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Biology
Bir Doku Dissosiyatör Kullanarak Stromal Vasküler Fraksiyonun Murin Beyaz Yağ Dokusundan Yarı Oto...
Bir Doku Dissosiyatör Kullanarak Stromal Vasküler Fraksiyonun Murin Beyaz Yağ Dokusundan Yarı Oto...
JoVE Journal
Biology
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JoVE Journal Biology
Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

Bir Doku Dissosiyatör Kullanarak Stromal Vasküler Fraksiyonun Murin Beyaz Yağ Dokusundan Yarı Otomatik İzolasyonu

Full Text
3,022 Views
06:08 min
May 19, 2023

DOI: 10.3791/65265-v

Kaede Saito1,2, Yuta Hiraike1,2,3, Misato Oguchi2, Toshimasa Yamauchi2

1Division for Health Service Promotion,The University of Tokyo, 2Department of Diabetes and Metabolic Diseases, Graduate School of Medicine,The University of Tokyo, 3The University of Tokyo Excellent Young Researcher Program,The University of Tokyo

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Overview

This study focuses on the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue, with the aim of obtaining preadipocytes and promoting adipocyte differentiation in vitro. A novel protocol utilizing a tissue dissociator for collagenase digestion is presented, which enhances reproducibility and reduces experimental variation.

Key Study Components

Research Area

  • Adipocyte differentiation
  • Energy expenditure in obesity treatments
  • Murine models for metabolic research

Background

  • Different functions of brown, beige, and white adipocytes
  • Need for reproducible methods in adipocyte research
  • Identification of critical transcription factors regulating differentiation

Methods Used

  • Semi-automated isolation using collagenase digestion
  • Murine adipose tissue
  • Tissue dissociators and enzymatic assays

Main Results

  • Improved methods for isolating SVF and differentiating adipocytes
  • Successful differentiation demonstrated via Oil Red O staining and mRNA analysis
  • Investigation into the effects of Rosiglitazone on brown fat-specific gene expression

Conclusions

  • This protocol facilitates reliable SVF isolation for adipocyte differentiation.
  • The findings hold significant implications for obesity and diabetes research.

Frequently Asked Questions

What is the significance of the stromal vascular fraction in adipocyte research?
The SVF is crucial for isolating preadipocytes, which are necessary for studying adipocyte development and differentiation.
How does the tissue dissociator improve the isolation process?
It reduces variability and contamination during collagenase digestion, leading to more reproducible results.
What specific outcomes were measured to confirm adipocyte differentiation?
Outcomes included Oil Red O staining and mRNA expression levels of adipogenesis regulators.
What are the potential applications of the differentiated adipocytes?
They can be used for functional and mechanistic analyses, including oxygen consumption rate analysis.
How might this research contribute to obesity treatments?
By promoting energy expenditure rather than suppressing intake, new obesity treatments can be developed based on these findings.
What role does the transcription factor NFIA play in this study?
NFIA is identified as a critical regulator of adipocyte differentiation and metabolism.

Bu protokol, preadipositleri elde etmek ve in vitro adiposit diferansiyasyonunu sağlamak için stromal vasküler fraksiyonun (SVF) murin yağ dokusundan yarı otomatik izolasyonunu tanımlar. Kollajenaz sindirimi için bir doku ayrıştırıcısı kullanmak deneysel varyasyonu azaltır ve tekrarlanabilirliği arttırır.

Enerjiyi ısı şeklinde dağıtan adipositin kahverengi ve bej alt tipleriyle ilgileniyoruz, beyaz adiposit ise genellikle enerji depolarken, amacımız enerji alımını baskılamak yerine enerji harcamasının teşvik edilmesine dayanan obezite ve diyabet için yeni bir tedavi geliştirmektir. Bir transkripsiyon faktörü olan NFI'yi, genom çapında bir açık kromatin analizi ile kahverengi adiposit farklılaşmasının kritik bir düzenleyicisi olarak tanımladık. NFI ve adipogenezin ana transkripsiyonel düzenleyicisi PPAR-gama, kahverengi yağa özgü arttırıcılar olarak birlikte lokalize olur.

Stromal vasküler fraksiyonun fare yağ dokusundan izole edilmesi sürecinde, yağ dokusunun manuel kıyma ve kollajenaz sindirimi deneysel değişkenliğe ve kontaminasyona yol açabilir. Yöntemimiz, SVF izolasyonunu kolaylaştıran, deneysel değişkenliği ve kontaminasyonu azaltan ve tekrarlanabilirliği artıran kollajenaz sindirimi için bir doku ayrıştırıcısı kullanır. Bireysel olarak farklılaştırılmış adiposit kullanılarak yapılan deneyler, alanımızdaki araştırmalar için vazgeçilmezdir.

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