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Construction method of in-vitro model for simulating mesenchymal stem cell transplantation

A technology of bone marrow mesenchyme and construction method, which is applied in the field of building an in vitro model of simulating bone marrow mesenchymal stem cell transplantation, and achieves the effect of avoiding interference

Inactive Publication Date: 2014-09-17
姜晓丹 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, no unit or individual has conducted research in this area

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  • Construction method of in-vitro model for simulating mesenchymal stem cell transplantation
  • Construction method of in-vitro model for simulating mesenchymal stem cell transplantation
  • Construction method of in-vitro model for simulating mesenchymal stem cell transplantation

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Embodiment 1

[0043] Example 1 The method for establishing an in vitro model of simulating bone marrow mesenchymal stem cell transplantation

[0044] (1) Acquisition and cultivation of cortical neurons:

[0045] Animal selection: 1 wistar inbred suckling mouse within 3 days after birth;

[0046] Operated on ice, decapitated, stripped the suckling mouse skull, exposed the brain tissue, and isolated the cerebral cortex in a petri dish containing cold DMEMF12 medium using microscopic instruments;

[0047] Transfer the cerebral cortex to a sterile penicillin bottle and cut it into pieces about 1mm×1mm×1mm in size, add 0.015g / mL papain 400μL, 50mM ethylenediaminetetraacetic acid (EDTA) 60μL, 1mg / mL into the bottle Digest 200 μL of cysteine ​​and 5.34ml of DMEM / F12 medium in a 37°C incubator for 10 minutes. Trypsin inhibitor terminates the digestion, use a pipette to blow off the cortex appropriately, filter, take the filtrate, and centrifuge;

[0048]Resuspend the centrifuged cell pellet with...

Embodiment 2

[0073] After the in vitro model was established, the establishment of the model and the identification of the formation of synapse-like structures between cortical neurons and bone marrow mesenchymal stem cells were carried out by immunofluorescence microscopy, ordinary light microscopy, fluorescence microscopy, atomic force microscopy and scanning electron microscopy.

[0074] For immunofluorescence images, see Figure 13-14 , the co-culture system was established for 6 days, and the axons of neurons showed a tendency to migrate towards bone marrow mesenchymal stem cells. Figure 13-14 as shown, Figure 13-14 Two trends of neurons growing toward bone marrow mesenchymal stem cells or growing along the surface of bone marrow mesenchymal stem cells in the co-culture system were shown by immunofluorescence technique. Among them, the nucleus is stained blue for DAPI, and the cytoplasm is red for positive expression of β-tubulin. Figure 13-14 The growth trend of the axons of neu...

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Abstract

The invention discloses a construction method of an in-vitro model for simulating mesenchymal stem cell transplantation, which comprises the following steps of: (1) selecting in-vitro neonatal rat cortical neurons; (2) selecting in-vitro rat mesenchymal stem cells; (3) pre-processing the in-vitro rat mesenchymal stem cells, and inoculating in the neonatal rat cortical neurons for co-culture; and (4) after the co-culture, treating the rat mesenchymal stem cells and the neonatal rat cortical neurons by a light microscope, a fluorescence microscope, an atomic force microscope and a scanning electron microscope; and determining an in-vitro model for simulating mesenchymal stem cell transplantation and a growth tendency, an interaction and formation of a synapsis-like structure between the cortical neurons and the mesenchymal stem cells on an ultrastructure. The construction method disclosed by the invention provides a simplified in-vitro model for the mesenchymal stem cell transplantation; meanwhile, the synapsis-like structure formed in the model provides an in-vitro evidence of a structure integration mechanism for the mesenchymal stem cell injury mechanism.

Description

technical field [0001] The invention relates to a method for constructing an in vitro model for simulating bone marrow mesenchymal stem cell transplantation. Background technique [0002] In recent years, bone marrow mesenchymal stem cells, as one of the seed cells for transplantation and treatment of central nervous system diseases, have received extensive research and attention in both basic experiments and clinical trials. As far as the current research is concerned, the mechanisms by which bone marrow mesenchymal stem cells participate in the repair of central nervous system diseases mainly include: cell replacement, factor secretion, and angiogenesis promotion. In order to further explore the interaction between bone marrow mesenchymal stem cells and neurons in the central nervous system, and to achieve the most simplified model to accurately explore the mechanism of bone marrow mesenchymal stem cells repairing central nervous system diseases, the experiment of in vivo ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0793C12N5/0775
Inventor 姜晓丹李鹏法志强张润孙海涛刘翼
Owner 姜晓丹
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