改良骨髓间充质干细胞体外培养的方法

发布时间：2018-04-27 03:17

本文选题：微环境 + 细胞培养　；参考：《郑州大学》2012年硕士论文

【摘要】：骨髓间充质干细胞(bone mesenchymal stem cells, BMSCs)也称骨髓基质来源干细胞,是存在于骨髓中除造血干细胞以外另一类具有多向分化潜能的干细胞。在一定诱导条件下,这类细胞可定向分化为多种造血以外的组织,特别是中胚层和神经外胚层来源的组织细胞,例如成骨细胞、成软骨细胞、脂肪细胞、腱细胞、肌肉细胞、神经细胞等。另外间充质干细胞(mesenchymal stem cells, MSCs)体外获取容易,创伤微小,具有无限增殖和多向分化的潜能,再加上BMSCs具有贴壁生长的特性,在体外易分离和扩增,还易于外源基因的转入和表达,所以BMSCs受到医学界的青睐,它成为一种理想的细胞治疗工具和基因治疗的靶细胞。开创了再生医学和组织工程的先河。然而间充质干细胞在骨髓中的含量很低,需要进行体外分离纯化和大量增殖。这是目前面临的主要问题。目的通过观察不同血清微环境对大鼠骨髓间充质干细胞的生长增殖状态的影响得到一种更适合BMSCs体外生长的环境,以利于改良骨髓间充质干细胞体外培养的方法,提高体外培养的获得率,以便于其体外扩增和纯化,更好的应用于临床治疗。方法自6-8W龄SD雄性大鼠股骨、胫骨和腓骨中分离提取骨髓间充质干细胞。采用全骨髓贴壁法培养。分别给于以下三种血清微环境进行培养：同种异体血清组：分离提取细胞后原代用含10%大鼠同种异体血清的培养基培养,以后换液传代时用含10%胎牛血清的培养基培养；胎牛血清组：分离提取细胞后原代用含10%胎牛血清的培养基培养,以后换液传代时仍用含10%胎牛血清的培养基培养；无血清组：分离提取细胞后原代用不含胎牛血清的DMEM培养基培养,以后换液传代时用含10%胎牛血清的培养基培养。在倒置显微镜下观察不同血清微环境下骨髓间充质干细胞的生长形态、细胞生长曲线,流式细胞仪检测不同血清微环境培养条件下的细胞表面分子CD45和CD90的表达。应用SPSS17.0软件对数据进行统计学分析。结果大鼠同种异体血清微环境下培养细胞形态较小,呈纺锤状,网状生长。10%胎牛血清微环境下培养细胞呈梭形成纤维样,集落式生长。无血清微环境下培养细胞稀少,只有稍许集落式生长。生长曲线中大鼠同种异体血清组倍增速度最快,其次为10%胎牛血清组,而无血清组未见明显倍增速率。流式细胞仪检测含血清培养基培养条件下各组第三代细胞的CD90阳性表达率均在80%以上,CD45阳性表达率在2%以下。而在无血清培养条件下CD90阳性表达率在74.09%,CD45阳性表达率却在6.09%。这说明无血清微环境下培养的细胞纯度明显低于含血清微环境下培养的细胞。结论在含体积分数为10%的大鼠同种异体血清微环境培养条件下所培养的细胞形态均一性好,融合度均高于其他组,且细胞首次传代的时间明显短于其他血清微环境。,且含体积分数为10%的大鼠同种异体血清和10%胎牛血清均适合骨髓间充质干细胞的生长,但在同种异体血清微环境下细胞增殖更快,而在无血清培养条件下未见明显增殖现象。根据以上结果表明同种异体血清微环境下更适于骨髓间充质干细胞的生长和纯化,更有利于体外培养方法的改良。
[Abstract]:Bone marrow mesenchymal stem cells ( MSCs ) are also known as bone marrow stromal - derived stem cells , which are derived from bone marrow stromal - derived stem cells . These cells can be directionally differentiated into cells other than hematopoietic stem cells in the bone marrow . These cells can be directionally differentiated into various hematopoietic tissues , such as osteoblasts , chondrocytes , adipocytes , tendon cells , muscle cells , nerve cells , etc .

Purpose

by observing the effect of different serum microenvironment on the growth and proliferation state of bone marrow mesenchymal stem cells in rats , a method is more suitable for the in vitro growth of bone marrow mesenchymal stem cells , thereby being beneficial to improving the in vitro culture method of the bone marrow mesenchymal stem cells , improving the obtaining rate of in vitro culture so as to facilitate in vitro amplification and purification , and better application to clinical treatment .

method

Bone marrow mesenchymal stem cells were isolated from femur , tibia and fibula from 6 - 8 W - old SD male rats . All three kinds of serum micro - environments were cultured : allogeneic serum group : culture medium containing 10 % rat allogeneic serum after isolation and extraction of cells , and culture medium supplemented with 10 % fetal bovine serum at the later passage ;
Fetal bovine serum group : culture medium containing 10 % fetal bovine serum after isolation and extraction of cells was cultured in culture medium containing 10 % fetal bovine serum at the time of subculture ;
The growth form , cell growth curve and flow cytometry of bone marrow mesenchymal stem cells in different serum microenvironment were observed under reversed microscope .

Results

The results showed that the rate of CD90 positive expression of the third generation cells was above 80 % and the expression rate of CD90 was lower than 2 % under the condition of serum - free culture .

Conclusion

The results showed that the growth and purification of bone marrow mesenchymal stem cells were more suitable for the growth and purification of bone marrow mesenchymal stem cells .

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

【参考文献】