山羊骨髓间充质干细胞体外分离培养与鉴定的实验研究
发布时间：2018-09-18 16:57
【摘要】： 背景与目的 骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)来源于中胚层,主要存在于全身的结缔组织与器官间质中,以骨髓中的含量最多。骨髓间充质干细胞具有自我更新的能力与多向分化、增殖的潜能。骨髓间充质干细胞因为具有可于自体获得、易培养、移植后能够长期存活并且不会出现排斥反应,可被外源基因转染并长期表达的优点,近年来成为组织工程、细胞治疗、基因治疗及器官移植的研究热点,在多学科多领域做为一种理想的种子细胞得到了广泛应用。但是骨髓间充质干细胞虽然在体内的来源比较广泛,但是数量极少,并且随着身体机能的下降与个体的衰老,数量会逐渐减少。而组织工程与细胞工程开展的首要条件是种子细胞的获取和培养传代,如何能够在体外获得足够数量和较高纯度的种子细胞极其重要。而且目前尚没有对骨髓间充质干细胞进行直接鉴定的方法。通过本研究拟建立一种体外分离培养、扩增骨髓间充质干细胞的理想方法,并且观察其生长特性,探讨其鉴定方法,为其在组织工程学与细胞工程学等学科的应用奠定基础。材料与方法 选用健康中国青山羊,骨髓穿刺法抽取髂骨骨髓,应用密度梯度离心法从中分离出骨髓间充质干细胞,结合贴壁筛选法进行培养,显微镜下观察细胞形态与生长情况,测定细胞成活率及传代细胞贴壁率,对第2、4、6代细胞采用连续计数法绘制出生长曲线,计算细胞倍增时间,免疫细胞化学染色法鉴定细胞表面抗原标志。结果 1.骨髓间充质干细胞原代细胞培养的形态观察：培养后24h可见部分细胞贴壁,贴壁细胞多呈椭圆形。48 h后可见更多的细胞贴壁,贴壁细胞有伸展变形现象,细胞伸出伪足,形态渐变为梭形、多角形。4～6d后可见数个细胞集落形成,显微镜下见贴壁细胞的细胞膜及细胞核清晰。随培养进行细胞集落进一步增多,细胞生长逐渐融成片状,12～14d时可长满培养瓶底80%。 2.骨髓间充质干细胞传代细胞培养的形态：细胞传代后6～8d即可长满瓶底,细胞融合率可达100%。传代细胞镜下呈均一长梭形,细胞膜及细胞核清晰,细胞密度较高时呈放射状、漩涡状排列。 3.骨髓间充质干细胞的鉴定结果：第3代骨髓间充质干细胞免疫组化染色,细胞表面抗原CD29,CD44呈阳性表达(胞浆呈黄褐色染色),CD34,CD45呈阴性表达。 4.骨髓间充质干细胞的生长曲线与倍增时间：生长曲线呈S型,1-3d为潜伏期,生长速度较为缓慢；2-3d后进入对数增长期,生长速度明显加快；6-7d后进入平台期,生长速度趋于稳定。第2、4、6代细胞平均倍增时间分别为(25.37±1.04)h,(27.45±0.91)h,(29.49±2.02)h,总体平均倍增时间为(27.44±2.18)h。结论 1.本研究采用密度梯度离心法从山羊骨髓中分离骨髓间充质干细胞,结合贴壁培养法进行体外培养扩增,细胞生长状态良好,体外增殖能力较强。 2.通过鉴定细胞表面抗原标志,结合形态学观察,证实研究中所分离培养细胞是骨髓间充质干细胞。
[Abstract]:Background and purpose
Bone marrow mesenchymal stem cells (BMSCs) are derived from the mesoderm and mainly exist in the connective tissue and organ stroma of the whole body, with the highest content in bone marrow. In recent years, it has become a research hotspot in tissue engineering, cell therapy, gene therapy and organ transplantation. It has been widely used as an ideal seed cell in many subjects and fields. Although mesenchymal stem cells have a wide range of sources in vivo, their number is very small, and the number will gradually decrease with the decline of body function and the aging of individuals. Daughter cells are extremely important, and there is no direct identification method for bone marrow mesenchymal stem cells. through this study, we intend to establish an ideal method of isolation and culture in vitro, amplification of bone marrow mesenchymal stem cells, and observe their growth characteristics, explore its identification methods, for its tissue engineering and cell engineering disciplines. Laying the foundation for application. Materials and methods
Bone marrow was extracted from iliac bone marrow of healthy Chinese goats by bone marrow puncture. Bone marrow mesenchymal stem cells were isolated by density gradient centrifugation and cultured by adherence screening. Cell morphology and growth were observed under microscope. Cell survival rate and adherence rate of passaged cells were measured. Cells of the 2nd, 4th and 6th passages were drawn by continuous counting method. The growth curve was made, the doubling time was calculated, and the surface antigen markers were identified by immunocytochemical staining.
1. Morphological observation of primary cell culture of bone marrow mesenchymal stem cells: Some cells adhered to the wall 24 hours after culture, and most of the adherent cells were elliptical. After 48 hours, more cells adhered to the wall, the adherent cells had stretching deformation, the cells extended pseudopodia, the morphology gradually changed into spindle shape, and several cell colonies formed after 4-6 days, microscopically. The cell membrane and nucleus of adherent cells were clear. With the further increase of cell colony, cell growth gradually melted into sheets. At 12-14 days, 80% of the bottom of the culture bottle could be grown.
