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Medicine composition for treating neuron degeneration disease

A composition and application technology, applied in drug combination, neurological diseases, gene therapy, etc., can solve the problems of long cycle, many steps, high failure rate, etc.

Inactive Publication Date: 2014-03-26
SHANGHAI GENECHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although Takahashi and Yamanaka's research has become a milestone in the field of stem cell research, in practice, it is necessary to induce pluripotent stem cells from somatic cells, and then differentiate them into target cells based on pluripotent stem cells. There are many steps in the whole process. , long cycle, high failure rate, and the potential danger of tumor formation in the differentiated cells, these problems are not conducive to the promotion and application of this technology

Method used

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  • Medicine composition for treating neuron degeneration disease
  • Medicine composition for treating neuron degeneration disease
  • Medicine composition for treating neuron degeneration disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Construction of Myt1L lentiviral vector:

[0115] (1) Using the cloned MYT1L cDNA as a template, carry out PCR reaction with amplification primers, and amplify to obtain a PCR product with a size of about 3.5 Kb.

[0116] (2) BamHI and AgeI double enzyme digestion is performed on the PCR product and the viral vector.

[0117] (3) T4 DNA ligase (purchased from Takara Company) was used to perform transformation reaction after ligation.

[0118] (4) Through the identification of transformants, positive clones were selected and sent for testing, and those whose sequencing sequence was consistent with the expected sequence were regarded as correct clones, and named as FUGW-MYT1L lentiviral vector.

[0119]

Embodiment 2

[0121] Packaging of Myt1L lentiviral vector:

[0122] (1) Prepare DNA solutions of three plasmids in the lentiviral packaging system (FUGW-MYT1L lentiviral vector 20 μg, pCMV-dR8.2 dvpr vector (purchased from Addgene) 15 μg, pCMV-VSV-G vector (purchased from Addgene) 10 μg, mixed with the corresponding volume of Opti-MEM and diluted evenly, adjusted the total volume to 2.5ml, and incubated at room temperature for 5 minutes.

[0123] (2) Take 100 μl of Lipofectamine2000 (purchased from invitrogen) reagent and mix and dilute it with 2.4 ml Opti-MEM (purchased from Invitrogen) in another tube, and incubate at room temperature for 5 minutes.

[0124] (3) Mix the diluted DNA described in (1) with the diluted Lipofectamine2000 described in (2), and mix by inverting gently within 5 minutes. Incubate at room temperature for 20 min.

[0125] (4) Transfer the mixture of DNA and Lipofectamine 2000 to the culture medium of 293T cells, mix well, and store at 37°C, 5% CO 2 cultured in ...

Embodiment 3

[0131] Myt1L lentivirus induced glial cells;

[0132] (1) When the glial cell U251 density reached 50%-60%, virus infection was carried out, and the cell culture medium was replaced with serum-free medium before infection.

[0133] (2) According to the MOI value of U251 cells (MOI=5), add an appropriate amount of MYT1L lentivirus (number of viruses added = number of cells x MOI value). Observe the cell state after 12 hours: if there is no obvious cytotoxic effect, replace the medium after continuing to culture for 24 hours; if there is obvious cytotoxic effect, replace the medium immediately.

[0134] (3) Medium replacement: Three days after U251 cells were infected with MYT1L lentivirus, the original medium was gradually replaced with Neurobasal medium (Invitrogen, 12348-017) containing factor N27 (Invitrogen, 17504-044,) . The replacement method is to keep 1 / 2 of the original medium each time, add 1 / 2 of the fresh Neurobasal medium with N27 factor, and replace the medium...

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PUM

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Abstract

The invention discloses a medicine composition for treating neuron degeneration disease. The invention provides a method for forming neurons by specifically inducing spongiocyte differentiation. The method disclosed by the invention specifically comprises the following steps: firstly establishing a Myt1L (Myelin transcription factor 1-like) gene on a virus vector and packaging to obtain pseudovirus particles with Myt1L; and infecting spongiocyte by the pseudovirus particles with the Myt1L, and culturing for a period of time, thus differentiating the spongiocyte into neuron cells. The neuron cells and the composition thereof have a potential value in treatment of neuron degeneration diseases, such as Parkinson disease.

Description

[0001] technical field [0002] The invention relates to the field of biotechnology, in particular to a method and composition for specifically inducing glial cells to transform into neuron cells. [0003] Background technique [0004] Stem cells have a very broad application prospect in the field of biomedicine, and embryonic stem cells have the potential of comprehensive differentiation, which has become a research hotspot. In 1997, British scientist Wilmut and others transferred the nucleus of sheep somatic cells into enucleated oocytes and developed them into mature individuals—the famous Dolly the sheep, thus confirming the possibility of somatic cells reversing into totipotent stem cells in practice. In 2006, Japanese scientists Takahashi and Yamanaka discovered that after four transcription factors (Oct-4, Sox-2, Klf4 and c-Myc) acted together on mouse skin cells, they could successfully induce reverse differentiation into three germ layers Differentiated pluripoten...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K38/17C12N7/04C12N15/11C12N15/63C12N5/0793A61P25/16
Inventor 高博盛一谢胜华吴庆徐述
Owner SHANGHAI GENECHEM
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