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Screening and identifying method and application of amniotic stem cell exocrine protein

An identification method and stem cell technology are applied in the field of screening, identification and application of the exocrine protein LOXL2 of amniotic stem cells, which can solve the problem that the exocrine proteome is not yet clear.

Active Publication Date: 2021-03-09
沈阳艾米奥生物工程技术研发中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the exocrine proteome of amnion epithelial stem cells and amnion mesenchymal stem cells has not yet been elucidated

Method used

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  • Screening and identifying method and application of amniotic stem cell exocrine protein
  • Screening and identifying method and application of amniotic stem cell exocrine protein
  • Screening and identifying method and application of amniotic stem cell exocrine protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] 1. Conditioned medium of amniotic mesenchymal stem cells and amniotic epithelial stem cells

[0045] Materials and Methods:

[0046] Protease Inhibitor (Biyuntian)

[0047] Trypsin BI

[0048] Ultrafiltration centrifuge tubes (Merck Millipore; Amicon Ultra-50, Ultracel-3k)

[0049] BCA Kit (Biyuntian)

[0050] Type IV collagenase (sigma)

[0051] Osteogenic / adipogenic / chondrogenic kit (Saiye Bio)

[0052] Step 1: Extraction, cultivation and identification of amniotic membrane mesenchymal stem cells (hAMSCs) and amniotic membrane epithelial stem cells (hAECs):

[0053] (1) Extraction and culture of amniotic mesenchymal stem cells and amniotic epithelial stem cells

[0054]The amnion tissues of healthy caesarean section women were collected, soaked in normal saline for 30 minutes, and washed repeatedly with PBS until there was no mucus. Cut the washed amniotic tissue into 5x5cm 2 Digest the small pieces in 10ml trypsin for 30-50min.

[0055] Extraction and cultur...

Embodiment 2

[0073] Mass spectrometry analysis of amniotic mesenchymal stem cells than amniotic epithelial stem cells expressed higher protein screening results verification.

[0074] Materials and Methods:

[0075] ELISA Kit (Shanghai Enzyme)

[0076] result:

[0077] Based on bioinformatics analysis, the exocrine proteins of hAMSCs and hAECs have positive and negative roles in the development, differentiation and maturation of various tissues. This may be related to the unique physiological function of the amniotic membrane tissue, which is the innermost layer of the fetal membrane and connects with the amniotic membrane layer covering the placenta and umbilical cord. In addition to producing amnion to protect embryo growth, it can also regulate the development of embryos through the secretory proteome to achieve regulation. In the present invention, the differentially expressed hAMSCs-CM and hAECs-CM proteins were enriched, respectively, focusing on functions such as skin development...

Embodiment 3

[0079] LOXL2 promotes keratinocyte migration and differentiation.

[0080] Materials and Methods

[0081] Recombinant protein CTHRC1 (nearshore protein, China)

[0082] Recombinant protein LGASL1 (nearshore protein, China)

[0083] Recombinant protein LOXL2 (Shenzhou, China)

[0084] TRIZOL (Invitrogen, USA)

[0085]Real-time reverse transcription kit and real-time quantitative PCR kit (TaKaRa, Japan)

[0086] Involucrin antibody (Abcam, USA)

[0087] CK10 antibody (Abcam, USA)

[0088] Step 1: LOXL2 promotes keratinocyte migration:

[0089] Scratch test was used to analyze the effect of the proteins screened in the previous step on cell migration. The keratinocytes were seeded into a 6-well plate, and when the confluence of the cells reached 90%, a 500 μm scratch was drawn horizontally and vertically with a 200 μL sterile pipette tip. Rinse 2-3 times with PBS to remove non-adherent cells. Keratinocytes were pre-cultured with LOXL2, CTHRC1 and LGASL1 (100ng / ml) for 3 ...

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Abstract

The invention relates to a method for screening and identifying exocrine proteins of amniotic stem cells and application, and the method identifies exocrine proteomes of amniotic epithelial stem cellsand amniotic mesenchymal stem cells, analyzes and integrates results, and further analyzes differential expression proteins of the amniotic epithelial stem cells and the amniotic mesenchymal stem cells. The positive factor LOXL2 for epithelial development and epidermal regeneration is screened out, and a detailed basis and a new thought are provided for subsequent research on the paracrine effectof the amniotic stem cells. LOXL2 provides a new idea for epithelialization of clinical large-area difficult-to-heal wounds, and provides a direction for better mining the characteristics of different amnion stem cell exocrine proteins. And a basis is provided for development and clinical application of specific cell-free dressings.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the screening identification and application of amniotic membrane stem cell exocrine protein LOXL2. Background technique [0002] Human amniotic membrane is the innermost layer of fetal membrane, which is mainly composed of amniotic epithelial stem cells of ectoderm and amniotic mesenchymal stem cells of mesoderm. Most of them are discarded with the placenta after the fetus is delivered. The study found that human amniotic membrane can be used as a new type of biological dressing in the adjuvant treatment of clinical acute and chronic wounds. Studies have reported that frozen or irradiated amniotic tissue still has significant effects in promoting wound healing, reducing inflammatory responses, and reducing the occurrence of scars in animal wound models or clinical wound healing. This proves that paraamniotic substances have great research value and clinical significance. However,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/573C12N5/071A61K38/44A61P17/02
CPCG01N33/573C12Y104/03013C12N5/0629A61P17/02A61K38/00C12N2501/71
Inventor 庞希宁张涛贺丹庞然赵峰
Owner 沈阳艾米奥生物工程技术研发中心有限公司
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