Cell-based gene therapy for neurodegenerative diseases
A technology of neurodegenerative diseases and cells, applied in nervous system diseases, gene therapy, animal cells, etc., can solve the problems of lack of safe and effective treatments for neurodegenerative diseases and type II diabetes
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Embodiment 1
[0331] Example 1: Insertion of transfected cells in an in vitro human blood-brain barrier (BBB) model and the output of transfected cells to GFP
[0332] The purpose of this experiment was to assess the ability of EPCs to integrate into the BBB.
[0333] Specifically, the BBB was prepared by coating cell culture inserts with 1:20 collagen for 1 hr at 37°C, followed by 2x10 5 hCMEC / D3 cells were seeded in OptiMEM to establish. Pipette 500 μl of Optimem into receiver wells (i.e., basolateral) and place the BBB at 37°C / 5% CO 2 Incubate for 72 hours to allow monolayer formation. On day 4, the medium was removed and replaced with HEPC.CB1 pl.CAG.GFP cells. The experimental groups were (1) only HEPC.CB1pl.CAG.GFP (GFP-EPC) positive control (n=3); and (2) hCMEC.D3(BBB)+HEPC.CB1pl.CAG.GFP cells The BBB experimental group (n=3). Specifically, HEPC.CB1 cells were suspended in OptiMEM to 1.2x10 7 / ml, and finally mix 200 μl of cell suspension with 1800 μl of OptiMEM to 10 6 cell...
Embodiment 2
[0338] Example 2: Homing of Transfected Cells to Cerebral Microvessels
[0339] Early precursors and mature cells of cerebral microvascular endothelial cells were transfected with the vectors described in Example 5. Both types of transfected cells were inserted into the blood-brain barrier in vitro and GFP fluorescence was measured. The results showed that the insertion of precursor cells into the BBB was significantly higher than that of mature cells (the insertion was very weak).
[0340] In order to test whether MAgEC cells (E10.5) will adhere to the blood vessel wall of the mouse brain in vivo and stay there for a long time, 10 6 MAgEC cells injected into BALB / c x DSRed - in the right common carotid artery of mice. After 24 hours, the brains were sectioned and imaged. image 3 Fluorescence microscopy detection of GFP-MAgEC 10.5 in mouse brain microvessels is depicted. The results showed that when EPCs were co-cultured with mature endothelial cells (MBrMECs), insertion...
Embodiment 3
[0341] Example 3: Homing of transfected EPCs to the blood-brain barrier
[0342] The aim of this experiment was to determine whether the inserted transfected EPCs were incorporated into the angiogenic network formed by BBB cells (MBrMEC cells were used as BBB substitutes). Specifically, the cells used were Hoescht pre-labeled MBrMEC (to generate the BBB), and GFP-transfected MAgEC10.5 as EPC. To assess angiogenesis and the interaction between EPCs and the BBB, both cell types were seeded on matrigel-coated wells or slides. Matrigel matrix was diluted two-thirds in optiMEM (without FBS) at 4°C. Allow Matrigel to polymerize at room temperature before seeding cells (2.5x10 4 cells / ml). At specific time points, endothelial cell rearrangement and vascularization were regularly observed under an inverted light phase-contrast microscope. Such as Figure 4 A- Figure 4 Depicted in B, synergy between co-cultured BBBs (Hoescht-labeled MBrMECs) and EPCs (GFP-MAgEC 10.5 cells) durin...
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