Application of gene modified mesenchymal stem cell in pulmonary fibrosis treatment

A technology of mesenchymal stem cells and gene modification, applied in the fields of biotechnology and gene therapy, can solve the problems of complex structure of recombinant HGF and the inability of cells to play anti-inflammation, anti-apoptosis, and promotion of proliferation

Inactive Publication Date: 2013-07-17
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, more than 90% of the cells died quickly due to lack of nutrients within a few days of implantation, and the cells could not play their anti-inflammatory, anti-apoptotic, and proliferation-promoting functions wel

Method used

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  • Application of gene modified mesenchymal stem cell in pulmonary fibrosis treatment
  • Application of gene modified mesenchymal stem cell in pulmonary fibrosis treatment
  • Application of gene modified mesenchymal stem cell in pulmonary fibrosis treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Preparation and purification of embodiment 1 recombinant adenovirus-hepatocyte growth factor (Ad-HGF)

[0034] The method is to clone the human HGF gene of the target gene into the multiple cloning site of the shuttle plasmid pXCJL1-cmv / pA to obtain pXCJL1-cmv / pA-HGF; It carries out intracellular homologous recombination, and obtains recombinant adenovirus Ad-HGF by packaging in HEK293 cells. After correct identification at the genomic level, amplification, purification, and titer determination are performed.

[0035] (1) Construction of the shuttle plasmid pXCJL1-cmv-HGF:

[0036] Firstly, the hepatocyte growth factor cDNA was amplified from the human placenta cDNA library by PCR method, and the PCR product was cloned into the BamHI and SalI restriction sites of pBluescript SK (pSK) vector, named pSK-HGF. After the plasmid pBLUESCRIPTSK-HGF was digested with restriction endonuclease Apa I, the end was blunted with the large fragment of DNA polymerase Klenow, and then...

Embodiment 2

[0042] Example 2 Isolation, cultivation, expansion and gene modification of bone marrow mesenchymal stem cells

[0043] (1) Isolation and culture of bone marrow mesenchymal stem cells

[0044] Heparin-anticoagulated normal human bone marrow was filtered with a 1ml syringe, added PBS to dilute the bone marrow, mixed well, carefully added the bone marrow cell suspension on the surface of the lymphocyte separation medium Ficoll according to the ratio of 1:1, centrifuged at 1600rpm for 30min; sucked out Dilute the mononuclear cell layer with twice the volume of PBS, mix well, and centrifuge at 2000rpm for 8min; wash the cells once more with PBS; resuspend the cells with complete medium (α-MEM+10%FBS) 5 cells / cm 2 The density was divided into flasks for culture; after 72 hours, the medium was changed to remove unattached cells. Change the medium every 3-4 days thereafter. When the cells grew to 80% confluence, they were trypsinized and passaged, which was recorded as the first p...

Embodiment 3

[0058] Example 3 HGF Modified MSC Biological Performance Detection

[0059]48h after MSCs were infected with 150MOI Ad-HGF, the expression level of HGF was detected by ELISA; the changes of cell surface molecules were detected by flow cytometry; the changes of cell proliferation ability were detected by Dy670 dye method;

[0060] (1) Detection of HGF expression level

[0061] After Ad-HGF infected MSCs, the expression of HGF was detected by ELISA. The results showed that after Ad-HGF infection, the expression level of HGF gradually increased until the 7th day (see attached figure 2 shown).

[0062] (2) Effect of HGF modification on MSC cell surface molecules

[0063] Flow cytometry was used to detect the expression of various surface molecules CD29, CD73, CD166, HLA-AB, CD105, HLA-DR, CD31, CD34 and CD45, and the results showed that infection with Ad-HGF had no effect on the expression of MSC surface molecules ( as attached image 3 shown).

[0064] (3) Effect of HGF mod...

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Abstract

Relating to the fields of biotechnologies and gene therapy, the invention provides application of a gene modified mesenchymal stem cell in pulmonary fibrosis treatment. The gene modified mesenchymal stem cells are obtained through: in-vitro isolated culture and amplification of a mesenchymal stem cell (MSC) deriving from bone marrow and an umbilical cord, and recombinant adenovirus Ad-HGF mediated in-vitro modification of the MSC by a hepatocyte growth factor (HGF). By transplanting the gene modified MSC to a C57 mouse to intervene in radiation induced lung injury and fibrosis, exudation of a plurality proteins including albumin, IgM and the like from an alveolar space can be reduced, local inflammatory responses of the lung can be alleviated, and expression of TNF-alpha, soluble ICAM-1 and multiple factors is inhibited, expression of the profibrotic factor TGF-beta, the collagen gene col1 alpha 1 and col 3 alpha 1 can be inhibited, and pulmonary tissue collagen fiber deposition is reduced. The expression results of endogenous HGF and its receptor cmet show that endogenous HGF expression can be induced and endogenous MSC can home to injured parts. Therefore, the employment of HGF modified MSC in treatment of lung injury and fibrosis brought by various pathogenic causes is of great significance.

Description

technical field [0001] The invention relates to the fields of biotechnology and gene therapy, in particular to the application of gene-modified mesenchymal stem cells in the treatment of pulmonary fibrosis. Background technique [0002] Mesenchymal stem cells (MSC) can be induced to differentiate into various cells in vitro and in vivo, and can improve the structure and function of various damaged tissues. MSCs have demonstrated their therapeutic potential in a variety of lung injury diseases, including pulmonary fibrosis. In a drug-induced animal lung injury model, the application of bone marrow MSCs through blood circulation can effectively reduce lung injury and fibrosis. At the same time, the following characteristics of MSC make it have unique advantages in the treatment of pulmonary fibrosis caused by lung injury: (1) No transplant rejection: MSC belongs to immune pardoned cells; (2) MSC can home to the injured lung , and can obtain the phenotype of alveolar epitheli...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K38/18A61K35/44A61K35/28A61P11/00C12N5/10
Inventor 王华王立生吴祖泽杨月峰肖凤君段海峰李庆芳张群伟
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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