人脂肪组织与脂肪瘤组织来源间充质干细胞生物学特性比较
发布时间：2018-09-02 09:39
【摘要】：目的:1.通过胶原酶消化法提取、分离人脂肪与脂肪瘤来源间充质干细胞并培养,对提取培养的细胞进行初步鉴定;2.对分离培养的人脂肪与脂肪瘤来源间充质干细胞的生物学特性进行对比,研究两种细胞的不同,有助于脂肪瘤间充质干细胞的进一步研究,并对探讨脂肪瘤发生的相关机制提供重要基础。方法:将同等体积的脂肪及脂肪瘤组织经胶原酶充分消化,对两种间充质干细胞进行分离、培养及扩增,镜下观察人脂肪间充质干细胞(human adipose derived mesenchymal stem cells,hASCs)及人脂肪瘤间充质干细胞(human lipoma derived mesenchymal stem cells,hLMSCs)的细胞形态及生长情况。经成脂成骨诱导后,对培养至14天后的hASCs及hLMSCs油红O染色及茜素红染色,观察hASCs及hLMSCs成脂成骨分化程度。流式细胞仪检测细胞免疫表型C34、CD45、CD90及CD133的表达情况,观察两种干细胞表面标志物表达情况。CCK8检测hASCs及hLMSCs的增殖情况。实时定量PCR检测hASCs及hLMSCs中HMGA2的表达量的不同。结果:1.通过胶原酶消化法分离提取了人脂肪及脂肪瘤来源的间充质干细胞。在MSCs培养基中培养时发现:(1)原代细胞开始贴壁时间:hASCs及hLMSCs均于24小时内完成贴壁;(2)提取数量及首次融合时间:原代细胞提取后发现每毫升人脂肪瘤组织中提取的干细胞数多于人脂肪组织中提取的干细胞数,但hASCs及hLMSCs首次融合的时间基本一致,即6d~8d细胞即可达到80%融合,并向一定方向生长;(3)原代培养的hASCs及hLMSCs两种细胞均成长梭形,并出现旋涡状排列生长;(4)两种细胞在成骨成脂诱导后均可分化为脂肪细胞及成骨细胞。(5)流式细胞仪检测hASCs及hLMSCs表面标志物CD45及CD90的表达量,表达类似。表明:hASCs及hLMSCs具有相似的特性;2.CCK8法检测、对比hASCs及hLMSCs的增殖能力,实验结果表明于96h~144h hLMSCs增殖能力明显高于hASCs。流式细胞仪检测hASCs及hLMSCs细胞表面标志物CD34与CD133表达情况。hASCs中CD34与CD133表达分别为2.14%及3.17%,hLMSCs中CD34与CD133表达分别为20.62%及16.04%。实时定量PCR检测hASCs及hLMSCs组的HMGA2表达量变化,可见hLMSCs组HMGA2表达量显著高于hASCs组表达量。结论:1.运用胶原酶消化法成功将hASCs及hLMSCs分离、培养及扩增,hASCs及hLMSCs均为贴壁生长,诱导后均具有成脂成骨能力,具有多向分化潜能,表达相似的细胞表面标志物,该两种细胞具有相似的生物学特性;2.表面标志物表达量及HMGA2表达量不同,对探讨脂肪瘤发生发展及良、恶性肿瘤发生发展提供重要基础。
[Abstract]:Purpose 1. Mesenchymal stem cells derived from human adipose and lipoma were isolated and cultured by collagenase digestion. The biological characteristics of mesenchymal stem cells derived from lipoma and human adipose cells were compared, and the differences between the two types of cells were studied, which was helpful to the further study of mesenchymal stem cells of lipoma. It provides an important basis for exploring the mechanism of lipoma. Methods: two kinds of mesenchymal stem cells were isolated, cultured and expanded by collagenase digestion in the same volume of fat and lipoma tissue. The morphology and growth of human adipose mesenchymal stem cells (human adipose derived mesenchymal stem cells,hASCs) and human lipoma mesenchymal stem cells (human lipoma derived mesenchymal stem cells,hLMSCs) were observed under microscope. After induced by adipogenic osteogenesis, the degree of hASCs and hLMSCs adipogenic osteogenesis was observed by hASCs and hLMSCs oil red O staining and alizarin red staining after cultured for 14 days. Flow cytometry was used to detect the expression of CD45, CD90 and CD133, and the proliferation of hASCs and hLMSCs were detected by CCK8. The expression of HMGA2 in hASCs and hLMSCs was detected by real-time quantitative PCR. The result is 1: 1. Mesenchymal stem cells derived from human adipose and lipoma were isolated by collagenase digestion. When cultured in MSCs medium, we found that: (1) the initial cell adhesion time was 1: hASCs and hLMSCs were completed within 24 hours; (2) the number of extracted cells and the first fusion time: after the primary cells were extracted, it was found that the cells were extracted from every milliliter of human lipoma tissue. The number of stem cells is more than the number of stem cells extracted from human adipose tissue. However, the time of first fusion of hASCs and hLMSCs was basically the same, that is, 6d~8d cells could reach 80% fusion and grow in a certain direction. (3) hASCs and hLMSCs cells in primary culture both grew fusiform. (4) the two kinds of cells could differentiate into adipocytes and osteoblasts after adipogenic induction. (5) the expressions of CD45 and CD90 on hASCs and hLMSCs surface markers were detected by flow cytometry, and the expression of CD45 and CD90 were similar. The results show that hLMSCs and hASCs have similar characteristics. 2. The proliferative ability of 96h~144h hLMSCs is higher than that of hASCs. 2. The proliferation ability of hASCs and hLMSCs is higher than that of 96h~144h hLMSCs. 2. The results of CCK8 method show that the proliferative ability of 96h~144h hLMSCs is higher than that of hASCs.. The expression of CD34 and CD133 in hASCs and hLMSCs cells were 2.14% and 3.17%, respectively. The expression of CD34 and CD133 were 20.62% and 16.04%, respectively. Real-time quantitative PCR was used to detect the changes of HMGA2 expression in hASCs and hLMSCs groups. It was found that the expression of HMGA2 in hLMSCs group was significantly higher than that in hASCs group. Conclusion 1. HASCs and hLMSCs were isolated successfully by collagenase digestion and cultured and amplified for adherent growth. Both of them had the ability of adipogenic osteogenesis, the potential of multiple differentiation and the expression of similar cell surface markers after induction. The two kinds of cells have similar biological characteristics. The difference of surface marker expression and HMGA2 expression provides an important basis for the development of lipoma, benign and malignant tumor.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R329.2
