立体诱导兔骨髓间充质干细胞成软骨分化及其SOX9与Ⅱ型胶原基因时空表达关系的研究

发布时间：2018-07-17 20:02

【摘要】：目的：分离培养兔骨髓间充质干细胞(Bone Marrow-derived Mesenchymal Stem Cells, BMSCs)。体外立体诱导骨髓间充质干细胞向成软骨方向分化,观察立体诱导过程中骨髓间充质干细胞中Sox9及11型胶原mRNA的含量随时间的表达关系,对组织工程化软骨进行基因水平上的初步研究。 方法：新西兰大白兔3只。股骨粗隆处备皮,用骨髓穿刺针抽取骨髓,采用全骨髓贴壁培养生长法培养骨髓间充质干细胞,使之得到分离纯化。传代至第3代备用。MTT法测细胞增值率,描绘增殖曲线,观察细胞生长状态。取3代细胞,调整细胞数量为1.5×106/离心管,150g离心后分为实验组(立体成软骨诱导培养组)与对照组(立体非成软骨诱导培养组)。实验组采用含有lOng/mlrhTGF-p,的成软骨诱导液培养,对照组采用不含TGF-β1的诱导液进行培养。分别在第4天,8天,12天,16天提取总RNA,行RT-PCR,检测Sox9及Ⅱ型胶原mRNA表达。 结果：全骨髓培养后骨髓间充质干细胞呈贴壁生长,随着换液骨髓中其他不贴壁生长的细胞逐渐被弃去,使得骨髓间充质干细胞得到进一步纯化。传代后细胞生长速度明显加快,并呈旋涡状生长。3代骨髓间充质干细胞生长良好,经历潜伏期、对数期、平台期。实验组细胞第2天形成小球,并逐渐长大。而对照组细胞无法形成小球,松散沉积于离心管底。RT-PCR检测实验组Sox9与Ⅱ型胶原表达含量均随时间逐渐升高,两者呈高度相关性。对照组Sox9表达随时间变化无明显统计学意义,Ⅱ型胶原表达未检出。 结论：全骨髓贴壁生长法可使骨髓间充质干细胞得到分离纯化。含有TGF-β1的诱导培养液可使骨髓间充质干细胞立体诱导成软骨分化,而不含TGF-β1的诱导培养液无法诱导成软骨分化。成软骨分化中,Sox9与Ⅱ型胶原表达逐渐增高,且两者呈相关性。TGF-β1可以上调Sox9的表达量,由于Sox9是Ⅱ型胶原转录因子,表明在本实验中立体诱导过程是通过上调Sox9表达从而促使成软骨分化的。
[Abstract]:Objective: to isolate and culture rabbit bone Marrow-derived mesenchymal stem cells (BMSCs). Bone marrow mesenchymal stem cells were induced to differentiate into chondrocytes in vitro. The expression of Sox9 and type 11 collagen mRNA in bone marrow mesenchymal stem cells was observed with time. A preliminary study on the gene level of tissue engineered cartilage was carried out. Methods: 3 New Zealand white rabbits. Bone marrow mesenchymal stem cells (BMSCs) were isolated and purified from femoral trochanter by bone marrow puncture needle. Bone marrow mesenchymal stem cells (BMSCs) were cultured by whole bone marrow adherent growth method. After passage to the third passage, the proliferation rate was measured by MTT method, the proliferation curve was depicted and the state of cell growth was observed. After centrifugation, the cells were divided into experimental group (stereotactic chondrogenic culture group) and control group (stereotactic non-chondrogenic culture group). The experimental group was cultured with chondrogenic medium containing lOng / ml rhTGF-pand the control group was cultured without TGF- 尾 1. Total RNAs were extracted on day 4, day 8, day 12, day 16, respectively. RT-PCR was performed to detect the expression of Sox9 and collagen 鈪，

本文编号：2130783