组织工程化髓核种子细胞优化获取的实验研究

发布时间：2018-04-19 13:26

本文选题：骨髓间充质干细胞 + 脂肪干细胞　；参考：《第四军医大学》2008年硕士论文

【摘要】： 椎间盘退变为腰背痛主要原因之一,其退变主要是由于椎间盘细胞减少和细胞外基质成份的改变,尽管并没明确椎间盘内细胞的性质,但是研究表明:髓核细胞与软骨细胞在某些方面有很多共性。近年来研究表明:细胞移植可以达到组织功能上的修复,所以此方法最有希望成为治疗椎间盘退变的最佳办法。因为干细胞可以向软骨样细胞方面分化,所以大部分学者主要通过改变细胞外环境等来研究骨髓间充质干细胞向软骨样细胞分化。最近研究表明:人体脂肪组织中的脂肪干细胞含量较丰富,而脂肪干细胞在不同的获取技术下可分化为软骨样细胞,如转基因和不同生长因子联合诱导脂肪干细胞向软骨样绍胞分化技术。所以我们设计这个试验就是为了探索比较体外培养的骨髓间充质干细胞(Bone Marrow Mesenchymal Stem Cells,BMSCs)和脂肪干细胞(Adipose Stem Cells,ASCs)变化,以及碱性成纤维绉胞生长因子(Basic Fibroblast Growth Factor,BFGF)对其代谢的影响。方法 1.抽取日本大耳白兔骨髓,用全骨髓培养法进行BMSCs的体外培养、扩增,取日本大耳白兔肩胛间的脂肪组织,在0.075%Ⅱ皎原酶溶液中下剪碎并消化1小时(37℃),所得的消化液过滤离心获取脂肪干细胞,进行体外单层培养扩增,所收集的骨髓间充质干细胞和脂肪干细胞分别用DMEM、DMEM/F12(2:1)、α—MEM培养,倒置显微镜观察骨髓间充质干细胞和脂肪干细胞的形态改变,分别比较兔骨髓间充质干细胞和脂肪干细胞的数量。 2.当细胞传三代后,胰蛋白酶消化收集的脂肪干细胞和骨髓间充质干细胞沉淀以2×10~5/ml接种型24cm~2的培养瓶里,用软骨诱导培养基(Chondrogenic Medium,CM)进行诱导,CM的组成:1.25mg/ml牛血清白蛋白、10ng/ml TGF-β1、100nmol/L地塞米松、50μg/mL维生素C、100μg/mL丙酮酸钠、40μg/mL脯氨酸、1%ITS-plus(10 mg/ml胰岛素,6.7 mg/ml亚硒酸钠,5.5 mg/ml转铁蛋白,2ng/ml乙醇胺)的DMEM/F12(2:1)培养液 3。兔骨髓间充质干细胞和脂肪干细胞分为4组后分别进行诱导培养三周,即A组,BMSCs+CM;B组,BMSCs+CM+5ng/ml BFGF;C组,ASCs+CM;D组,ASCs+CM+5ng/mlBFGF,测定培养液上清羟脯氨酸含量和~(35)SO_4~(2-)基掺入量。结果 1.α—MEM培养基比DMEM、DMEM/F12(2:1)制备的BMSCs和ASCs所需时间大大缩短。 2.单层培养:单层培养的BMSCs和ASCs增殖较快贴壁较牢且生成细胞集落,然而在CM诱导后,细胞增殖较漫,贴壁不牢,且细胞没有集落生成。 3.诱导后的BMSCs和ASCs的形状趋向于类圆形,B组的细胞数量比A组的细胞多,D的细胞数量比C组的细胞多,B组比A组表达的羟脯氢酸增加、~(35)SO_4~(2-)摄入量增加,D组分别比C组表达羟脯复酸增加、~(35)SO_4~(2-)摄入量增加。而D组表达羟脯氢酸和~(35)SO_4~(2-)摄入量与B组表达羟脯氢酸和~(35)SO_4~(2-)摄入量无差别。结论 α-MEM培养基比DMEM、DMEM/F-12更适合兔骨髓间充质干细胞和脂肪干细胞的单层培养,添加BFGF的软骨诱导液可以增加诱导后兔骨髓间充质干细胞和脂肪干细胞的代谢,脂肪干细胞可作为髓核组织工程种子细胞的替代物。
[Abstract]:The degeneration of intervertebral disc is one of the main causes of low back pain. Its degeneration is mainly due to the reduction of intervertebral disc cells and the changes in the components of the extracellular matrix. Although the nature of the intervertebral disc cells is not clear, the study shows that the nucleus pulposus cells have many similarities with the chondrocytes in some aspects. In recent years, the study showed that the cell transplantation could reach the group. This method is the most promising way to treat intervertebral disc degeneration. Because stem cells can differentiate into chondroid cells, most scholars mainly study the differentiation of bone marrow mesenchymal stem cells into chondroid cells by changing the extracellular environment. Recent research shows that human adipose tissue is in human body. The fat stem cells are rich in fat stem cells, and fat stem cells can differentiate into chondroid cells under different acquisition techniques, such as transgenic and different growth factors combined to induce adipose stem cells to differentiate into cartilage like cells. So we designed this experiment to explore bone marrow mesenchymal stem cells (Bone Ma) than in vitro culture. The changes in rrow Mesenchymal Stem Cells, BMSCs) and fat stem cells (Adipose Stem Cells, ASCs), and the effect of basic fibroblast crepe growth factor (Basic Fibroblast Growth) on its metabolism.
Method