本文编号：2218967
[Abstract]:Purpose 1. Mesenchymal stem cells derived from human adipose and lipoma were isolated and cultured by collagenase digestion. The biological characteristics of mesenchymal stem cells derived from lipoma and human adipose cells were compared, and the differences between the two types of cells were studied, which was helpful to the further study of mesenchymal stem cells of lipoma. It provides an important basis for exploring the mechanism of lipoma. Methods: two kinds of mesenchymal stem cells were isolated, cultured and expanded by collagenase digestion in the same volume of fat and lipoma tissue. The morphology and growth of human adipose mesenchymal stem cells (human adipose derived mesenchymal stem cells,hASCs) and human lipoma mesenchymal stem cells (human lipoma derived mesenchymal stem cells,hLMSCs) were observed under microscope. After induced by adipogenic osteogenesis, the degree of hASCs and hLMSCs adipogenic osteogenesis was observed by hASCs and hLMSCs oil red O staining and alizarin red staining after cultured for 14 days. Flow cytometry was used to detect the expression of CD45, CD90 and CD133, and the proliferation of hASCs and hLMSCs were detected by CCK8. The expression of HMGA2 in hASCs and hLMSCs was detected by real-time quantitative PCR. The result is 1: 1. Mesenchymal stem cells derived from human adipose and lipoma were isolated by collagenase digestion. When cultured in MSCs medium, we found that: (1) the initial cell adhesion time was 1: hASCs and hLMSCs were completed within 24 hours; (2) the number of extracted cells and the first fusion time: after the primary cells were extracted, it was found that the cells were extracted from every milliliter of human lipoma tissue. The number of stem cells is more than the number of stem cells extracted from human adipose tissue. However, the time of first fusion of hASCs and hLMSCs was basically the same, that is, 6d~8d cells could reach 80% fusion and grow in a certain direction. (3) hASCs and hLMSCs cells in primary culture both grew fusiform. (4) the two kinds of cells could differentiate into adipocytes and osteoblasts after adipogenic induction. (5) the expressions of CD45 and CD90 on hASCs and hLMSCs surface markers were detected by flow cytometry, and the expression of CD45 and CD90 were similar. The results show that hLMSCs and hASCs have similar characteristics. 2. The proliferative ability of 96h~144h hLMSCs is higher than that of hASCs. 2. The proliferation ability of hASCs and hLMSCs is higher than that of 96h~144h hLMSCs. 2. The results of CCK8 method show that the proliferative ability of 96h~144h hLMSCs is higher than that of hASCs.. The expression of CD34 and CD133 in hASCs and hLMSCs cells were 2.14% and 3.17%, respectively. The expression of CD34 and CD133 were 20.62% and 16.04%, respectively. Real-time quantitative PCR was used to detect the changes of HMGA2 expression in hASCs and hLMSCs groups. It was found that the expression of HMGA2 in hLMSCs group was significantly higher than that in hASCs group. Conclusion 1. HASCs and hLMSCs were isolated successfully by collagenase digestion and cultured and amplified for adherent growth. Both of them had the ability of adipogenic osteogenesis, the potential of multiple differentiation and the expression of similar cell surface markers after induction. The two kinds of cells have similar biological characteristics. The difference of surface marker expression and HMGA2 expression provides an important basis for the development of lipoma, benign and malignant tumor.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R329.2
【参考文献】
相关期刊论文 前4条
1 刘培焱;王崇;;CD248/内皮唾酸蛋白在慢性肝损伤过程中通过血小板衍生生长因子调节机制显著促进肝星状细胞增殖[J];临床肝胆病杂志;2016年10期
2 何福果;陈晏林;林晓;赵涌;;CD105与CD248在星形细胞胶质瘤组织中的表达及其意义[J];中国生物制品学杂志;2015年06期
3 陆微;杨刚龙;薛鹏;刘昌梅;关锋;;整合素α6在不同转移潜能膀胱癌细胞中的定量差异表达分析[J];生物化学与生物物理进展;2015年05期
4 李敬来;CD45—淋巴细胞活化的重要调节分子[J];国外医学(免疫学分册);2002年01期
，本文编号：2218967
本文链接：https://www.wllwen.com/yixuelunwen/jichuyixue/2218967.html
