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In-vitro induction regulatory macrophage and preparation method and application

A macrophage and regulatory technology, applied in the medical field, can solve the problems of M1-type pro-inflammatory macrophage differentiation and instability, and achieve the effect of good therapeutic effect and stability.

Active Publication Date: 2016-01-27
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the published literature, R-Mф induced in vitro is induced by IL-10, TGF-beta, glucocorticoid and immune complex combined with IL-1 receptor antagonist or TLR, but all of them are unstable and prone to M1 Disadvantages of pro-inflammatory macrophage differentiation

Method used

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  • In-vitro induction regulatory macrophage and preparation method and application
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  • In-vitro induction regulatory macrophage and preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0034] In this embodiment, the preparation method for inducing regulatory macrophages in vitro includes the following steps:

[0035] (1) Isolation and purification of rat bone marrow-derived macrophages

[0036] Rat bone marrow cells were collected with a medium containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 U / ml penicillin, 100 μg / ml streptomycin, 2 mML-glutamine, 20 μM β-mercaptoethanol (the above reagents were purchased from Sigma, St.Louis, MO, USA) RPMI1640 (Gibco) culture medium. Recombinant rat macrophage colony-stimulating factor (M-CSF; PeproTech, Rocky Hill, NJ, USA, final concentration 50 ng / ml) was added to the medium to adjust the cell number to 2×10^6 / mL. Cultivate in 37℃, 5% CO2 incubator for 7 days, and change the medium every 2 days to obtain adherent cells.

[0037] (2) All-trans-retinoic acid (atRA) combined with tumor transforming factor-β (TGF-β)

[0038] Induce the polarization of macrophages into regulatory macrophages (ATR-Mф)...

Embodiment 2

[0054] 1. Subconjunctival injection of ATR-Mф promotes healing of corneal alkali burn injury

[0055] A rat corneal alkali burn model was established according to our previous research method (InvestOphthalmolVisSci.2011; 52:9108–9115, PlosOne.2012; 7:e30842), and a Whatman filter paper piece with a diameter of 4 mm was cut out using a corneal trephine. After dripping 5 ul of 1M sodium hydroxide solution on each piece of filter paper with a micro-sampling gun, place the filter paper on the center of the cornea of ​​the SD rat's right eye for 60 seconds, then rinse the cornea and conjunctival sac with 60ml of normal saline for 1 minute. After the model was established, check under the slit lamp every day (single-blind). The observation items mainly include: corneal epithelial defect, corneal ulcer, corneal perforation, corneal neovascularization and other related complications. The corneal epithelial defect after staining with 0.1% fluorescein was observed under the cobalt blu...

Embodiment 3

[0058] 2. Local subconjunctival injection of ATR-Mф significantly inhibited the occurrence of corneal transplant rejection in rats (see attached Figure 4 )

[0059] The allograft corneal rejection model was established according to the method of Thomas Ritter et al. (InvestOphthalmolVisSci, 2007; 48:1043-1052), Wistar rats were used as transplant donors, SD rats were used as recipients, the diameter of the graft was 3.5 mm, and the diameter of the implant bed was 3.0 mm , 10 / 0 nylon suture, interrupted suture 8 stitches.

[0060] The corneal transparency, edema, and neovascularization were checked under slit lamp every day after operation, and the scores and photos were taken to determine the time of rejection. According to the internationally recognized Holland's criteria (Cornea.1991; 10(5):374-80), the rejection criteria for corneal transplantation are established. When the sum of the scores of corneal graft transparency, edema, and new blood vessels exceeds 6 points, it ...

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Abstract

The present invention discloses an in-vitro induction regulatory macrophage and a preparation method and application, the preparation method comprises the following steps: rat bone marrow cells are taken and cultured, during culture, a recombinant rat macrophage colony stimulating factor is added into a culture medium to obtain wall adherent growth macrophages; in the culture medium, all-transretinoic acid and tumor transforming growth factor-beta are added, the culture is continued, and the macrophage is induced for polarization to obtain the regulatory macrophage. The in-vitro induction regulatory macrophage can be used for treatment of ocular autoimmune diseases. The all-transretinoic acid and TGF-beta are used for in-vitro induction of R-M phi, and the disadvantages of being unstable and prone to M1-type pro-inflammatory macrophage differentiation in the prior art can be avoided, stability of R-M phi can be well kept, and R-M phi anti-inflammatory and immunomodulatory stability can be effectively maintained.

Description

technical field [0001] The invention belongs to the field of medicine, and in particular relates to inducing regulatory macrophages in vitro and its preparation method and application. Background technique [0002] Ocular immune inflammatory disease generally refers to a class of diseases whose pathogenesis is mainly based on ocular immune inflammatory response. Due to the unique structure and physiological functions of the eye, the pathogenesis of various eye diseases is mainly based on immune response, including ocular chemical injury, corneal transplant rejection, uveitis, etc. [0003] With the rapid development of my country's enterprises, especially the foundry industry and chemical industry, serious eye chemical injury accidents caused by hot substances, acid or alkaline chemicals splashing into the eyes are increasing day by day. At present, there are 3 million to 3.5 million corneal blind patients in my country More than 30% are caused by ocular surface injuries suc...

Claims

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Application Information

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IPC IPC(8): C12N5/0786A61K35/15A61P27/02A61P37/06
Inventor 苏文如梁丹郑颂国戴烨
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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