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Application of ID1 and ID3 in reprogramming of fibroblasts into Schwann cells and promotion of peripheral nerve regeneration

A fibroblast and cell technology, applied in the field of biomedicine, can solve the problem that the role of ID protein molecules has not been reported.

Pending Publication Date: 2021-05-25
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the role of ID protein molecules in adult cell reprogramming has not been reported

Method used

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  • Application of ID1 and ID3 in reprogramming of fibroblasts into Schwann cells and promotion of peripheral nerve regeneration
  • Application of ID1 and ID3 in reprogramming of fibroblasts into Schwann cells and promotion of peripheral nerve regeneration
  • Application of ID1 and ID3 in reprogramming of fibroblasts into Schwann cells and promotion of peripheral nerve regeneration

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Expression of ID1 and ID3 in post-traumatic fibroblasts

[0044] Full-thickness excision injury model of mouse skin: choose 8-12 weeks, body weight about 20g, C57 female mice, conventional skin preparation and depilation one day before trauma, intraperitoneal injection of 1% chloral hydrate anesthesia (5ml / kg), in mice Gently lift the back skin of the mouse on the midline of the back, and injure it with a full-thickness skin puncher with a diameter of about 1 cm.

[0045] Collect the specimens from full-thickness skin defect wounds of mice on days 1, 3, 5, 7, 9, 12, 15, and 20, fix with 4% paraformaldehyde, embed in paraffin, and section 5 μm for ID1 and ID3 protein immunization For histochemical staining, PBS was used instead of the primary antibody as a negative control group. For the experimental results, see Figure 1A, B, Section staining showed that ID1 and ID3 were expressed in wound neonatal granulation tissue fibroblasts from day 3.

[0046] In vitr...

Embodiment 2

[0049] Example 2 ID1 or ID3 induced fibroblasts into Schwann cells

[0050] Human and mouse dermal fibroblasts were inoculated into 24-well plates. After the cell confluence reached 40%, human and mouse lentiviral vectors Vector-mCherry and ID1- For mCherry and ID3-mCherry, the number of virus particles / number of cells (MOI) is 100. After transfection, after the cells are completely confluent, they are subcultured. After the cells are amplified for one generation, the mCherry control fibroblasts are sorted by flow cytometry. cells, ID1 overexpressing fibroblasts and ID3 overexpressing fibroblasts.

[0051] Colony formation assay was used to detect the proliferation ability of ID1 and ID3 overexpression cells: human and mouse control, ID1 overexpression and ID3 overexpression fibroblasts were divided into 1×10 3 The number of cells / well was seeded in a 6-well plate with DMEM+10% fetal bovine serum+double antibody, and the medium was changed every 3 days. After 12 days of cultu...

Embodiment 3

[0054] Example 3 Schwann cells induced by ID1 or ID3 promote axonal growth of dorsal root ganglion neurons

[0055] Isolation of dorsal root ganglion neurons in rats: Select 8-12 weeks old, SD female mice with a body weight of about 200 g, and kill them by dislocation of the neck, separate the dorsal root ganglion of the 4th to 5th segment, and apply type I collagenase (0.25% )+0.01% trypsin, 37°C shaker for 40min, after passing through a 75um cell sieve, the obtained cells were cultured in Neurobasal TM Medium+2% B27+L-glutamine (2mM), cultured for 1 week, the surviving cells are dorsal root ganglion neurons.

[0056] Co-cultivate dorsal root ganglion neurons and induced Schwann cells: use the control, ID1 and ID3 overexpressed fibroblasts after flow cytometry sorting in Example 2, according to 1×10 4 The number of cells / well was inoculated in a 24-well plate with pre-set cell slides. The culture conditions were: DMEM+10% fetal bovine serum+double antibody. After 24 hours, ...

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Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of ID1 and ID3 in reprogramming of fibroblasts into Schwann cells and promotion of peripheral nerve regeneration, the application uses ID1 or ID3 reprogrammed Schwann cells or ID1 or ID3 overexpressed virus vectors, post-traumatic nerve regeneration can be promoted, trauma healing can be promoted, trauma healing quality can be improved, and the application is an effective way for solving the problem of refractory trauma caused by local nerve regeneration or peripheral neuropathy after trauma.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of ID1 / ID3 in inducing reprogramming of fibroblasts into Schwann cells to promote nerve regeneration. Background technique [0002] Wound repair is a complex biological process, including a variety of orderly cell biological processes such as re-epithelialization, angiogenesis and so on. Among them, innervation has a great impact on wound healing. Previous studies have shown that pathological or trauma-induced innervation disorders can delay wound healing or even lead to non-healing. In addition, the recovery of pain, temperature and touch in local tissues after trauma is crucial to improving the quality of life of patients. Therefore, improving local nerve repair after trauma has important clinical significance for treating refractory wounds and improving the quality of wound healing. [0003] Previous studies have shown that Schwann cells play an...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61K48/00A61P25/00A61P25/02
CPCA61K38/1709A61K48/005A61P25/00A61P25/02A61K2300/00
Inventor 陈泽林史春梦王钰沈谷芳谭旭
Owner ARMY MEDICAL UNIV
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