建立永生化兔骨髓间充质干细胞及其成骨分化能力的初步研究

Q1 Health Professions Animal models and experimental medicine Pub Date : 2024-11-26 DOI:10.1002/ame2.12513

Yao Zhang, Chang Xu, Yun Huang, Dongmei Tan, Wenping Luo, Yan Zhang, Yi Tan

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摘要

背景：稳定和标准化的间充质干细胞来源是骨修复组织工程研究和应用的先决条件。我们的目的是建立新西兰兔骨髓间充质干细胞的稳定细胞系，并探索其成骨分化能力：方法：通过逆转录病毒表达 SV40 大 T 抗原（LTA），分离并永生原代兔骨髓间充质干细胞（RBMSCs）。为了评估细胞在体外的成骨分化能力，我们使用 ALP 染色和定量法以及茜素红染色法研究了碱性磷酸酶（ALP）的表达水平和骨形态发生蛋白 9（BMP9）诱导的永生化细胞中的钙沉积。使用微型计算机断层扫描（μCT）和组织学检查评估了细胞异位骨的形成：结果：我们利用 SV40 LTA 建立的永生化细胞系（我们称之为 iRBMSCs）无致瘤性并能保持长期增殖活性。我们进一步发现，BMP9（MOI = 30）在体外能有效诱导 iRBMSCs 的成骨分化能力，在体内与 GelMA 水凝胶协同诱导 iRBMSCs 的成骨分化：结论：我们证实 iRBMSCs 是一种用于骨缺损修复工程的稳定细胞系。

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Establishment of immortalized rabbit bone marrow mesenchymal stem cells and a preliminary study of their osteogenic differentiation capability

Background

A stable and standardized source of mesenchymal stem cells is a prerequisite for bone repair tissue engineering research and application. We aimed to establish a stable cell line of bone marrow mesenchymal stem cells from New Zealand rabbits and explore their osteogenic differentiation capacity.

Methods

Primary rabbit bone marrow mesenchymal stem cells (RBMSCs) were isolated and immortalized via retroviral expression of SV40 Large T antigen (LTA). To assess the osteogenic differentiation capacity of the cells in vitro, we studied the alkaline phosphatase (ALP) expression level and calcium deposition in bone morphogenetic protein 9 (BMP9)-induced immortalized cells using ALP staining and quantification, as well as alizarin red staining. Ectopic bone formation by the cells was assessed using micro-computed tomography (μCT) and histological examination.

Results

The immortalized cell line we established using SV40 LTA, which we termed iRBMSCs, was non-tumorigenic and maintained long-term proliferative activity. We further discovered that BMP9 (MOI = 30) effectively induced the osteogenic differentiation capacity of iRBMSCs in vitro, and there was a synergy with GelMA hydrogel in inducing osteogenic differentiation of the iRBMSCs in vivo.