1. the bone marrow of Japanese big ear white rabbit was extracted and cultured in vitro by full bone marrow culture. The fat tissue between the scapula of Japanese big ear was amplified, and the fat tissue was extracted from the scapula of Japanese big ear white rabbit. The fat stem cells were obtained by filtration and centrifugation in the 0.075% II clear enzyme solution and digested for 1 hours (37 degrees C). The bone marrow was cultured and amplified in vitro, and the collected bone marrow was collected. Mesenchymal stem cells and adipose stem cells were cultured with DMEM, DMEM/F12 (2:1) and alpha MEM respectively. The morphologic changes of bone marrow mesenchymal stem cells and fat stem cells were observed by inverted microscope, and the number of rabbit bone marrow mesenchymal stem cells and fat stem cells were compared respectively.
2. when the cells were passed on the three generation, the trypsin digested fat stem cells and bone marrow mesenchymal stem cells were precipitated in the culture bottle of 2 x 10~5/ml inoculated 24cm~2, and the cartilage induced medium (Chondrogenic Medium, CM) was induced, and the composition of CM was: 1.25mg/ml bovine serum white egg white, 10ng/ml TGF- beta 1100nmol/L dexamethasone, 50 mu g/mL vitamin. C, 100 mu g/mL sodium pyruvate, 40 micron proline, 1%ITS-plus (10 mg/ml insulin, 6.7 mg/ml sodium selenite, 5.5 mg/ml transferrin, 2ng/ml ethanolamine) DMEM/F12 (2:1) culture
3. rabbit bone marrow mesenchymal stem cells and fat stem cells were divided into 4 groups to be induced and cultured for three weeks, namely, group A, BMSCs+CM; group B, BMSCs+CM+5ng/ml BFGF; C group, ASCs+CM; D group, ASCs+CM+5ng/mlBFGF, determine the content of hydroxyproline and ~ (35) SO_4~ (2-) content in the culture liquid supernatant.
Result
1. a - MEM medium than in DMEM, DMEM/F12 (2:1) prepared by BMSCs and ASCs required time shortened.
2. single layer culture: the proliferation of BMSCs and ASCs in monolayer culture was fast and cell colonies were fast, but after CM induction, the proliferation of cells was more diffuse, the adhesion was not strong, and the cells were not formed.
3. the shape of BMSCs and ASCs tended to be round, the number of cells in group B was more than that in the A group, and the number of D cells was more than that in the C group. The intake of hydroxyl proline was increased in the B group, and the intake of ~ (35) SO_4~ (2-) was increased. (35) SO_4~ (2-) and hydroxyproline acid intake and expression of ~ (35) B group SO_4~ (2-) were no difference.
conclusion
The basal cell culture of rabbit bone marrow mesenchymal stem cells (MSCs) and adipose stem cells (adipose stem cells) is more suitable for the culture of rabbit bone marrow mesenchymal stem cells and adipose stem cells. The addition of BFGF cartilage inducer can increase the metabolism of rabbit bone marrow mesenchymal stem cells and fat stem cells, and fat stem cells can be used as substitutes for seed cells of nucleus pulposus tissue engineering. The DMEM/F-12 is more suitable for the culture of rabbit bone marrow mesenchymal stem cells and adipose stem cells.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

【参考文献】