In-vitro glial scar forming model and establishment method and application thereof

Abstract

The invention belongs to the technical field of cytobiology and provides an establishment method of an in-vitro glial scar forming model. The establishment method of the in-vitro glial scar forming model comprises the steps that purified meninx (meningo) fiber cells and purified astroglia cells are planted in adjacent small cells on a cavity glass slide respectively with a certain amount ratio, and a culture medium is added for culture; after the cells grow in an adherent mode, a partition board between the small cells is removed, and culture continues to be conducted; when it is observed that the meninx (meningo) fiber cells and the astroglia cells grow and come close to each other, mechanical scratching is conducted at the junction of the two kinds of cells so as to damage the cells, and finally, a glial scar structure is formed. The in-vitro glial scar forming model obtained through the method can well simulate the whole forming process of an in-vitro glial scar, the corresponding glial scar form structure and the change relevant to the cytobiology and the molecular biology appear, and an in-vitro cell-level experimental model is provided for studying of the glial scar forming mechanism and relevant treatment.

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 on astroglial 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 entire glial scars form 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 in vitro. 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 Dev Neurosci. 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 nervous tissue, and there is no correlation when the central nervous system is damaged factors involved

(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 To study the effect of an inhibitory substance

(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 the glial scar inhibits axon growth.Glia.2008; 56:1691-709.); This method requires special silicone rubber cell culture dishes, and its supply and price are not satisfactory

(5) Utilizing 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, Teng X, 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 is the result of the direct action of bioactive molecules, but when the central nervous system is injured, the change in the level of bioactive molecules is not the first factor in the formation of glial scars, but the secondary result. Glial scar formation is then induced; therefore, 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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