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Culture method for transforming human oral mucosal stem cells into astrocytes

An astrocyte and culture method technology, applied in the fields of biology and new medicine, can solve the problems of astrocytes with long time, inconsistent induction factors, and inability to use clinically, and achieves short induction time, repair of nerve defects, The effect of improving nerve function

Active Publication Date: 2020-06-09
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The CN102703387A patent is for the direct isolation and cultivation of astrocytes, using animal-derived cerebral cortex tissue, and using high-concentration calf serum during the cultivation process. The obtained cells are only used for scientific research and cannot be used for clinical
[0006] CN102559593A discloses a method for differentiating human embryonic stem cells into neurons, including the separation and purification of human embryonic stem cells, monolayer adherence and pre-induction of human embryonic stem cells, and induction of neurons, wherein the pre-induction time is 2 days, and the induction is 10 days; the uniformity of cell differentiation is 85%; this induction method is not suitable for astrocytes because the induction factors are inconsistent
[0007] CN102899285A discloses a method for inducing embryonic stem cells to differentiate into nerve cells in vitro, which can induce embryonic stem cells to differentiate into spinal motor neurons and oligodendrocytes, but does not induce astrocytes
There is a risk of tumor formation when embryonic stem cells are transplanted into the body, and the sources of human pluripotent stem cells are also different, and it takes a long time to induce astrocytes
However, autologous stem cells are derived from individuals and are not mature cell lines. They need to be cultured from primary cultures, which is very difficult to operate and may easily lead to experimental failure. Those skilled in the art generally do not use autologous stem cells to induce astrocytes.

Method used

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  • Culture method for transforming human oral mucosal stem cells into astrocytes
  • Culture method for transforming human oral mucosal stem cells into astrocytes
  • Culture method for transforming human oral mucosal stem cells into astrocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: A culture method for transforming human oral mucosal stem cells into astrocytes

[0045] Include the following steps:

[0046] (1) Culture of hOMSC

[0047] The human oral mucosa slices were washed twice with normal saline, cut into pieces with sterilized scissors, placed in low-sugar DMEM medium for culture, and added with a final concentration of 100U / mL penicillin and 100ug / mL streptomycin, 2mM gluten Aminoamide and 10% fetal bovine serum were used to obtain primary cells.

[0048] When the primary cells reached 70% confluence, they were digested with the digestive solution, centrifuged at 1500rpm for 5min, and 8000-10000 cells / cm 2 Density was inoculated and subcultured to obtain the third generation of human oral mucosal stem cells with consistent purity. Microscopic pictures are shown in the appendix figure 1 .

[0049] (2) Induction stage

[0050] The obtained third passage human oral mucosal stem cells were placed in NIM medium at 37°C, 5% CO 2 Th...

Embodiment 2

[0053] Example 2: Identification of hOMSCs

[0054] The cultured third-generation hOMSC cells were used for identification, and the surface markers of stem cells were identified by flow cytometry. Cell markers detected by flow cytometry include CD73, CD105, CD45, CD34 and nestin. The specific experimental steps are as follows:

[0055] (1) Collect the hOMSCs to be detected, wash the cells with PBS, and adjust the concentration of the cell suspension to 1-5×10 with PBS, 10% FCS, and 1% sodium azide 6 cells / mL, transferred to a Falcon tube.

[0056] (2) Dilute the conjugated primary antibody to 0.5ug / mL and add it to the Falcon tube. Incubate at 4°C for 30 min in the dark.

[0057] (3) Wash the cells three times, centrifuge at 400g for 5 min, and then resuspend in 1 mL of LPBS, 10% FCS, and 1% sodium azide.

[0058] (4) Test on the machine and analyze the experimental results.

[0059] The results are attached Figure 3-7 As shown, the expression rate of the third-generat...

Embodiment 3

[0060] Example 3: Identification method of astrocytes

[0061] (1) RT-PCR method:

[0062] First, use the RNeasy Plant Mini Kit (purchased from Qiagen, product number: 74904) to extract RNA from human oral mucosal stem cells (hOMSC) and human oral mucosal stem cell-induced astrocytes (hOMSC-Astroglia). For the extraction method, see the kit instructions . Use the PrimeScript RT reagent Kit with gDNA Eraser kit (purchased from TaKaRa, Cat. No.: RR047A) to reverse transcribe the two RNAs to form cDNA. For the operation method, see the kit instructions. The two cDNAs were used as templates, and fluorescent quantitative PCR reagents were purchased from Sano Company. The detected genes included NGF, GDNF, BDNF, GFAP, and EAAT2, and the β-atcin gene was used as an internal reference gene. Quantitative PCR primer sequences are listed in Table 1. Three replicates were set up for each PCR reaction. The program of RT-PCR was set as: 94°C for 3min, (94°C for 15s, 58°C for 30s, 72°C f...

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Abstract

The invention provides a culture method for transforming human oral mucosal stem cells into astrocytes. The method includes the steps of culture of the human oral mucosal stem cells, induction and differentiation. The autologous oral mucosa stem cells are induced and differentiated into the astrocytes, and the autologous oral mucosa stem cell induced astrocytes have no ethical problems when used clinically and reduce immune rejection response; and the astrocytes induced by the oral mucosal stem cells have high purity, the purity can reach 95% or more, and the induction differentiation time is8-10 days.

Description

technical field [0001] The invention relates to a culture method for transforming human oral mucosa stem cells into astrocytes, belonging to the technical field of biology and new medicine. Background technique [0002] Astrocytes are the most widely distributed type of cells in the mammalian brain. They have always been considered as the support cells of the nervous system, playing the roles of nutrition and protection, ion buffering, and transmitter metabolism. [0003] CN104870634A discloses the regeneration of functional neurons used in the treatment of diseases and injuries in the nervous system, and the use of special transcription factors can convert the astrocytes of the cerebral cortex into functional neurons, therefore, the in vivo method can treat Alzheimer's disease and neurological diseases such as cerebral infarction. However, in the treatment of central nervous system injuries (such as: Parkinson's disease, multiple sclerosis, epilepsy, amyotrophic lateral sc...

Claims

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Application Information

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IPC IPC(8): C12N5/079
CPCC12N5/0622C12N2500/32C12N2501/01C12N2501/11C12N2501/115C12N2501/13C12N2501/999C12N2506/097
Inventor 刘明录韩庆梅卢永灿王立新金海锋张传鹏许淼冯建海强邦明
Owner SHANDONG XINRUI BIOTECH CO LTD
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