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A preparation method of a recombinant eye conjunctival epithelial patch containing goblet cells

A technology of conjunctival epithelium and goblet cells, which is applied in the field of preparation of recombined conjunctival epithelial sheets to achieve the effects of uniform stratification of conjunctival epithelium, promotion of proliferation, increase of density and ability to secrete mucin

Active Publication Date: 2011-12-07
SHANDONG EYE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still many problems to be solved in the construction of the recombinant conjunctival epithelial patch containing goblet cells

Method used

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  • A preparation method of a recombinant eye conjunctival epithelial patch containing goblet cells
  • A preparation method of a recombinant eye conjunctival epithelial patch containing goblet cells
  • A preparation method of a recombinant eye conjunctival epithelial patch containing goblet cells

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preparation example Construction

[0019] The preparation method of the present invention: includes 1) the culture preparation of the conjunctival epithelial membrane and 2) the step of inducing differentiation and maturation of goblet cells, characterized in that the above-mentioned 2) step of inducing the differentiation and maturation of goblet cells is in the conjunctival epithelial cell culture medium Add a γ-secretase inhibitor with a final concentration of 100-400nm / L in the medium.

[0020] 1) The cultivation of the conjunctival epithelial membrane of the present invention can be carried out by using existing methods, and the preferred method refers to the method described in the Chinese invention patent: preparation method of amniotic membrane composite limbal stem cell membrane (201110036633.4), and the specific steps are as follows :

[0021] a. Preparation of amniotic membrane nested culture mold:

[0022] Such as figure 1 , according to the conventional method to prepare 5.0cm × 5.0cm size of ste...

Embodiment 1

[0031] Example 1 Induction of Eye Recombinant Conjunctival Epithelial Patch Containing Goblet Cells in Vitro

[0032] 1. Preparation of amniotic membrane nested culture mold:

[0033] Preparation of amniotic membrane nested culture mold:

[0034] Such as figure 1 , according to the conventional method to prepare 5.0cm × 5.0cm size of sterile amniotic membrane 3 amniotic membrane epithelium, the amniotic membrane 3 epithelial side up in a 6-well culture plate; On the end face of the sleeve 2 made of polypropylene material, the embedded culture chamber 1 is buckled on the amniotic membrane sheet 3 of the sleeve 2 to form an amniotic membrane nested culture mold. Wherein used sleeve 2 external diameter 24mm, high 16mm.

[0035] 2. Preparation of mouse embryonic fibroblast feeder layer:

[0036] Treat adherent mouse embryonic fibroblasts with 0.01mg / ml mitomycin C solution and incubate at 37°C for 2 hours; wash the cells carefully with 10ml PBS phosphate buffer; add 1ml 0.25% ...

Embodiment 2

[0041] Example 2 Induction of rabbit eye recombinant conjunctival epithelial patch containing goblet cells in vitro

[0042] 2. Preparation of amniotic membrane nested culture mold:

[0043] Preparation of amniotic membrane nested culture mold:

[0044] Such as figure 1 , according to the conventional method to prepare 5.0cm × 5.0cm size of sterile amniotic membrane 3 amniotic membrane epithelium, the amniotic membrane 3 epithelial side up in a 6-well culture plate; On the end face of the sleeve 2 made of polypropylene material, the embedded culture chamber 1 is buckled on the amniotic membrane sheet 3 of the sleeve 2 to form an amniotic membrane nested culture mold. Wherein used sleeve 2 external diameter 24mm, high 16mm.

[0045] 2. Preparation of mouse embryonic fibroblast feeder layer:

[0046] Treat adherent mouse embryonic fibroblasts with 0.01mg / ml mitomycin C solution and incubate at 37°C for 2 hours; wash the cells carefully with 10ml PBS phosphate buffer; add 1ml...

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Abstract

The invention relates to a preparation method of a recombinant conjunctival epithelial diaphragm containing goblet cells, which induces differentiation of goblet cells by a gamma-secretase inhibitor. The preparation method provided by the invention takes autologous conjunctiva or allogenic conjunctiva as a raw material for culturing and preparing the recombinant conjunctival epithelial diaphragm containing goblet cells. Compared with the original preparation methods of conjunctival epithelial diaphragms, the culture method provided by the invention can promote proliferation of conjunctival epithelial cells at the early stage, so that the formed conjunctival epithelial multiple layers are more uniform; an amniotic membrane nested type culture mold is used as a culture support, the culture surface of the amniotic membrane is flat, the differentiated goblet cells exist among the conjunctival epitheliums, have functions and are convenient in clinical application, and the amniotic membrane is not liable to rupture; and the density and ability to secrete mucoprotein of the goblet cells in the recombinant conjunctival epithelial diaphragm can also be increased, thus more meeting the clinical demand.

Description

technical field [0001] The invention belongs to the technical field of eye surface conjunctival epithelium preparation, and in particular relates to a preparation method of a recombined eye conjunctiva epithelium sheet containing goblet cells. Background technique [0002] Conjunctival goblet cells are single-cell mucus glands located in the conjunctival epithelium. Their main function is to secrete mucin, which is the main component of the innermost layer of the tear film and plays an important role in maintaining ocular surface function. However, if the goblet cells are damaged, the composition of tears will be abnormal, causing keratoconjunctival xerosis, which may cause discomfort in mild cases and blindness in severe cases. Therefore, after extensive damage to the conjunctiva, even if various methods such as lip mucosal transplantation and amniotic membrane transplantation are used to overcome the problem of symblephane, the normal goblet cell density and function canno...

Claims

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Application Information

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IPC IPC(8): A61L27/38C12N5/071
Inventor 周庆军谢立信王瑶杨玲玲田乐
Owner SHANDONG EYE INST
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