resistance genes.The enzyme of pcDNA3-HGF is cut the pcDNA3-HGF of the pcDNA3-HGF3.Bam HI+Apa I double digestion of identifying electrophorogram 1.lambda DNA/EcoRI+Hind III Markers2.BamH I single endonuclease digestion, and the band about big or small 2.2kb is pcDNA3 blank plasmid Fig. 3 of HGFcDNA3.Bam H I single endonuclease digestion.Former generation VEGF of expressing of Skeletal Muscle Cell behind expression (.) the transfection pcDNA3-HGF of VEGF behind the former generation Skeletal Muscle Cell transfection liver cell growth factor gene (.) cell conditioned medium Fig. 4 behind the transfection pcDNA3.The pHGF expression product to former generation Human umbilical vein endothelial cells propagation stimulating activity (.) transfection pcDNA3 contrast serum-free cell conditioned medium to the proliferation function of endotheliocyte (.) both have significant difference (P＜0.01) Fig. 5 to the stimulating activity * statistical procedures of people's umbilical cord endotheliocyte for the serum-free cell conditioned medium of transfection pcDNA3-HGF.Immunohistochemical method is analyzed muscle Fig. 6 of muscle (B) the pcDNA3 transfection of expression level (A) the pcDNA3-HGF transfection of HGF in the plasmid injection site muscle.The animal of HE dyeing (A) transfection pcDNA3-HGF of ischemic hind leg skeletal muscle tissue section has the animal of a large amount of neovascularization (B) transfection pcDNA3
Embodiment: 1, the structure people HGF cDNA of the structure of plasmid and preparation (1) plasmid obtains by separating among the human placenta cDNA library, and subclone is to the multiple clone site of carrier for expression of eukaryon pcDNA3 then.See accompanying drawing 1 and Fig. 2.(2) preparation of plasmid
Large scale fermentation bacterium amplification plasmid, centrifugal recovery thalline adopts alkaline denaturation to extract recombinant plasmid, the chromatography large scale plasmid purification, agarose gel electrophoresis and ultraviolet spectrophotometry are determined purity and the content of DNA.2, the cultivation of Skeletal Muscle Cell and the newborn Wistar rat skeletal muscle of DNA transfection (1) cell former is commissioned to train foster
Wistar rat in two days is born, peel off a rear flank limb skin after the whole body sterilization, take off this hind leg and put in the ware, peel off its muscle in a bottle, fully wash with PBS, and shred, add 0.125% tryptic digestion 3-4 time, 1500 rev/mins are centrifugal 8 minutes; as far as possible collect discrete cell, behind Skeletal Muscle Cell feeding liquid suspension cell, be inoculated in the culturing bottle, hatch for 37 ℃.After 75 minutes suspension cell is moved in another culturing bottle, to remove easily adherent non-myocyte's composition.(2) the plasmid DNA transfection Skeletal Muscle Cell adopts the lipofectin method, and each transfection is with 5 micrograms of DNA+20 microlitre liposomes, and cell count is 3 * 10
5Transfection was changed behind the liquid 12 hours, and 24 hours, 36 hours and sampling in 48 hours detected the expression level of VEGF with the ELISA method.
Detect VEGF in can be thereon behind the former generation Skeletal Muscle Cell transfection pcDNA3-HGF clear, show HGF can be by stimulating the VEGF secretion indirectly performance promote the effect of blood vessel hyperplasia.See Fig. 3.3, the HGF expression product is measured the stimulating activity of human umbilical vein endothelial cell propagation
Former being commissioned to train of people's umbilical cord endotheliocyte supported and the proliferation activity analysis: fully wash people's umbilical cord chamber with PBS under the aseptic condition, with an end ligation, inject 20 milliliters of O.125% trypsinase and sealings of 37 ℃ of preheatings from the other end, umbilical cord is inserted among 37 ℃ of preheating PBS, digested 15 minutes, Digestive system moves in the centrifuge tube, and 1200 rev/mins centrifugal 10 minutes, DMEM re-suspended cell with containing 80%FBS is inoculated in the culturing bottle.
Function analysis to former generation people umbilical cord endotheliocyte: 96 orifice plate cell count that every hole adds are 6000, and the expression supernatant liquor added amount of HGF in Chinese hamster ovary celI accounts for 5%, 10%, 15%, 25% and 50% of cumulative volume respectively.Mtt assay is measured cell-proliferation activity.
As shown in Figure 4: the expression supernatant liquor of HGF in Chinese hamster ovary celI has the activity of tangible stimulation people umbilical cord endothelial cell proliferation of former generation, and this test has confirmed that fully HGF is that the powerful mitogenesis of vascular endothelial cell is former.4, the acute posterior-limb ischemia animal model of revascularization evaluation (1)
The Wistar male rat, body weight 200-250 gram.Vetanarcol intraperitoneal anesthesia (50 mg/ml), left side hind leg arteria iliaca externa section far away and branch thereof cut off after with the ligation of thin operation silk thread, cause acute hind leg occlusive vascular disease model.
