摘要
目的：脂肪间充质干细胞是一类具有多向分化潜能的干细胞,在特定培养条件下可分化为骨、软骨、脂肪、神经、心肌、内皮细胞等。实验拟采用高密度、多种生长因子体外诱导兔脂肪间充质干细胞向软骨细胞分化。方法：实验于2007-01/12在解放军兰州军区总医院实验中心完成。①实验材料：7d龄乳兔6只,雌雄不限,体质量150-200g,由兰州生物制品研究所动物中心提供,实验过程中对动物处置符合动物伦理学标准。②实验方法：分离乳兔脂肪间充质干细胞,体外扩增至第3代,高密度诱导组按Zuk等高密度微团培养法调整细胞密度为1×10^10L^-1高密度培养,加入10μg/L转化生长因子β1、50μg/L胰岛素样生长因子、100μmol/L地塞米松、50mg/L维生素C诱导成软骨;低密度诱导组按5×10^8L^-1低密度培养,并加入与高密度诱导组相同的诱导剂诱导培养;高密度常规培养组按1×10^10L^-1高密度加入常规培养基培养;对照组按5×10^8L^-1的密度接加入常规培养基培养。③实验评估：诱导7、14、21d后观察细胞形态学变化,应用MTT法检测细胞生长曲线,RT-PCR、免疫细胞化学法检测Ⅱ型胶原表达,阿尔新蓝染色检测糖氨多糖的合成。结果：①兔脂肪间充质干细胞经诱导后生长速度减慢,由长梭形细胞变为短梭多角细胞,呈铺路石状。常规培养的脂肪间充质干细胞潜伏期为一二天,3d为对数增殖期,第5天以后进入生长平台期。诱导的脂肪间充质干细胞潜伏期为1-3d,4d为对数增殖期,第6天进入平台期。②RT-PCR、免疫细胞化学法检测显示,高密度诱导组诱导14、21dⅡ型胶原阳性表达,阿尔新蓝染色见胞浆和细胞基质有棕黄色颗粒。低密度诱导组组诱导14、21dⅡ型胶原弱阳性表达,阿尔新蓝染色仅少量细胞基质着色。高密度常规培养组和对照组Ⅱ型胶原与阿尔新蓝染色均为阴性。结论：高密度培养的脂肪间充质干细胞经多种生长因子诱导可以向软骨细胞分化。
AIM： Adipose-derived mesenchymal stem cells possess the potential of multi-directional differentiation and can differentiate into osteocyte, chondrocyte, adipocyte, neural cells, myocardial cells and endothelial cells. The study investigated rabbit＇s adipose-derived mesenchymal stem cells differentiating into chondrocytes by high-density and multi-growth factor culture in vitro.
METHODS： Experiments were performed at Experimental Center, Army General Hospital of Lanzhou Military Area Command of Chinese PLA from January to December 2007. ①Six rabbits aged 7 days, of either sex, 150-200 g, were provided by Animal Center of Lanzhou Biological Product Institute. Animal intervention met the animal ethical standards. ②Adipose-derived mesenchymal stem cells were isolated from rabbit＇s adipose tissue and amplified to the 3rd generation. Adipose-derived mesenchymal stem cells were cultured in a condition of high density （1×10^10L^-1） according to the method ofZuk et al induced with transforming growth factor beta l （TGF-β 1） （10 μg/L）, insulin-like growth factor （IGF） （50 μg/L）, dexamethasone （100μmol/L） and Vitamin-C （50 mg/L） in the high-density induction group. Adipose-derived mesenchymal stem cells were cultured in a condition of low density （5 × 10^8·L^-1） induced with the same inductor as above mentioned in the low-density induction group. Cells in the high-density conventional culture group were cultured in conventional medium at 1×10^-1· L^-1, whereas those in the control group were incubated in conventional medium at 5×10^8 L^-1.③The morphological changes in the cells were observed at 7, 14, 21 days after induction. Cell growth curve was measured by MTF. The expression of collagen type II was detected with RT-PCR and immunocytochemical method. Aggrecan synthesis was detected by alcian blue staining.
RESULTS：①Adipose-derived mesenchymal stem cells induced by high density culture and multi-growth factors were slowly changed their shape from long spindle cells into short spindle and paving stone-shaped cells. The latent phase of the primary culture cells was 1-2 days, followed by 1 day of logarithmical proliferation; the plateau began at day 5. The latent phase of induced cells was 1 3 days, followed by 1 day of logarithmical proliferation; the plateau began at day 6. ②RT-PCR and immunocytochemical method showed positive expression of collagen type II at days 14 and 21 and alcian blue staining showed buffy granula in kytoplasm in the high-density induction group. Weakly positive expression of collagen type II was detected at days 14 and 21, and alcian blue staining showed a few buffy granula in kytoplasm in the low-density induction group, collagen type II and alcian blue staining were negative in the high-density conventional culture group and control group.
CONCLUSION： Adipose-derived mesenchymal stem cells can be differentiated towards chondrocytic phenotype induced by high-density, multi-growth factor culture.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第16期3092-3096,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
青海省重点科技项目(2005-N-116)~~
