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Culture medium for neural stem cell proliferation and capable of inducing neural stem cell to differentiate to dopaminergic neuron and application and proliferation inducing method

A technology of neural stem cells and proliferation medium, which is applied in the field of culture medium for the proliferation of neural stem cells and induction of their differentiation into dopaminergic neurons, which can solve the problems of high price, cumbersome steps, and inconvenient transportation, so as to increase the differentiation ratio and increase the proliferation Speed, low cost effect

Inactive Publication Date: 2017-03-15
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although the differentiation ratio of the patent CN104031882A is relatively high, it requires two stages of induction and differentiation, and the steps are cumbersome, and a variety of expensive foreign commercial products are used in the medium, and B27, as a serum substitute, needs to be separated separately for low temperature Frozen storage brings a lot of inconvenience to transportation
[0007] In addition, the existing neural stem cell proliferation medium is generally DMEM / F12 plus GIBICO’s B27 additive and growth factors, or GIBICO’s Neurobasal medium containing 2% B27 with growth factors added, such as patent CN103045538A, patent CN105296429A and patent CN1536073A , but this type of medium has a low proliferation rate for neural stem cells, and the composition of Neurobasal medium is complex and unclear, B27 transportation is inconvenient, etc., which bring a lot of inconvenience to the cultivation and proliferation of neural stem cells

Method used

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  • Culture medium for neural stem cell proliferation and capable of inducing neural stem cell to differentiate to dopaminergic neuron and application and proliferation inducing method
  • Culture medium for neural stem cell proliferation and capable of inducing neural stem cell to differentiate to dopaminergic neuron and application and proliferation inducing method
  • Culture medium for neural stem cell proliferation and capable of inducing neural stem cell to differentiate to dopaminergic neuron and application and proliferation inducing method

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1: Isolation, culture and subculture of primary neural stem cells

[0039] 1. Isolation and culture of primary neural stem cells

[0040] Pregnant C57BL / 6J mice with 12.5 days of pregnancy were killed by neck dissection. After disinfecting with alcohol, the abdominal cavity was opened along the midline of the abdomen to expose the uterus on both sides. Put it into a petri dish with ice PBS solution, remove the uterine wall and fetal membrane in turn, carefully separate to obtain brain tissue, and manually obtain fetal mouse brain cells. Wash once with ice-cold DMEM / F12, add 0.01% trypsin, digest on a constant temperature culture shaker at 37°C for 30min, and terminate the digestion with DMEM / F12. Filter through a 100-mesh sieve, collect in a centrifuge tube, centrifuge at 1000r / min for 3min, remove the supernatant, add the prepared proliferation medium to make a single cell suspension, count the cells, and add them to a 6-well plate for primary culture. Change...

Embodiment 2

[0050] Embodiment 2: Neural stem cell proliferation culture test

[0051] Starting with the single cell suspension of fetal mouse brain-derived neural stem cells from the same source as in Example 1, they were respectively inoculated into the (1) medium in Example 1 and the DMEM / F12+B27+EGF+bFGF medium in the patent CN103045538A, Culture and proliferation were carried out under the same conditions, and the results were shown in figure 2 . Depend on figure 2 It can be seen that under the culture medium of the present invention, the proliferation speed of neural stem cells is obviously faster than that of the existing patented culture medium as compared.

[0052] In addition, taking the media (2), (3) and (4) in Example 1 as test objects, the proliferation speed of neural stem cells is also significantly faster than that of the prior patent.

Embodiment 3

[0053] Embodiment 3: induction medium of the present invention

[0054] Based on DMEM / F12 medium, including the following components: 10 μg / mL human insulin, 20 nM progesterone, 0.4 μg / mL hydrocortisone, 100 nM dexamethasone, 10 nM estradiol, 100 ng / mL transferrin, 10ng / mL EGF, 10ng / mL FGF, 20ng / mL CTGF, 20ng / mL B-NGF, 10ng / mL VEGF, 0.1% soybean trypsin inhibitor, 2μg / mL L-glutathione, 5μg / mL oleic acid . 2mM glutamine, 100ng / mL laminin, 40ng / mL fibronectin, 0.1% hyaluronic acid, 2μg / mLα2 macroglobulin, 200nmol / L angiotensin II, 0.005U / L erythropoietin.

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Abstract

The invention relates to the medical field, and discloses a culture medium for neural stem cell proliferation and capable of inducing neural stem cell to differentiate to dopaminergic neuron and an application and proliferation inducing method. The proliferation culture medium is a culture medium using DMEM / F12 as the base. The culture medium comprises insulin human, progesterone, hydrocortisone, dexamethasone, estradiol, transferrin, EGF, FGF, CTGF, B-NGF, VEGF, a soybean pancreatin inhibitor, L-glutathione, oleic acid, Tween 80, phosphatidic acid, sodium selenite, panax notoginseng saponins, resveratrol, lithium chloride, cholesterol, WNT3a, and glutamine. A novel neural stem cell culture medium is formed by using a plurality of suitable components with low cost and extensive sources, the proliferation speed of the neural stem cells is remarkably improved, and other induced differentiation components are added on the basis of the neural stem cell, so the neural stem cell is induced to be differentiated to the dopaminergic neuron, and the differentiation proportion is remarkably improved.

Description

technical field [0001] The invention relates to the field of medicine, in particular to a culture medium for neural stem cells to proliferate and induce their differentiation into dopaminergic neurons, as well as its application and proliferation inducing method. Background technique [0002] Neural stem cells (neural stem cells, NSCs) have the ability to differentiate into neurons, astrocytes and oligodendrocytes, can self-renew, and are sufficient to provide a large number of brain tissue cells. It has been found that after various types of central nervous system injury, NSCs are activated and are conducive to tissue damage repair and functional recovery. Neural stem cells have become the necessary seed cells in biomedical engineering and in vitro model research. Therefore, studying the biology of NSCs is of great significance for the repair of nervous system damage and the treatment of degenerative diseases. [0003] Neurons are the structural and functional units of th...

Claims

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Application Information

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IPC IPC(8): C12N5/0797C12N5/0793
CPCC12N5/0619C12N5/0623C12N2500/24C12N2500/30C12N2500/32C12N2500/35C12N2500/36C12N2501/11C12N2501/113C12N2501/115C12N2501/119C12N2501/165C12N2501/32C12N2501/33C12N2501/998C12N2506/08
Inventor 陈海佳葛啸虎王一飞万丽张维敏
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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