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Recombinant adipose-derived stem cells to express gene BDDhFVIII and preparation method and application thereof

An adipose stem cell and gene technology, applied in the field of recombinant adipose stem cells and their preparation, can solve the problems of undetermined selection of viral vectors and target cells, slow progress of gene therapy for hemophilia A, and the like, and achieve the effect of good application prospects.

Pending Publication Date: 2019-08-16
NORTH CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
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Problems solved by technology

A review of domestic and foreign literature found that gene therapy for hemophilia B is relatively mature and has been successfully applied clinically, but the progress of gene therapy for hemophilia A is slow, and the selection of viral vectors and target cells has not yet been determined

Method used

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  • Recombinant adipose-derived stem cells to express gene BDDhFVIII and preparation method and application thereof
  • Recombinant adipose-derived stem cells to express gene BDDhFVIII and preparation method and application thereof
  • Recombinant adipose-derived stem cells to express gene BDDhFVIII and preparation method and application thereof

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preparation example Construction

[0030] The invention provides the preparation method of the recombinant adipose stem cell, comprising the following steps: constructing an adenovirus vector expressing the BDDhFVIII gene; extracting the adipose stem cell; infecting the adipose stem cell with the adenovirus vector expressing the BDDhFVIII gene to obtain the recombinant adipose stem cell.

[0031] In the present invention, the construction of the adenoviral vector expressing the BDDhFVIII gene preferably uses the adenoviral vector to package the BDDhFVIII gene to obtain the adenoviral vector expressing the BDDhFVIII gene. In the present invention, the BDDhFVIII gene is the gene of human blood coagulation factor VIII deleted in the B region, which is the sequence after 2657-5141 of the deleted gene FVIII; Factor VIII gene concordance. In the present invention, the construction method of the adenoviral vector expressing the BDDhFVIII gene refers to the HB-infusion seamless cloning kit of Hanheng Biotechnology (Sha...

Embodiment 1

[0040] Extraction of Adipose Tissue Stem Cells

[0041] Take 2 commercially available SD rats, male or female, weighing about 120 g. The SD rats were intraperitoneally anesthetized. Place the rats below the neck in 75% alcohol for 10 minutes for disinfection. After disinfection, move the SD rat into the biological safety cabinet; carefully separate the inguinal fat tissue on both sides of the SD rat with tweezers, put the separated fat into a petri dish containing 1% double antibody and wash it 3-4 times , wash the blood vessels and residual substances in it as much as possible, then transfer the adipose tissue into a small beaker, and cut it into pieces (until it becomes a paste), the time is about 35 minutes. Transfer the shredded adipose tissue into a 50ml centrifuge tube, add 2 times the volume of 0.1% (v / v) type Ⅰ collagenase to it, seal the tube with a parafilm and put it in a 37°C water bath for 1.3h for digestion. Take out the manual shock 4 times, each time 13min. ...

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Abstract

The invention provides recombinant adipose-derived stem cells to express a gene BDDhFVIII and a preparation method and application thereof and belongs to the technical field of genetically engineereddrugs. The recombinant adipose-derived stem cells are prepared by infecting adipose-derived stem cells with an adenovirus vector that expresses the gene BDDhFVIII. The preparation method comprises thesteps of constructing the adenovirus vector that expresses the gene BDDhFVIII; extracting adipose-derived stem cells; infecting the adipose-derived stem cells with the adenovirus vector that expresses the gene BDDhFVIII to obtain the recombinant adipose-derived stem cells. The recombinant adipose-derived stem cells are suitable for expressing clotting factor VIII safely and persistently and havea good application prospect in the treatment of hemophilia A.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering drugs, and in particular relates to a recombinant fat stem cell expressing BDDhFVIII gene and its preparation method and application. Background technique [0002] Hemophilia A is the most common hereditary bleeding disorder clinically, which is caused by the defect or abnormal function of the gene encoding blood coagulation factor Ⅷ (FⅧ). The blood coagulation factor activity level in the plasma of patients with severe hemophilia A is less than 1% (normal range 50% to 150%), and they are prone to spontaneous, difficult to stop bleeding after minor trauma or trauma, and severe bleeding after surgery. The bleeding sites are mainly deep muscles and weight-bearing joints. If the treatment is not thorough, chronic pain and joint deformities are likely to form. If central nervous system hemorrhage occurs, the mortality rate is extremely high. The incidence of hemophilia A is about 1 / 10,00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/861A61K35/28A61P7/04
CPCC12N5/0667C12N15/86C07K14/755A61K35/28A61P7/04C12N2510/02C12N2710/10043C12N2710/10343C12N2510/00A61K48/005A61K38/37C12N2750/14141G01N1/4044C12N2750/14041C12N2750/14143
Inventor 闫振宇谢燕燕王霖虹朱益文
Owner NORTH CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY
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