When undergoing surgery at five different sites direct injection of three main muscle of buttocks totally 200 microgram plasmids: a part of model mouse injection pcDNA3 empty plasmid; Another part model mouse injection pcDNA3-HGF.Per injection should slowly be carried out with anti-overflow.About 2.5 milliliters of the total amount of liquid of every animal injection.(2) expression of HGF in skeletal muscle tissue
Get the muscle at injection plasmid pcDNA3-HGF and pcDNA3 position, do paraffin section after 10% formalin fixed, carrying out immunohistochemical methods detects: one anti-ly is mouse-anti people HGF monoclonal antibody, two anti-are biotin labeled sheep anti-mouse igg, three anti-are the avidin of alkali phosphatase enzyme mark, and chromogenic substrate is AP-Red.As shown in Figure 5: in the muscle tissue of injection pcDNA3-HGF recombinant plasmid, can see the obvious expression of HGF.(3) immunohistochemical analysis of blood capillary proliferation
Get the transverse section of the main muscle of ischemic hind leg and do paraffin section, HE dyeing, opticmicroscope is counting blood vessel number down.
The animal model of the rat hindlimb acute ischemia that adopts in this experiment, the ability that self forms collateral circulation after surgery alone in 10 days is very weak, sees Fig. 6.By comparison, the acute ischemia hind leg of injection HGFF genetic animal group had a large amount of neovascularity to generate (packed red blood cell in the tube chamber is blood vessel) in 10 days after surgery, had fully confirmed the short vascularization effect of HGF and the validity of transgenosis.
Claims (2)
1. an eukaryotic expression recombinant plasmid that is used for the treatment of ischemic disease is characterized in that this recombinant plasmid carrier liver cell growth factor gene, and liver cell growth factor gene is subjected to the regulation and control of CMV promotor.
2. recombinant plasmid according to claim 1, it is characterized in that this recombinant plasmid carrier liver cell growth factor gene, can be behind amplification and purifying in bacterium by freeze-drying, can be made into injection liquid or baste etc., this recombinant plasmid has short angiopoietic effect, can be used for treating ischemic disease, as peripheral vascular disease and myocardial ischemia etc.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 99111446 CN1284563A (en) | 1999-08-17 | 1999-08-17 | Recombined carrier carrying human liver cell growth factor gene and its application in treating ischemic diseases |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 99111446 CN1284563A (en) | 1999-08-17 | 1999-08-17 | Recombined carrier carrying human liver cell growth factor gene and its application in treating ischemic diseases |
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| CN1284563A true CN1284563A (en) | 2001-02-21 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100441226C (en) * | 2002-04-26 | 2008-12-10 | 中国人民解放军军事医学科学院放射医学研究所 | Method of speeding wound repair and preventing complications |
| CN107686858A (en) * | 2016-08-03 | 2018-02-13 | 湖北生物医药产业技术研究院有限公司 | Determine whether plasmid medicine has method, apparatus and its application of bioactivity |
| CN108611367A (en) * | 2018-04-24 | 2018-10-02 | 北京诺思兰德生物技术股份有限公司 | The gene therapy recombinant vector that one kind is mediated by plasmid vector |
| WO2020143515A1 (en) * | 2019-01-07 | 2020-07-16 | 北京诺思兰德生物技术股份有限公司 | Human hepatocyte growth factor mutant and uses thereof |
-
1999
- 1999-08-17 CN CN 99111446 patent/CN1284563A/en not_active Withdrawn
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100441226C (en) * | 2002-04-26 | 2008-12-10 | 中国人民解放军军事医学科学院放射医学研究所 | Method of speeding wound repair and preventing complications |
| CN107686858A (en) * | 2016-08-03 | 2018-02-13 | 湖北生物医药产业技术研究院有限公司 | Determine whether plasmid medicine has method, apparatus and its application of bioactivity |
| CN108611367A (en) * | 2018-04-24 | 2018-10-02 | 北京诺思兰德生物技术股份有限公司 | The gene therapy recombinant vector that one kind is mediated by plasmid vector |
| WO2020143515A1 (en) * | 2019-01-07 | 2020-07-16 | 北京诺思兰德生物技术股份有限公司 | Human hepatocyte growth factor mutant and uses thereof |
| CN113383012A (en) * | 2019-01-07 | 2021-09-10 | 北京诺思兰德生物技术股份有限公司 | Human hepatocyte growth factor mutant and application thereof |
| US12264186B2 (en) | 2019-01-07 | 2025-04-01 | Beijing Northland Biotech Co., Ltd. | Human hepatocyte growth factor mutant and uses thereof |
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