Pharmaceutical for prevention and treatment of ophthalmic disease induced by increase in vasopermeability
A technology of vascular permeability and medicine, which is applied in the field of medicine for the prevention or treatment of eye diseases, and can solve the problems of no research reports
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Embodiment 1
[0073] Example 1: Regulation and control of GDNF by all-trans retinoic acid
[0074] Often in in vitro studies of astrocytes, the choice of cells used is an important issue. Astrocytes isolated from the human brain can be obtained from the American Cell Bank or the Research Society, but from the nature of the first-generation culture of nervous system cells with more differentiated forms, the cell proliferation is slow, and it is not suitable for gene expression. expression analysis. Therefore, the present inventors conducted an experiment using U373MG cells of human glioma cells, in which GFAP (glial fibrillary acidic protein, glial fibrillary acidic protein) known as a marker of astrocytic differentiation was positive, And it has stable properties as a cell line. U373MG cells are readily available in the market.
[0075] U373MG cells were cultured at high density, treated with 10nM or 100nM all-trans retinoic acid (atRA), mRNA was extracted from the cells for quantitative...
Embodiment 2
[0086] In Example 1, the expression of GDNF also increased (concentration-dependently) as the concentration of atRA increased, and co-culture with vascular endothelial cells was carried out in order to examine whether this change had a physiological response. Such as image 3 As shown, a double chamber (double chamber) using a penetrating hole (Transwell) was constructed. Such as image 3 As indicated, U373MG cells (astrocytes) as effector cells were cultured in the outer chamber, treated with atRA and further cultured for 24 hours. Vascular endothelial cells obtained from bovine brain were cultured in a separately prepared inner chamber, forming a dense cell sheet in advance.
[0087] The outer compartment of U373MG cells was combined with the inner compartment of inner tube endothelial cells to start co-culture, and the barrier function as a connection function of vascular endothelial cells was evaluated. By simultaneously proliferating and culturing two types of cells, t...
Embodiment 3
[0090] From the results of co-culture with vascular endothelial cells in Example 2, it has been clarified that the increased expression of GDNF by atRA has a function on vascular endothelial cells. In the results of preliminary experiments, it has been confirmed that Am580 induces the expression of GDNFmRNA in the same concentration-dependent and time-dependent manner as atRA (refer to the following Figure 6 ). The vascular endothelial cells used in Example 2 are the first-generation cultured cell lines obtained from bovine brain, which has the advantage of being able to reproduce in vitro conditions closer to those in the body. However, unlike so-called cell lines, first-generation cultured cell lines have a limited number of divisions and a slow division rate, and they are not suitable for the experimental system required for repeated verification. Therefore, MDCK (Madin Darby Canine Kidney) cells derived from dog renal tubular cells (which are easily available in the mark...
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