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学科主题: 骨科学
题名:
rAAV介导TGF-β联合sox9双基因共转染诱导骨髓干细胞成软骨分化及调控其代谢作用的实验研究
作者: 陶可
答辩日期: 2016-05-11
导师: 林剑浩
专业: 骨科学
授予单位: 北京大学
授予地点: 北京大学第二临床医学院
学位: 博士
关键词: 重组腺相关病毒 ; 骨髓间充质干细胞 ; 基因治疗 ; 软骨缺损 ; 人性别决定区Y框蛋白9 ; 转化生长因子-β ; 软骨分化
其他题名: Effects of combined rAAV-mediated TGF-β and sox9 gene transfer and overexpression on the metabolic and chondrogenic activities in human bone marrow derived mesenchymal stem cells
分类号: R329
摘要:

目的:

关节软骨损伤后自我修复能力非常有限,经基因修饰的间充质干细胞(MSCs)可稳定分化为软骨细胞,是治疗软骨缺损最有前景的种子细胞。

方法/结果:

< >hMSCs单层(2D)培养模型或hMSCs微球(3D)培养模型中,3天或21天后,细胞增殖、DNA含量和HE染色单位面积下平均细胞数检测发现,单独或联合转染rAAV-hTGF-β或rAAV-FLAG-hsox9能有效促进hMSCs的细胞增殖;

hMSCs21ELISART-PCRrAAV-hTGF-βrAAV-FLAG-hsox9hMSCs

< >rAAV-hTGF-β或rAAV-FLAG-hsox9能有效抑制hMSCs微球的软骨肥大化和终末分化。

结论:

rAAV-hTGF-β单独或联合rAAV-FLAG-hsox9转染能明显促进hMSCs增殖,诱导hMSCs向软骨细胞分化和细胞外基质合成,抑制hMSCs成骨细胞分化和肥大化;当rAAV-hTGF-β联合rAAV-hsox9在最高浓度同时共转染时,达最佳效果。

英文摘要:

Objective:

Articular cartilage has a limited potential for self-healing. Transplantation of genetically modified progenitor cells like bone marrow-derived mesenchymal stem cells (MSCs) is an attractive strategy to conveniently activate the chondrogenic differentiation processes as a means to improve the intrinsic repair capacities of damaged articular cartilage.

 

Methodology:

In this study, we examined the potential benefits of co-overexpressing the pleiotropic transformation growth factor beta (TGF-β) with the cartilage-specific tranion factor SOX9 via gene transfer with recombinant adeno-associated virus (rAAV) vectors upon the chondroreparative processes and biological activities of human MSCs (hMSCs). Freshly isolated hMSCs were transduced over time with separate rAAV vectors carrying either TGF-β or sox9 in chondrogenically-induced aggregate cultures to evaluate the efficacy and duration of transgene expression and to monitor the effects of rAAV-mediated genetic modification upon the cellular activities (proliferation, matrix synthesis) and chondrogenic differentiation potency compared with control conditions (lacZ treatment, sequential transductions).

 

Principal Findings:

1. Significant, prolonged TGF-β/sox9 co-overexpression was achieved in chondrogenically-induced hMSCs upon co-transduction via rAAV for up to 21 days;

2. TGF-β/sox9 co-overexpression enhanced proliferative, biosynthetic, and chondrogenic activities relative to control treatments, especially when co-applying the candidate vectors at the highest vector doses tested;

3. Optimal co-administration of TGF-β with sox9 also advantageously reduced hypertrophic differentiation of the cells in the conditions applied here.

 

Conclusions:

The present findings demonstrate the possibility of modifying MSCs by combined therapeutic gene transfer as potent future strategies for implantation in clinically relevant animal models of cartilage defects and futher improvement of repair of articular cartilage lesions in vivo.

语种: 中文
相关网址: 查看原文
内容类型: 学位论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/124609
Appears in Collections:北京大学第二临床医学院_学位论文

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作者单位: 北京大学第二临床医学院

Recommended Citation:
陶可. rAAV介导TGF-β联合sox9双基因共转染诱导骨髓干细胞成软骨分化及调控其代谢作用的实验研究[D]. 北京大学第二临床医学院. 北京大学. 2016.
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