2. The morphology of bone marrow mesenchymal stem cells: the cells could grow to the bottom of the bottle 6-8 days after passage, and the fusion rate could reach 100%. Under the passage microscope, the cells were uniform spindle-shaped, the cell membrane and nucleus were clear, the cells were radial and arranged in a vortex when the density was high.
3. Identification of bone marrow mesenchymal stem cells: Immunohistochemical staining of the third generation of bone marrow mesenchymal stem cells showed positive expression of cell surface antigen CD29 and CD44 (cytoplasm was yellow-brown staining), negative expression of CD34 and CD45.
4. The growth curve and doubling time of bone marrow mesenchymal stem cells: the growth curve was S-shaped, 1-3 days was incubation period, and the growth rate was slower; 2-3 days later entered the logarithmic growth period, the growth rate was significantly accelerated; 6-7 days later entered the plateau phase, the growth rate tended to be stable. The average doubling time of the 2nd, 4th and 6th generation cells were (25.37 + 1.04) h, (27.45 + 0.91) h, respectively. H, (29.49 + 2.02) h, the overall average doubling time was (27.44 + 2.18) H. conclusion.
1. Bone marrow mesenchymal stem cells (BMSCs) were isolated from goat bone marrow by density gradient centrifugation and cultured in vitro with adherent culture. The cells grew well and had strong proliferation ability in vitro.
2. By identifying the markers of cell surface antigen and morphological observation, it was confirmed that the cells isolated and cultured in this study were 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】：2010
【分类号】：R329
[Abstract]:Background and purpose
Bone marrow mesenchymal stem cells (BMSCs) are derived from the mesoderm and mainly exist in the connective tissue and organ stroma of the whole body, with the highest content in bone marrow. In recent years, it has become a research hotspot in tissue engineering, cell therapy, gene therapy and organ transplantation. It has been widely used as an ideal seed cell in many subjects and fields. Although mesenchymal stem cells have a wide range of sources in vivo, their number is very small, and the number will gradually decrease with the decline of body function and the aging of individuals. Daughter cells are extremely important, and there is no direct identification method for bone marrow mesenchymal stem cells. through this study, we intend to establish an ideal method of isolation and culture in vitro, amplification of bone marrow mesenchymal stem cells, and observe their growth characteristics, explore its identification methods, for its tissue engineering and cell engineering disciplines. Laying the foundation for application. Materials and methods
Bone marrow was extracted from iliac bone marrow of healthy Chinese goats by bone marrow puncture. Bone marrow mesenchymal stem cells were isolated by density gradient centrifugation and cultured by adherence screening. Cell morphology and growth were observed under microscope. Cell survival rate and adherence rate of passaged cells were measured. Cells of the 2nd, 4th and 6th passages were drawn by continuous counting method. The growth curve was made, the doubling time was calculated, and the surface antigen markers were identified by immunocytochemical staining.
1. Morphological observation of primary cell culture of bone marrow mesenchymal stem cells: Some cells adhered to the wall 24 hours after culture, and most of the adherent cells were elliptical. After 48 hours, more cells adhered to the wall, the adherent cells had stretching deformation, the cells extended pseudopodia, the morphology gradually changed into spindle shape, and several cell colonies formed after 4-6 days, microscopically. The cell membrane and nucleus of adherent cells were clear. With the further increase of cell colony, cell growth gradually melted into sheets. At 12-14 days, 80% of the bottom of the culture bottle could be grown.
2. The morphology of bone marrow mesenchymal stem cells: the cells could grow to the bottom of the bottle 6-8 days after passage, and the fusion rate could reach 100%. Under the passage microscope, the cells were uniform spindle-shaped, the cell membrane and nucleus were clear, the cells were radial and arranged in a vortex when the density was high.
3. Identification of bone marrow mesenchymal stem cells: Immunohistochemical staining of the third generation of bone marrow mesenchymal stem cells showed positive expression of cell surface antigen CD29 and CD44 (cytoplasm was yellow-brown staining), negative expression of CD34 and CD45.
4. The growth curve and doubling time of bone marrow mesenchymal stem cells: the growth curve was S-shaped, 1-3 days was incubation period, and the growth rate was slower; 2-3 days later entered the logarithmic growth period, the growth rate was significantly accelerated; 6-7 days later entered the plateau phase, the growth rate tended to be stable. The average doubling time of the 2nd, 4th and 6th generation cells were (25.37 + 1.04) h, (27.45 + 0.91) h, respectively. H, (29.49 + 2.02) h, the overall average doubling time was (27.44 + 2.18) H. conclusion.
1. Bone marrow mesenchymal stem cells (BMSCs) were isolated from goat bone marrow by density gradient centrifugation and cultured in vitro with adherent culture. The cells grew well and had strong proliferation ability in vitro.
2. By identifying the markers of cell surface antigen and morphological observation, it was confirmed that the cells isolated and cultured in this study were 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】：2010
【分类号】：R329
【参考文献】
相关期刊论文 前10条
1 周德存;骨髓间充质干细胞分离培养和应用[J];安徽医学;2003年04期
2 张文鹏;叶发刚;y囇澡，
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