摘要
目的建立一种体外分离和培养大鼠脂肪干细胞的方法,观察其增殖的规律,并探讨其造血因子的分泌情况。方法无菌技术下切取双侧腹股沟皮下脂肪垫,加入0.1%Ⅰ-型胶原酶消化获取细胞,接种入含10%胎牛血清的DMEM培养液,倒置显微镜下连续观察细胞形态变化,并用流式细胞学鉴定CD90、CD34、CD44、CD45、CDl06和CDllb的表达,ELISA法检测造血因子的表达,并作统计学分析。结果原代培养显示培养的脂肪干细胞24 h左右开始贴壁,8 d左右可达90%融合,基本上为梭形成纤维样细胞形态,从第3代开始细胞稳定增殖,15代以前具有活跃的增殖能力。流式细胞学鉴定显示CD44、CD90、CDl06为阳性表达,CD34、CD45、和CDllb为阴性表达,脂肪干细胞能持续的表达G-CSF、GM-CSF、M-CSF、SCF、IL-6,统计学分析提示各代细胞因子的分泌呈缓慢下降趋势。结论成功地建立了一种分离培养大鼠脂肪干细胞的方法,其生长稳定、增殖较快,可以作为组织工程的种子细胞,其稳定的表达多种造血因子,找到了一条促进造血干细胞增殖的新途径。
Objective To establish the method of isolating and culturing adipose-derived stem cells from SD rat adipose tissue in order to observe its generation in vitro and study the hematopoietic cytokines of its secreted.Methods The subcutaneous adipose tissue was obtained from iliac region of rat under the aseptic condition and was digested with 0.1% fetal bovine serum and observed by insertedmicroscope constantly to see its changes.The expression of CD90,CD34,CD44,CD45,CD106 and CD 11b were assayed by flow cytometry,hematopoietic cytokines were assayed by ELISA.That data were collected and analyzed statistically.Results The primary culture showed that adipose-derived stem cells began to adhesion in 24 hours and about 90% fuse in 8 days approximately with fibroblast-like shape.Adipose-derived stem cells had the capability to proliferate successively from passage 3 and the capability last until passage 15.CD44,CD90,CD106 were positive while CD34,CD45,and CD 11b were negative by flow cytometry The hematopoietic cytokines G-CSF,GM-CSF,M-CSF and IL-6 were secreted by adipose-derived stem cells continually.Statistical analysis showed that the generation of cytokine secretion showed a slow downward trend.Conclusion The method to isolation and culture of adipose-derived stem cells was successfully established and the cells can grow stably and proliferate rapidly in vitro.Adipose-derived stem cells could be used as seed cells for tissue engineering as they can secrete hematopoietic cytokines G-CSF,GM-CSF,M-CSF,SCF,and IL-6 continually.Anew ways was found of promote hematopoietic stem cells proliferation.
出处
《贵州医药》
CAS
2009年第7期579-582,共4页
Guizhou Medical Journal
基金
贵州省省长基金项目[黔省专合字(2006)54]
国家自然科学基金基项目(30760276)
关键词
脂肪干细胞
细胞培养
造血因子
Adipose-derived stem cells Cell culture Hematopoietic cytokines
