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Method for inducing PC-12 cells to be differentiated into neurons

A technology for cell differentiation and inducing differentiation, applied in the field of biomedicine, to achieve the effects of increasing the differentiation rate, improving neuron characteristics, and increasing the length of protrusions

Active Publication Date: 2017-09-01
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In summary, there are many ways to induce PC-12 cells to become neurons, and what kind of method can better induce PC-12 cells to become neurons needs to be improved

Method used

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  • Method for inducing PC-12 cells to be differentiated into neurons
  • Method for inducing PC-12 cells to be differentiated into neurons
  • Method for inducing PC-12 cells to be differentiated into neurons

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Comparison of several different conditions for inducing PC-12 differentiated neurons

[0029]Opti-mem complete medium containing 0.5% (v / v) fetal bovine serum (FBS) and / or 1% (v / v) horse serum (HS), and 0.5% (v / v) fetal bovine serum (FBS) and / or 1% (v / v) horse serum (HS) RPMI 1640 complete medium to culture PC-12 cells (inoculate 3mL about 3.0*10 4 / mL of PC-12 cells, a total of 1.0*10 5 cells), at 37°C, 5% (v / v) CO 2 Differentiation was induced with NGF at a final concentration of 50ng / mL under the condition, and the culture was continued. To quantify the differentiation of PC-12 cells, we took seven days as a cycle to statistically analyze the cell protrusion length and differentiation rate (use the analysis software ImagePro Plus to measure the protrusion length).

[0030] The result is as figure 1 As shown, the statistical results of the protrusion length show that the PC-12 cells cultured in the complete opti-mem medium containing 0.5% (v / v) fetal bo...

Embodiment 2

[0031] Example 2: Effect of NGF on inducing PC-12 cells to differentiate into neurons

[0032] Opti-mem complete medium containing 0.5% (v / v) fetal bovine serum (FBS) and RPMI 1640 complete medium containing 0.5% (v / v) fetal bovine serum (FBS) were used as basic culture conditions to explore different The influence of concentration NGF (final concentration is 25ng / ml, 50ng / ml, 100ng / ml) on PC-12 cell differentiation at different time points (culture 2 days, 4 days, 6 days); NGF in PBS (concentration of 0.01M, pH 7.4) was compared with different concentrations of NGF.

[0033] The result is as figure 2 As shown, among them, figure 2 A~2C are comparisons of neurite lengths of PC-12 cells in Opti-MEM and 1640 groups at 2, 4, and 6 days; Figure D is PC-12 cells cultured in Opti-MEM+0.5%FBS and 1640+0.5%FBS Morphological comparison of nerve growth factor added under different conditions. It can be seen from the figure that when the concentration of NGF reaches 50ng / ml, it has...

Embodiment 3

[0034] Example 3: Improvement of Opti-MEM medium for PC-12 cell adhesion after differentiation

[0035] Opti-mem complete medium containing 0.5% (v / v) fetal bovine serum (FBS) and RPMI 1640 complete medium containing 0.5% (v / v) fetal bovine serum (FBS) were used as basic culture conditions. The concentration is 50ng / ml NGF induces the differentiation of PC-12 cells, and detects the adhesion of the cells at different time points (0.5h, 1h, 2h, 3h, 4h, 5h, 6h) in the next time period (7 days as a cycle) , the detection method is as follows: add 50 μl Matrigel (Matrigel) to 96-well plate, 37 ℃, 5% (v / v) CO 2 Incubate for 1h, wash with PBS twice, then add 1.0*10 5 / mL density of PC-12 cell suspension 100uL, at 37°C, 5% (v / v) CO 2 Cultivate for 0.5, 1, 2, 3, 4, 5, 6 hours. Half of the wells in each group were not washed with PBS, and the other half of the wells were washed with warm (37°C water bath, water bath for 30 minutes) PBS to remove unadhered cells, and then the absorban...

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Abstract

The invention discloses a method for inducing PC-12 cells to be differentiated into neurons. The method comprises the following steps: adding an induction medium and an NGF (nerve growth factor) into PC-12 cells, and performing induced differentiation to form neurons, wherein the induction medium is an opti-mem complete medium. According to the method disclosed by the invention, induction for inducing neurite length and form of cells to be similar to neurons can be obviously improved, the cell adhesion ability is excellent, induced cell proliferation is slow, and the PC-12 cell induction efficiency is greatly improved. The method disclosed by the invention can be widely applied to research models in neurobiology and neuropharmacology and can also be widely applied to neurobiological, neuropharmacological and toxicologic study.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to a method for inducing PC-12 cells to differentiate into neurons. Background technique [0002] PC-12 cells are cell lines cloned from adrenal chromaffin cell tumors, and are often obtained from calves, rats, cats, pigs, etc. It can not only secrete catecholamine hormones such as epinephrine and norepinephrine, but also has the characteristics of nerve cells, such as the existence of various voltage-dependent sodium, potassium, calcium ion channels and receptor activation channels, and chromaffin cells are used as secretory hormones. Classic cell models in the study of mechanisms and membrane ion channels. It is neurogenic, and there are nerve growth factor NGF receptors on the membrane. After being induced by physiological levels of NGF, they stop dividing, grow neurites, and differentiate into cells with sympathetic neuron characteristics. They have nerve cell characteristics and can...

Claims

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Application Information

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IPC IPC(8): C12N5/0793
CPCC12N5/0619C12N2501/13C12N2506/30
Inventor 肖飞胡仁东孙中轻曹巧玉林熙武征苏志坚罗焕敏郑青
Owner JINAN UNIVERSITY
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