摘要
目的:分离纯化人胎肾中的间充质样干细胞,在化学因子作用下诱导其定向分化,并对其生物学特性进行初步鉴定。方法:实验于2005-10/2006-01在暨南大学医学院血液病研究所完成。①利用二步离心法、密度梯度离心法及细胞差速贴壁生长特性分离纯化人胎肾间充质样干细胞。细胞来源为自然流产胎儿,供者知情同意。②流式细胞仪检测其细胞周期和表面标志。③添加常规诱导液诱导其向脂肪分化犤以脂肪诱导分化液(DMEM/F12+体积分数为0.1的胎牛血清+1μmol/L地塞米松+5u/mL胰岛素+200μmol/L消炎痛)进行培养犦、成骨细胞分化(完全培养基中加入0.1μmol/L地塞米松,10mmol/Lβ-磷酸甘油,50μmol/L维生素C进行培养)并加以鉴定。结果:从人胎肾中成功分离纯化得到间充质样干细胞:①P6代细胞有79.1%的细胞处于静止期/DNA合成前期。②表达CD29,CD44,CD105和CD166,不表达造血细胞标志CD15,CD34,CD45。③不表达与移植物抗宿主病相关的HLA-DR,CD80,CD86,CD40,CD40L。④在向成骨分化的诱导液中培养后,间充质样干细胞发生形态变化,碱性磷酸酶染色阳性,出现钙结节;在脂肪诱导分化液内培养后,细胞形态发生变化,油红O染色阳性。结论:人胎肾中含有间充质样干细胞,人胎肾间充质样干细胞具有多向分化潜能,且免疫原性弱,可以作为组织工程和细胞治疗种子细胞的来源之一,但目前存在较难分离纯化,细胞产量不大的不足。
AIM： To isolate and purify the mesenchymal-like stem cells （MSCs） in human fetal kidney, and induce their oriented differentiation by chemical factors as well as primarily identify their biological characteristics. METHODS： The experiment was conducted at the Institute of Hematology, Medical College of Jinan University from October 2005 to January 2006. （1）Two-step centrifugation, density gradient centrifugation and characteristics of cell different adherence growth were used to isolate and purify the MSCs of human fetal kidney. The cells were obtained from aborted fetus, the providers of which all knew and agreed.（2）Flow cytometer was adopted to inspect the cell cycle and surface markers. （3）Regular inductive medium was added to induce the MSCs to differentiate to lipid [cultured with adipogenic inductive agents （DMEM/FI2 ＋ 0.1 of fetal bovine serum ＋ 1 μmol/L decaspray ＋ 5μ/mL insulin ＋ 200 μmol/L antinfan）] and osteoblast （The complete medium was added into 0.1 μmol/L decaspray, 10 mmol/L β-glycerol phophate, 50 μmoL/L vitamin C for culture）, which were then identified. RESULTS： MSCs obtained from successful separation and purification of human fetal kidney：（1）79.1% of cells of passage 6 were at metabolic stage/ presynthetic phase. （2）The MSCs expressed CD29, CD44, CD105 and CD166, but not antigens of hematopoietic CD15, CD34 and CD45. （3）No antigen in the expression of MSCs is related with graft-versus-host disease （GVHD） such as HLA-DR, CD80, CD86, CD40 and CD40L.（4）Exposure of these cells to osteogenic inductive agents resulted in morphological changes of MSCs, positive expression after alkaline phosphatase staining and hydroxyapatite nodules. Incubation with adipogenic inductive agents resulted in morphological changes and positive results when staining with Oil Red O. CONCLUSION： There are MSCs in human fetal kidney, which have multi-differentiation potency and weaker immunogen, and it is one of the sources of tissue engineering and cell therapy. However, at present it is difficult to isolate and purity the MSCs, and the output is insufficient.
出处
《中国临床康复》
CAS
CSCD
北大核心
2006年第25期17-20,共4页
Chinese Journal of Clinical Rehabilitation
基金
国务院侨办重点学科建设基金(2005)
广东省自然科学重点基金(021195)~~
