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MSC-TNF alpha-AB stem cell, preparation method and application thereof

A technology of MSC-TNF and stem cells, applied in the field of MSC-TNFα-AB stem cells and its preparation, can solve the problems of high recurrence rate, ineffective treatment, large side effects, etc., and achieve the effect of avoiding allergic reactions

Active Publication Date: 2016-11-30
连祺周
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no report on the combination of MSC and TNF α antibody at home and abroad. However, the traditional treatment of osteoarthritis has a high recurrence rate, large side effects and ineffective therapeutic effect. Therefore, the effective combination of MSC and TNF α antibody can make TNF α antibody can be activated by the inflammatory factor TNF α in MSC, which is expected to be the key point of gene therapy for osteoarthritis

Method used

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  • MSC-TNF alpha-AB stem cell, preparation method and application thereof
  • MSC-TNF alpha-AB stem cell, preparation method and application thereof
  • MSC-TNF alpha-AB stem cell, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1. Preparation of Osteoarthritis Inflammatory Infiltration Mouse Model

[0053] Dissolve the purified natural collagen in 11M acetic acid to make a solution with a concentration of 1mg / mL, and mix it with an equal amount of Freund’s adjuvant or incomplete adjuvant to form a stable emulsion; use 1mL of the emulsion to inject 4 to 6 sites Intradermally on the back of the mouse, small ulcers will appear at the injection site (heal after 7-10 days); 21 days later, dissolve 15 mg of collagen in 15 mL of 1M acetic acid and inject it into the abdominal cavity of the mouse for another immunization.

[0054] 14 to 60 days after the initial injection, joint redness and swelling appear, usually in the distal joints, and the rear foot ankle joint is the most common site of accumulation. The pathological changes in the acute phase can be divided into two phases. After 10 days of immunization, there are only some fibrous deposits in the joints and no changes in the synoviocyt...

Embodiment 2

[0055] Example 2. Design and synthesis of TNF α-AB antibody fragments, lentivirus preparation, MSC infection

[0056] The packaging plasmid required for this experiment was purchased from addgene company. The plasmid map is shown in Figure 3, and Figure 3 includes Figure 3A , Figure 3B and Figure 3C ; The shuttle plasmid pGFP was purchased from Guangzhou Funeng Gene Co., Ltd., see the plasmid map Figure 4 ; The information of the above plasmids is shown in Table 1; lentiviral packaging cells 293FT were purchased from Invitrogen (catalog number R700-07); MSCs were extracted from the bone marrow of C57 / BL6 mice; C57 / BL6 mice were purchased from the Experimental Animal Center of the University of Hong Kong.

[0057] The target fragment sequence TNF α-AB required in this experiment is shown in SEQ ID NO:1.

[0058] The reagents required for this experiment are listed in Table 1.

[0059] Table 1 Lentiviral packaging plasmid and shuttle plasmid information

[0060]

[0...

Embodiment 3

[0101] Example 3. Detection of the constructed new stem cell line MSC-TNF α-AB can be specifically regulated and initiated by the inflammatory factor TNF α

[0102] This example is to detect the expression of TNF α-AB after adding the inflammatory factor TNF α in MSC cell culture in vitro.

[0103] 3.1 MSC-TNF α-AB cell culture:

[0104] MSC-TNF α-AB cell culture medium is: in DMEF / F12 medium containing 10% fetal bovine serum (FBS), 100U / mL penicillin streptomycin, 1% non-essential amino acids, 0.1% β-mercaptoethanol, 5ng / mL of EGF, 5ng / mL of FGF.

[0105] 1) Before aseptic culture, autoclave the materials required in the laboratory; irradiate with ultraviolet light for 30 minutes; wear special lab coats and gloves between cells, and wipe hands with 75% alcohol; observe the state of cells under a microscope, and the cell density is confluent If it reaches more than 70%, it needs to be passed down, turn off the ultraviolet lamp, turn on the fan, and at the same time wipe the...

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Abstract

The invention relates to the technical field of stem cell therapy, in particular to an MSC-TNF alpha-AB stem cell, a preparation method and application thereof. The MSC-TNF alpha-AB stem cell is a recombinant mesenchymal stem cell, which is integrated with a TNF alpha antibody gene. The invention also provides a promoter which enables positive activation expression of TNF alpha-AB by the MSC-TNF alpha-AB stem cell in the presence of the inflammatory factor TNF alpha. According to the invention, overexpression of the TNF alpha-AB gene in the mesenchymal stem cell is carried out by an in vitro lentivirus method, the constructed MSC-TNF alpha-AB stem cell can be activated by the TNF alpha inflammatory factor in an inflammatory environment, and be combined to an inflammation site more stably, thereby strengthening the anti-inflammation and immunoregulation capacity of MSC, and effectively alleviating the inflammation response of osteoarthritis.

Description

technical field [0001] The invention relates to the technical field of stem cell therapy, in particular to a MSC-TNFα-AB stem cell and its preparation method and application. Background technique [0002] Osteoarthritis (OA), also known as degenerative osteoarthritis, is the degeneration of articular cartilage and the formation of bony spurs at the edge of joints. As time goes on, the development of osteoarthritis is getting faster and faster. Inflammation can promote the degeneration of cartilage, and cartilage degeneration can stimulate the development of inflammation. Inflammation is mainly through the destruction of cell matrix and chondrocytes through inflammatory factors, and the main signaling pathways include MAPK, nuclear transcription factor signaling pathway, Wnt signaling pathway, etc. The methods of treating osteoarthritis include drug therapy, non-drug therapy such as physical therapy, physical exercise, weight control, orthopedics and surgery. The current mai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/113C12N15/867A61K39/395A61K35/28A61P19/02A61P29/00
CPCA61K35/28A61K39/395A61K48/005A61K2039/5156C07K16/241C12N15/86C12N2740/15043A61K2300/00
Inventor 连祺周
Owner 连祺周
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