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建立一种用于分离和鉴定人牙周膜来源间充质干细胞的技术。

Establishing a technique for isolation and characterization of human periodontal ligament derived mesenchymal stem cells.

作者信息

Banavar Spoorthi Ravi, Rawal Swati Yeshwant, Paterson Ian Charles, Singh Gurbind, Davamani Fabian, Khoo Suan Phaik, Tan Eng Lai

机构信息

Oral Diagnostic and Surgical Sciences, School of Dentistry, International Medical University, Kuala Lumpur, Malaysia.

Department of Surgical Sciences, Marquette University, USA.

出版信息

Saudi Dent J. 2021 Nov;33(7):693-701. doi: 10.1016/j.sdentj.2020.04.007. Epub 2020 Apr 19.

Abstract

UNLABELLED

Mesenchymal stem cells (MSCs) are extensively used in tissue regenerative procedures. One source of MSCs is the periodontal ligament (PDL) of teeth. Isolation of MSCs from extracted teeth is reasonably simple, being less invasive and presenting fewer ethical concerns than does the harvesting of MSC's from other sites. The objectives of this study were to isolate and characterize the PDL stem cells (PDLSC) from healthy adults' extracted teeth and then to characterize them by comparing them with bone-marrow derived MSCs (BMMSC).

METHODS

The PDL tissue was scraped from the roots of freshly extracted teeth to enzymatically digest using collagenase. The cells were sub-cultured. Flow-cytometric analysis for the MSC surface-markers CD105, CD73, CD166, CD90, CD34, CD45 and HLA-DR was performed. To confirm the phenotype, total RNA was extracted to synthesize cDNA and which was then subjected to RT-PCR. The gene-expression for Oct4A, Sox2, NANOG and GAPDH was determined by gel-electrophoresis. To assess their multilineage potential, cells were cultured with osteogenic, chondrogenic and adipogenic medium and then stained by Alizarin-red, Alcian-blue and Oil-Red-O respectively. MSCs from the bone-marrow were processed similarly to serve as controls.

RESULTS

The cells isolated from extracted teeth expanded successfully. On flow-cytometric analysis, the cells were positive for CD73, CD90, CD105, CD166 and negative for CD34, CD45 and HLA-DR. The PDLSCs expressed Oct4A, Sox2, and NANOG mRNA with GAPDH expression. Cells cultured in the osteogenic, chondrogenic and adipogenic media stained positive for Alizarin-red, Alcian-blue and Oil- Red-O respectively. The surface marker expression and the trilineage differentiation characteristics were comparable to those of the BMMSCs.

CONCLUSIONS

The periodontal ligament tissue of extracted teeth is a potential source of therapeutically useful MSCs. Harvesting them is not invasive and are a promising source of MSC as the PDLSCs showed characteristics similar to those of the highly regarded MSC's derived from bone-marrow.

摘要

未标记

间充质干细胞(MSCs)广泛应用于组织再生程序。MSCs的一个来源是牙齿的牙周膜(PDL)。从拔除的牙齿中分离MSCs相当简单,与从其他部位获取MSCs相比,侵入性较小且伦理问题较少。本研究的目的是从健康成年人拔除的牙齿中分离和鉴定牙周膜干细胞(PDLSC),然后通过与骨髓来源的MSCs(BMMSC)进行比较来对其进行表征。

方法

从新鲜拔除牙齿的根部刮取PDL组织,用胶原酶进行酶消化。将细胞传代培养。对MSCs表面标志物CD105、CD73、CD166、CD90、CD34、CD45和HLA-DR进行流式细胞术分析。为了确认表型,提取总RNA以合成cDNA,然后进行RT-PCR。通过凝胶电泳测定Oct4A、Sox2、NANOG和GAPDH的基因表达。为了评估它们的多向分化潜能,将细胞分别用成骨、成软骨和成脂培养基培养,然后分别用茜素红、阿尔辛蓝和油红O染色。对来自骨髓的MSCs进行类似处理以作为对照。

结果

从拔除牙齿中分离的细胞成功扩增。流式细胞术分析显示,细胞CD73、CD90、CD105、CD166呈阳性,CD34、CD45和HLA-DR呈阴性。PDLSCs表达Oct4A、Sox2和NANOG mRNA以及GAPDH。在成骨、成软骨和成脂培养基中培养的细胞分别对茜素红、阿尔辛蓝和油红O染色呈阳性。表面标志物表达和三系分化特征与BMMSCs相当。

结论

拔除牙齿的牙周膜组织是具有治疗用途的MSCs的潜在来源。获取它们的侵入性小,并且是有前景的MSCs来源,因为PDLSCs显示出与备受关注的源自骨髓的MSCs相似的特征。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e1e1/8589598/ad9bd4ec8912/gr1.jpg

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