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Use of neurocyte differentiated from stem cell induced by isobavachin

A technology of isopsoraleae, nerve cells, applied in the field of cell biology and pharmacology

Inactive Publication Date: 2008-10-01
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] So far, there are no domestic and foreign research reports on the effect of monomeric compounds of psoraleae extract, especially isopsoralen A, on inducing the directed differentiation of stem cells into nerve cells

Method used

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  • Use of neurocyte differentiated from stem cell induced by isobavachin
  • Use of neurocyte differentiated from stem cell induced by isobavachin
  • Use of neurocyte differentiated from stem cell induced by isobavachin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Study on Induction of Embryonic Stem Cells (embryonic stem cells, ES cells) into neural cells in vitro by isopsoralen

[0029] 1. ES cell support culture:

[0030] Mouse ES cell D3 cell line (ES-D3 cell) in the basal medium (containing high glucose DMEM, non-essential amino acids, 10% fetal bovine serum, leukemia inhibitory factor), press 1 × 10 6 Cells / ml were inoculated on feeder cells (mouse fibroblasts), and subcultured for 2 to 3 days.

[0031] 2. Culture of isopsoralen-induced ES cells to differentiate into neurons:

[0032] ES-D3 cells were digested with trypsin, and made into a single-cell suspension with differentiation medium (containing high-sugar DMEM, non-essential amino acids, 10% fetal bovine serum, and no leukemia inhibitory factor), at about 600 cells / 30 μL Inoculate on the inner surface of the lid of the petri dish, turn the lid over to form a hanging drop, and culture it in an incubator for 3 days. Thereafter, the hanging drops that had ...

Embodiment 2

[0034] Example 2: Identification of Isopsoralen Induced ES Cells into Neural Progenitor Cells in Vitro by Immunofluorescence Experiment

[0035] In the early stage of ES cell differentiation, the three germ layer structures are constantly differentiating, and the formation of neural precursor cells is a key step in the differentiation of cells into nerve cells. Nestin is a marker protein of neural progenitor cells, and we investigated the differences of neural progenitor cells induced by isopsoralen in the early stage of differentiation based on this marker. The results showed that on day 8+0, there were obvious differences in nestin immunostained positive cells induced by isopsoralen A, and the quantitative results of flow cytometry showed that isopsoralen A could significantly increase the differentiation of ES cells See Figure 2 for the proportion of nestin-positive neural progenitor cells derived. Figure 2 shows that isopsoralen can significantly increase the proportion o...

Embodiment 3

[0036] Example 3: Identification of Isopsoralen Inducing ES Cells to Differentiate In Vitro into Nerve Cells by Immunofluorescence Experiment

[0037]Cell samples from the isopsoralen-induced group differentiated for 8+6 days were collected, fixed with pure cold methanol for 10 minutes, then blocked with bovine serum for 30 minutes, and then added primary antibodies: (1) glial cell-specific protein monoclonal mouse anti-GFAP, diluted 1:200, (2) neuronal cell-specific protein monoclonal mouse anti-β-tubulin III, diluted 1:100, incubated overnight at 4°C. Secondary antibody was added the next day: FITC-Conjugated goat anti-Mouse IgG or Rhodamine-Conjugated goatanti-Rabbit IgG, diluted 1:1000, and incubated at 37°C for 1.5h. After mounting in non-fluorescent glycerol, observe with a fluorescence microscope and take pictures for recording.

[0038] see image 3 , the results showed that isopsoralen A induced ES cells to differentiate for 8+6 days, and glial cell-specific glial f...

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Abstract

The invention provides a single type nerve cell which is formed by in vitro directed differentiation of sobavachin-induced stem cells (comprising embryonic stem cells, neural stem cells and bone marrow mesenchymal stem cells). The single type nerve cell is applied in the preparation of drugs used for stem cell transplantation therapy of neurodegenerative diseases, in the preparation of cell differentiation agents used for repairing and reconstructing of damaged nerve cells and is also applied in the construction of a efficacy screening and evaluation model. The invention develops a new usage of the isobavachin, provides a physical basis for the prevention and treatment functions of the isobavachin traditional Chinese medicine and also provides a basis for the drug regulation and control regenerative medicine or the tissue engineering.

Description

technical field [0001] The invention belongs to the field of cell biology and pharmacology, and relates to the use of an isopentenyl traditional Chinese medicine monomer compound, in particular to the use of isopsoralen A in inducing the directional differentiation of stem cells in vitro, which can be used for drug treatment of neurodegeneration Disease and Repair Cellular Differentiation Agents for Regenerative Medicine Reconstructing Damaged Nerve Tissue. Background technique [0002] Psoralen is the fruit of Psoralea corylifolia, a leguminous plant. It has the functions of invigorating the kidney and yang, consolidating essence and reducing urination, warming the spleen and stopping diarrhea, stopping bleeding and sweating, and absorbing qi and relieving asthma. The chemical composition of psoralen contains coumarin, flavonoids, monoterpene phenols, volatile oil, saponins, polysaccharides, lipids and other components. The flavonoids that have been isolated and purified i...

Claims

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Application Information

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IPC IPC(8): A61K31/352A61P25/28A61L27/38C12N5/06C12N5/079
Inventor 楼宜嘉朱丹雁王志强王欢欢吴佳莹梅宇钦许敏华俞永平欧丽丽
Owner ZHEJIANG UNIV
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