An in vitro glial scar formation model and its construction method and application

Abstract

The invention belongs to the technical field of cell biology. The invention provides a method for constructing an in vitro glial scar formation model, which comprises purifying the purified cerebellar (spinal) membrane fibroblasts and astrocytes in a certain quantitative ratio. Planted in adjacent small chambers on chamber slides, culture medium was added; when the cells adhered to the wall, the partitions between small chambers were removed, and the culture was continued; when brain (spinal) membrane fibroblasts and When these two types of cells grow and approach each other, astrocytes use a sharp instrument to make mechanical scratches at the junction of the two types of cells to damage the cells, eventually forming a glial-like scar structure. The in vitro glial scar formation model obtained by the present invention can better simulate the whole process of in vivo glial scar formation, and the corresponding morphological structure of the glial scar and the changes of related cell biology and molecular biology appear, which is called glial scar formation. Mechanism studies and related therapeutic studies provide experimental models at the cellular level in vitro.

Description

technical field [0001] The invention belongs to the technical field of cell biology, and specifically relates to a model that can simulate the formation of glial scars after central nervous system injury in vitro and its construction method, and provides in vitro cellular level for research on the mechanism of glial scar formation and related treatment research. experimental model. Background technique [0002] When the central nervous system is injured, it will cause the quiescent astrocytes to transform into reactive astrocytes, and protrude star-shaped thick processes around the injury site; at the same time, the brain (spinal) membrane fiber cells will also migrate to the injury site. , distributed between the injured tissue and reactive astrocytes, arranged tightly; reactive astrocytes will extend finger-like processes to the brain (spinal) membrane fibroblasts, and combine with each other; the two types of cells secrete together And form a special extracellular matrix...

AI Technical Summary

Problems solved by technology

So far, there are several culture models of glial scars as follows: (1) Use nitrocellulose sheets to insert into the cortex, then remove them, and culture them on the tissue adhered to the surface (Rudge JS, Silver J. Inhibition of neurite outgrowth onastroglial scars in vitro.J Neurosci.1990; 10:3594-3603.); Although this method can obtain the corresponding components of glial scars in vitro, it is not a structure formed by in vitro cell culture, so the formation of the entire glial scar process cannot be observed

(2) Using astrocytes and brain (spinal) membrane fibroblasts to directly co-culture to produce substances and related effects similar to glial-limiting membrane (Ness R, David S. Leptomeningeal cells modulate the neurite growth promoting properties of astrocytes invitro. Glia. 1997; 19:47-57. Struckhoff G. Cocultures of meningeal and astrocytic cells–a model for the formation of the glial-limiting membrane. Int J DevNeurosci. 1995; 13:595-606.) ; This method can observe the activation process of astrocytes, but in the process of simulating the formation of glial boundary membrane, it may be closer to the process of normal development of nerve tissue, and there is no relevant factor involved in the damage of the central nervous system

(3) Use 3D glue to solidify the inhibitory proteoglycan and other substances of glial scars, and observe the effect on the growth of neurons and other cells (Gilbert RJ, McKeon RJ, Darr A, Calabro A, Hascall VC, Bellamkonda RV.CS-4, 6is differentially upregulated in glial scar and is a potent inhibitor of neurite extension. Mol Cell Neurosci. 2005; 29(4):545-58.); but this method does not involve cells forming glial scar, so it is only for a certain The role of inhibitory substances is studied

(4) Culture astrocytes and cerebrospinal fibroblasts on a silicone rubber petri dish, and then form cell wounds through appropriate mechanical stretching, which can make astrocytes appear star-shaped and aggregate into clusters , and the marker expression of glial scar was significantly enhanced (Wanner IB, Deik A, Torres M, Rosendahl A, Neary JT, Lemmon VP, Bixby JL.A new in vitro model of theglial scar inhibits axon growth.Glia.2008; 56 : 1691-709.); This method requires special silicon rubber cell culture dishes, and its supply and price are not satisfactory

(5) Using the action of β-transforming growth factor, glial cells and cerebrospinal fibroblasts aggregate into clusters and the characteristic markers of glial scars appear (Kimura-Kuroda J, TengX, Komuta Y, Yoshioka N , Sango K, Kawamura K, Raisman G, Kawano H. An in vitro model of the inhibition of axon growth in the lesion scar formed after central nervous system injury. Mol Cell Neurosci. 2010; 43:177-87.); of course this method It is the result caused by the direct action of bioactive molecules, but in the process of glial scar formation caused by central nervous system injury, the change of the level of bioactive molecules is not the first factor, but the secondary result, and then induces Glial scar formation; so this method cannot fully reproduce the entire process of glial scar formation

[0006] From the above-mentioned experimental models of glial scars that have appeared in vitro, it can be seen that there are still many shortcomings. At present, an ideal model of glial scar formation in vitro has not been successfully constructed.

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